key: cord- -y wjp aj authors: yuan, shishan; mickelson, daniel; murtaugh, michael p.; faaberg, kay s. title: erratum to “complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains” date: - - journal: virus res doi: . /s - ( ) - sha: doc_id: cord_uid: y wjp aj two full-length porcine reproductive and respiratory syndrome virus (prrsv) genomes, strain vr- and its cell culture passaged descendent respprrs vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. of the nucleotide changes, resulted in conservative changes and produced non-conservative changes. the results suggest that key amino acids in orf may contribute to the phenotype of respprrs, which includes increased growth rate on ma- cells and decreased virulence in swine. the results provide a genetic basis for future manipulation of a prrsv reverse genetics system. porcine reproductive and respiratory syndrome virus (prrsv) was first recognized as a 'mystery swine disease' in the united states in (keffaber, ). since the virus was identified in europe (lelystad virus; wensvoort et al., ) and in the usa (vr ; benfield et al., ; collins et al., ) , prrsv has become a serious pathogen of swine herds world-wide (rossow et al., ) . prrsv, along with lactate dehydrogenase-elevating virus (ldv), equine arteritis virus (eav) and simian hemorrhagic fever virus (shfv), is a member of the family arteriviridae in the order nidovirales (cavanagh et al., ; plagemann and moennig, ) . prrsv causes respiratory disease in young swine and production of mummified, weakborn or aborted piglets in pregnant sows through an unresolved mechanism. clues to the genetic basis of pathogenicity can be gleaned from comparison of the nucleotide sequence of a virulent parent viral strain with that of the cell-passaged attenuated variant. one such pair is the prototype north american isolate, strain vr- , and resp-prrs, its ma- cell adapted attenuated descendent. in this article, we report the complete nucleotide sequence of both strains of prrsv and have deduced and characterized their nucleotide and protein differences. several key amino acid changes between parental and vaccine strains were identified using this approach, but many other nucleotide and amino acid changes also occurred in genomic regions of unknown function. the nucleotide sequences were also compared to strain b, which is claimed to be a virulent field revertant of respprrs (allende et al., ) . a full comparison of all three prrsv strains shows that the origin of b ('michelle' strain) is unclear. prrsv strains vr- and respprrs have been described in previous reports nelsen et al., ; yuan et al., ) . strain b (michelle) was described by allende et al. ( ) . for growth curve analysis, ma- cells were infected with each prrsv strain at an m.o.i. of . . after h of adsorption, the unattached virus was washed off of the cells and ml of fresh cell medium (emem/ % fbs or dmem/ % swine serum) was added. the culture was placed at °c, % co and . -ml aliquots of virus were removed and replaced with . ml fresh medium at , , , , , , , , and h. viral plaque assays of each time point were completed on ma- cells with a -ml agar overlay (an equal volume of mixture of % low gelling point agarose (fmc seaplaque) and ×mem medium). incubation at °c, % co for - h revealed the presence of plaques, which were quantitated for each time point. prrsv was harvested from infected ma- cells on day post-infection (p.i.). after pelleting of cellular debris at rev./min, the supernatants were layered onto a ml . m sucrose cushion in an sw ultracentrifuge tube and centrifuged at rev./min for h. the pelleted virions were resuspended in ste buffer ( ml; mm nacl/ mm tris ph . / mm edta, °c) and transferred to a microcentrifuge tube. another ml of ste was used to collect any remaining disrupted virions and the two fractions of viral suspension were pooled and stored at − °c. viral rna was isolated using the qiaamp viral rna kit (qiagen) and stored at − °c after quantitation by optical density and native rna agarose gel electrophoresis. approximately mg of purified viral rna in ml was added to ml of mm reverse primer / a-p ( %-ggt-cgttgacaagttggtcatctaccggttt-atcctcgga), incubated at °c for min and placed on ice. first strand cdna synthesis was then completed as described previously (yuan et al., ) . the reverse transcribed product was purified (microcon , amicon) and the cdna was eluted in ml of rnase-free water. multiple adenosine or guanosine residues were added to the %-end of purified prrsv cdna ( mg) using a terminal deoxynucleotide transferase procedure described by the manufacturer (tdt; new england biolabs), diluted to ml and stored at − °c. first round pcr was completed as described previously using mm qt ( %-ccagt-gagcagagtgacgaggactcgagctca-agcttttttttttttttttt) or qc ( %-cca-gtgagcagagtgacgaggactcgagct-caagcccccccccccccccccc) and mm qo ( %-ccagtgagcagagtg-acg) as the forward primers and mm / a-p as the reverse primer (frohman, ; yuan et al., ) . an identical second round of pcr was then completed using ml first round pcr product and qi ( %-gaggactcgagctcaagc) and / a-p ( %-ccttcggcaggcggggagta-gtgtttgaggtgctcagc; mm each) as the primer pair. automated sequencing reactions were completed with taq dyedeoxy terminator cycle sequencing kit (applied biosystems) using a pe thermocycler (perkin-elmer) at the univer-sity of minnesota advanced genetic analysis center. comparison of the parental and vaccine genomes and deduced orf amino acid comparison was completed using computer software included in the lasergene package (dnastar inc., madison, wi), wisconsin package version . and (genetics computer group (gcg), madison, wi). genbank accession numbers used for sequence analysis include the complete vr- (u , nelsen et al., ) , respprrs (af ; yuan et al., ) and b (af ) sequences. primer extension experiments had suggested that nucleotides were unaccounted for in the published sequence of strain vr- . in order to determine the unresolved bases, we derived several clones of pcr products generated from different passages of vr- and respprrs (fig. ) . initially, several standard %-race was performed on passage from the original vr- field isolate brain homogenate, passage of strain vr- and passage of respprrs vaccine strain (frohman, ) . three different %-terminal base patterns were elucidated, with added ambiguity due to the poly t tract added during the %-race procedure, immediately preceding an apparent base prrsv sequence gacguauagguguuggc. interestingly, heterogeneity at the %-end was detected early during passaging of the original prrsv field isolate, yet no heterogeneity was seen after viral plaque purification to produce strain vr- fig. . %-terminal nucleotides of strains vr- and respprrs. vr- (vr) or respprrs (r) from different passages were subjected to rapid amplification of cdna ends (race) and products were cloned and sequenced as described in section . vr- represents sequence submitted to genbank prior to elucidation of the % terminal nucleotides. strain vr- -infected brain homogenate was passaged two times on two separate occasions (vr- a, vr- b), or plaque purified and passaged (vr- ) or (vr- ) times. respprrs was passaged four (r- ) or (r- ) times. bold letters indicate previously unresolved bases and letter in italics refer to the non-viral nucleotides (t or c) arising from the race procedure. ( fig. ). further analysis of pcr clones derived from %-race experiments using poly (g) addition discriminated the remaining % terminal bases of strains vr and respprrs. only one additional base, corresponding to an adenine residue, was identified for strain vr- ( fig. ) . two additional bases, ta, were detected for the % terminal bases of four pcr clones derived from vaccine strain respprrs. the number of subsequent passages shown in fig. ( , , , , ) indicates the relative stability of the cloned virus strains. these results are discordant with a recent report with respect to the first nucleotide of vr and resp-prrs. oleksiewicz and investigators reported a %-terminal u in strain vr- and lack of such in strain respprrs (oleksiewicz et al. ) , whereas we observed a %-terminal u only in the respprrs vaccine strain. the complete leader sequence of respprrs vaccine strain shares . % identity with another prrsv vaccine strain, primepac prrs (shen et al., ) (data not shown). we have determined that the genome size for vr- is bases (u ) and that of respprrs, with an extra %-terminal u, is bases (af ). respprrs leader sequence acquired two additional mutations when passaged for another eight passages at high m.o.i. (ua base and uc at base ). from the sequence data, we cannot determine exactly when the %-terminal thymidine residue of strain respprrs was acquired during passaging. however, the possibility exists that strain respprrs has developed individual genomes of variable nucleotide sequence due to strain evolution over the course of more than passages. the respprrs sequence which we derived from -to -fold coverage of the genome (af ) is somewhat different from the sequence of re-spprrs recently reported (allende et al., ) . complete genome analysis of the parental strain vr- and its cell-culture adapted descendant, strain respprrs, revealed that nu-cleotides had changed during viral strain attenuation. the changes appeared throughout the genome ( fig. a) , except for orf and the % untranslated region (utr), and no single orf exhibited less than the % identity between parental and vaccine strains (fig. b) . of the altered nucleotides, resulted in silent mutations such that the encoded protein was not changed. one of these silent mutations resided in the %-leader, resided in orf , and one silent change was detected in orf . the rest of the nucleotide changes resulted in amino acid changes in each of the remaining identified orfs. detailed analysis of the nucleotide changes, corresponding amino acid changes and potential coding domains are listed in table . for ease of understanding, nucleotide numbering in table is based on the sequence of vr- ( - ). the %-terminal uracil residue of strain respprrs is numbered as zero, and all site designations are one less than the value of the sequence deposited in genbank. the resolution of the % terminal sequence indicated that the % ends of both of these prrsv strains coded for a aa peptide. however, no conclusive evidence has been generated to demonstrate the presence of this potential leader protein. the leader sequence of resp-prrs suggested some virus heterogeneity. in one case (table , mutation a), u a (vr- respprrs) coded for a conservative change at amino acid (f y). in the other case (table , mutation b), the c u nucleotide change, located in the third base of the st codon in this polypeptide sequence, is silent. rna folding predictions (mfold; zucker, ) suggested that each of these nucleotide changes resulted in minor alterations in nucleotide-pairing in stems of secondary structure. the %-terminal bases, an a residue in strain vr- and an at dinucleotide sequence in vaccine strain respprrs, are predicted to extend % from a long stable hairpin located immediately downstream between nucleotides and (data not shown). transversion ( ) g c a analysis of complete genome sequences and classification of amino acid changes were completed using gcg computational biology computer programs. predicted domains of viral proteins was based on hopp-woods analysis of peptide structure (jameson and wolf, ) were also completed using gcg. predicted orf non structural proteins (nsp) were derived from genome comparison of strain vr- with prrsv strain b (allende et al., ) and with equine arteritis virus van dinten et al., ) . b represent two separate sequence analyses of respprrs leader (fig. ) . full-genome schematic the nucleotide differences between vr- and respprrs reveals several changes occurred in orf a, a cluster of changes were seen in the %-terminus of orf b, and discrete changes were seen in all envelope glycoproteins (orfs - ) and in the membrane protein (orf ). when respprrs is similarly compared to strain b, many nucleotide changes are seen throughout the genome. (b) each region of the prrsv genome was analyzed for the number of nucleotide changes and the corresponding orf percent identity between vr- and respprrs. several prrsv orf replicase protein domains have been identified (fig. ; snijder and meulenberg, ; allende et al., ; . from the analysis of both vr- and respprrs genomes, it was deduced that only four of the orf specific nucleotide changes occurred in these identified domains. one mutation, a c u transition, which would alter amino acid of orf from a serine residue in strain vr- to a phenylalanine in the attenuated strain respprrs (s f), resulted in a nonconservative change located in the putative orf cleavage product, nsp (den boon et al., ; allende et al., allende et al., , nelsen et al., ) . a second recognizable mutation, a u c transition, resulted in a change from tyrosine to histidine within the helicase domain. the final orf recognizable mutation was located in the coronavirus-like domain. in this instance, the transversion from g c resulted in amino acid undergoing a non-conservative change from glycine to alanine. a glycine residue at this position, except for strain respprrs, has been shown to be conserved in all nidoviruses analyzed to date . all other amino acid changes located within orf are in regions of unknown function. however, the cluster of amino acid mutations located near the carboxyl terminal end suggests that the replicase region was altered during passage to result in a more fit virus for replication in cell culture, as evidenced by the in vitro one-step growth curve comparison shown in fig. . the growth curve comparison appears to be the reverse of virus strain replication in swine (m. roof, unpublished data). prrsv orfs - have been shown to code for four glycoproteins (gp - ), the membrane protein (m) and the nucleocapsid protein (n), respectively. sequence analysis of strains vr- and respprrs indicated that there were nucleotide changes in this region, and all but one of which resulted in amino acid alterations. there were four amino acid mutations within gp . one amino acid change (aa , lf) was located within the predicted signal sequence of gp , but the nucleotide mutation (g u ) also altered amino acid from an aspartic acid to a tyrosine of a putative orf a protein (wu, christopher-hennings, and nelson, unpublished data) . the remaining three gp changes (aa , a s; aa , k r; aa , v m) were clustered in an eight amino acid region in the middle of the protein sequence, predicted to be located on the exterior of the virion. the three amino acid fig. . viral growth curves of prrsv strains vr- () and respprrs () reveal that respprrs has enhanced growth kinetics in vitro. results are representative of three separate experiments. the number of plaques were determined in triplicate for two separate viral dilutions and were found to deviate by less than % in each individual experiment. vealed that this mutation had reverted back to a vr- -like sequence while maintaining all other orf and mutations and the avirulent viral phenotype (data not shown). the other m protein mutation was shown to be the result of two nucleotide changes within the codon for amino acid , altering the sequence from an arginine to a glycine (r g). curiously, both carboxyl terminal gp and m mutations resulted in a r g change. in , prrsv strain b was isolated from a nebraska herd and has since been shown to be virulent (allende et al., (allende et al., , . full-length genome sequencing revealed that this strain was % identical to vr- and respprrs (allende et al., ) . whether the isolate is a naturally occurring prrsv field variant, or directly related to the original vr- field isolate (isolated in ) or the respprrs vaccine strain (released in ) is not known. to address this question, a full-length genome comparison of strain resp-prrs to strain b was completed, as it has been suggested that strain b is a field revertant of respprrs (allende et al., ) (fig. a) . as can be seen in fig. a , strain b differs from respprrs at many nucleic acid residues. all three strains were analyzed in detail. we compared identical and non-identical nucleic acid residues between strains vr- and respprrs, strains vr- and b, between respprrs and b, and between all three strains. we observed instances in which all three viral genomes contained the identical base, instances in which strains vr- and respprrs were identical but different from strain b, cases in which strains vr- and b were identical but different from strain respprrs, and instances in which strains respprrs and b were identical but different from vr- . importantly, no instances were observed in which the three strains were all different from one another. if strain b were in fact derived from a field reversion of respprrs, the data would imply that for nucleotide mutations b reverted back to the exact vr- nucleotide sequence and in no modifications that occurred in gp (aa , ge; aa , g s; aa , a t) and the two in gp (aa , c y; aa , v a) also are predicted to lie on the exterior of the virion. all of these mutations but one are semi-or non-conservative changes. in the putative viral attachment protein gp , two amino acids were altered during virus attenuation. one gp change occurred within the proposed signal sequence (aa , r q) and the other change (aa , rg) is predicted to be located in the virion interior . gp has been shown to be disulfide linked to the m protein mardassi et al., ) and studies with a similar virus have revealed that this disulfide bond is critical to infectivity . while no mutations occurred in the extravirion region of gp , one mutation did occur within the m protein (aa , q e) predicted to be located on the outer surface of the virus. although this m protein mutation was conservative, mutations in this critical region of the prrsv viral attachment heterodimer could be important. however, analysis of the sixth in vivo backpassage of respprrs re-instance would it have mutated to either of the two possible remaining bases. the likelihood of such a probability is exceedingly low. we also considered the possibility that strain b resulted from a viral recombination event between strain respprrs and an unidentified but closely related field isolate. however, no region of considerable length was clearly identified as being derived from strain respprrs, which one would expect if viral recombination had taken place. therefore, the origin of b remains unclear. an attenuated prrsv strain, respprrs, was found to differ in sequence from the parental strain, vr- , by only nucleotides. this represents a . % variation during passages in ma- cells. this rate of variation is comparable to other rna viruses (steinhauer and holland, ) and suggests that prrsv is not susceptible to inherently high spontaneous mutation rates in the absence of immunological and environmental pressures. this may explain the relative stability of strain vr- when passaged alone in cell culture, as this report delineates. however, prrsv has been shown to undergo high frequency recombination in the presence of two or more virus strains (yuan et al., ) and appears to evolve rapidly in the presence of biological pressure. attenuation can result from changes in many areas of viral genomes and the nucleotide mutations described include alterations in several key prrsv regions. thus, no definitive site of prrsv attenuation can be identified simply from sequence analysis. twelve of the nucleotide changes in strain respprrs were silent. however, one or more of these silent mutations could change the secondary or tertiary structure of prrsv rna and thus alter the stability of the genome. in other viruses, such as picornaviruses, viral rna folding may play a critical role in host protein association (meerovitch et al., ) and in neurovirulence (pilipenko et al., a,b; westrop et al., ) . the % and % ends of viral sequences have been correlated with attenuation in other viruses, such as poliovirus (westrop et al., ) , and shown to be important in viral replication and transcription for coronaviruses, another member of the nidovirus order (williams et al., ) . only two mutations occurred in the %-end of prrsv during attenuation of strain vr- to respprrs, and their relative roles in attenuation must be further investigated. no change occurred in the %-end of the prrsv genome. viruses may also acquire attenuated phenotypes as a result of mutations in viral proteases (ni et al., ) , protease cleavage sites (tozser et al., ) , within the polymerase gene (pelosi et al., ; skiadopoulos et al., ) , by altering viral proteins to decrease virion stability (bailly et al., ) or to interfere with the virus/host interaction (mccright et al., ) , as well as by other mechanisms. the comparison of vr- to its vaccine correlate, resp-prrs, revealed that the possibility exists for one or more of these mechanisms to be involved in attenuation. a recent publication suggested that nine specific amino acid changes may be important in attenuation of respprrs (allende et al., ) , possibly based on the assumption that prrsv attenuation may be localized by amino acid comparison of a non-parental genome ( b) with a parental (vr- ) and its vaccine offspring (respprrs). we have determined that our sequence of the respprrs strain is somewhat different from what has been previously reported (allende et al., ) , which brings additional complexity to the derivation of potential prrsv attenuation sites. in addition, the %-terminal nucleotides for both vr- and respprrs are markedly different from those reported for strain b. in our view, prrsv attenuation sites may be genotype specific and variable, possibly resulting from a complicated interplay of genomic regions and viral and host specific factors. postulation of attenuation sites can lead to interesting hypothesisbased experiments. however, a reverse genetics system for prrsv must be produced and genetically altered at specific nucleotides in order to discern sites of attenuation. north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype a recombinant human parainfluenza virus type (piv ) in which the nucleocapsid n protein has been replaced by that of bovine piv is attenuated in primates characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) recommendations of the coronavirus study group for the nomenclature of the structural proteins, mrnas, and genes of coronaviruses isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs processing and evolution of the n-terminal region of the arterivirus replicase orf a protein: identification of two papainlike cysteine proteases the envelope proteins of lactate dehydrogenase-elevating virus and their membrane topography disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity on beyond classic race (rapid amplification of cdna ends) the antigenic index: a novel algorithm for predicting antigenic determinants reproductive failure of unknown etiology intracellular synthesis, processing, and transport of proteins encoded by orfs to of porcine reproductive and respiratory syndrome virus theiler's viruses with mutations in loop i of vp lead to altered tropism and pathogenesis a cellular protein that binds to the %-noncoding region of poliovirus rna: implications for internal translation initiation porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents molecular basis of attenuation of neurovirulence of wild-type japanese encephalitis virus strain sa determination of %-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs a herpes simplex virus dna polymerase mutation that specifically attenuates neurovirulence in mice conservation of the secondary structure elements of the %-untranslated region of cardioand aphthovirus rnas conserved structural domains in the %-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus: a new group of positive stranded rna viruses porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterized by marked neurovirulence determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp gene with a unique insertion identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated cold-passage (cp ) human parainfluenza virus candidate vaccine the molecular biology of arteriviruses identification of a novel structural protein of arteriviruses rapid evolution of rna viruses effect of serine and tyrosine phosphorylation on retroviral proteinase substrates proteolytic processing of the open reading frame b-encoded part of arterivirus replicase is mediated by nsp serine protease and is essential for virus replication mystery swine disease in the netherlands: the isolation of lelystad virus genetic basis of attenuation of the sabin type oral poliovirus vaccine a phylogenetically conserved hairpin-type % untranslated region pseudoknot functions in coronavirus rna replication recombination between north american strains of porcine reproductive and respiratory syndrome virus on finding all suboptimal foldings of an rna molecule the authors wish to thank chris nelsen and faith klebs for excellent technical expertise. boehringer ingelheim vetmedica, inc., provided financial support for the research. key: cord- -m flptcv authors: bossé, ynuk title: the strain on airway smooth muscle during a deep inspiration to total lung capacity date: - - journal: j eng sci med diagn ther doi: . / . sha: doc_id: cord_uid: m flptcv the deep inspiration (di) maneuver entices a great deal of interest because of its ability to temporarily ease the flow of air into the lungs. this salutary effect of a di is proposed to be mediated, at least partially, by momentarily increasing the operating length of airway smooth muscle (asm). concerningly, this premise is largely derived from a growing body of in vitro studies investigating the effect of stretching asm by different magnitudes on its contractility. the relevance of these in vitro findings remains uncertain, as the real range of strains asm undergoes in vivo during a di is somewhat elusive. in order to understand the regulation of asm contractility by a di and to infer on its putative contribution to the bronchodilator effect of a di, it is imperative that in vitro studies incorporate levels of strains that are physiologically relevant. this review summarizes the methods that may be used in vivo in humans to estimate the strain experienced by asm during a di from functional residual capacity (frc) to total lung capacity (tlc). the strengths and limitations of each method, as well as the potential confounders, are also discussed. a rough estimated range of asm strains is provided for the purpose of guiding future in vitro studies that aim at quantifying the regulatory effect of di on asm contractility. however, it is emphasized that, owing to the many limitations and confounders, more studies will be needed to reach conclusive statements. the beneficial effect of a deep inspiration (di) on respiratory mechanics has long been an important topic of discussion among lung physiologists [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the mechanism accountable for the respiratory relief that is normally afforded by a di is still not clear [ , ] . several mechanisms are likely involved and each mechanism may contribute to various extents in different individuals. however, one mechanism that arguably received the most attention is the transient change of length (i.e., strain or stretch) that airway smooth muscle (asm) undergoes during the di . indeed, the expansion of lung volume during a di modifies the caliber of the airways (table ) ; the word "caliber" herein refers to the cross-sectional area of the airway lumen. the increase in caliber during a di results from increasing radial stress that stems from the tethering force of the lung parenchyma. since asm is embedded within the airway wall in an orientation that is nearly orthogonal to the long axis of the airways [ , ] , an increase in airway caliber necessarily implies that asm is transiently elongated. the question of whether, and to what extent, strain affects the contractility of asm is easier to investigate in vitro. this is because in vitro experiments can specifically control or monitor the length of asm. several types of asm preparations derived from resected or postmortem lung specimens are used to study asm mechanics. they include rings, spirals and segments of bronchi; strips and segments of tracheas; precision cut lung slices; and strips of lung parenchyma. our understanding of asm mechanics evolves quickly due to the use of these in vitro preparations. they can truly address questions that would otherwise be impossible to investigate in vivo. nowadays, the tools to study asm mechanics in vitro can recreate the dynamic lung movements that occur during breathing maneuvers . they thus emulate the in vivo environment within which asm normally resides and operates. this was a steppingstone in the field of asm mechanics. it has provided instrumental insights regarding the response of asm to stress and strain. these in vitro studies demonstrated that perturbing the length of asm greatly affects its contractile capacity. this effect is certainly relevant to the bronchodilator effect of a di. however, the reported in vitro results are also plagued with a lack of consistency. the main concern relates to the size of the effect, i.e., the magnitude by which strain decreases the contractile capacity of asm. however, the discrepancies go as far as being contradictory, as some studies reported that strain increases both airway responsiveness in vivo [ ] [ ] [ ] and the contractile capacity of asm in vitro [ ] . the controversy also extends to studies performed with isolated asm cells, where it was shown that strain can both decrease and increase surrogates for asm contractility [ , [ ] [ ] [ ] [ ] [ ] . all together, these findings are conducive to alternative conclusions regarding the potential contribution of asm strain to the bronchodilator effect of di. the purpose of the present review is not to discuss the variety of factors that may be responsible for the inconsistencies reported in in vitro studies, but rather to focus on one possible source of error, namely the magnitude by which asm is strained during a di. i attempted, based on published studies on human subjects in vivo, to determine the physiological range of strains that asm undergoes in vivo during a di from functional residual capacity (frc) to total lung capacity (tlc). the first section describes the methods that can be used to estimate asm strain. the second section discusses the limitations associated with those methods and to which extent they may affect the accuracy of the reported strain. finally, the third section describes the confounders that affect asm strain and that inevitably contribute to its variability. [ ] v d (using co as the tracer gas) healthy baseline exercise, co inhalation ( - %) or both all . % [ ] v d (using co as the tracer gas) healthy baseline always from tlc, expired to a given lung volume, and then inhaled different allocated tidal volume to subsequently measure v d on the subsequent expiration. all . % [ ] whole-body plethysmography the complexity of the question in hand stems from the difficulty to assess asm strain in vivo. although refining technologies are currently emerging, no direct measurement of asm strain during a di has yet been performed in vivo. however, many technologies provide reliable measures of luminal volume, airway caliber and resistance. these measurements at different lung volumes then allow approximation of the extent to which asm is strained during a di from frc to tlc. this section describes the methods that may be used to assess airway wall strain in vivo in humans. the major strengths of each method are also briefly stated. . dead volume. the measurement of dead volume (v d ) is the more archaic [ ] but still useful way to estimate the luminal volume of the airway tree at any given lung volume. the change in v d from one lung volume to the other then allows estimation of the strain undergone by the airway tree during such an excursion of lung volume ( table ). the measurement relies on the fact that the last part of the inspired air in a breath does not reach the zone of gas-exchanging units. that last part is rather contained in the v d (also called the dead space), which includes the upper airways down to the terminal bronchi. since air in the v d does not mix with the alveolar gas, the composition of gas at the mouth during an expiration suddenly changes when it transitions from the air that was contained in the v d to the air that was contained in the alveolar space. the volume of air that comes out of the lungs before this transition of air composition is v d . because diffusion inevitably occurs at the boundary between v d and alveolar volume, and that the amount of diffusion is time-dependent, appropriate timing during the respiratory maneuver is essential to obtain an accurate measure of v d . fowler was the first to use the single-breath nitrogen (n ) washout to measure v d [ ] . this technique has been adopted and is still used [ ] [ ] [ ] . alternatively, some have used co as the tracer gas to estimate v d [ ] . johns and coworkers have demonstrated that a breath-by-breath analysis of v d is possible using co as the tracer [ ] . by progressively changing the lung volume, one can then obtain a slope of the relationship between v d and end-inspiratory lung volume. this slope has been abbreviated dv d and has been used to assess airway distensibility (i.e., the ease by which the airways dilate in response to increasing lung volume) [ ] . dv d is advantageous because it can be measured quickly in a noninvasive manner. . although x-rays in humans may provide enough resolution to detect abnormal size of the trachea [ ] , it is not sufficient to quantitate the differences that may occur during an excursion of lung volume from frc to tlc. the resolution can be improved by contrasting agents. for example, dionosil oily inserted through the cricothyroid ligament under local anesthesia, has been used successfully to measure the changes in diameter of large and relatively smaller airways during a full inspiration in man [ ] . fluoroscopy is somewhat better, as it allows motion recording. it has been used without contrast agents to monitor changes in airway caliber during breathing maneuvers. it was to assess the degree of collapsibility of the trachea during coughing in controls and in cases of acquired tracheomalacia [ ] . fluoroscopy may be appropriate to diagnose and assign a degree of tracheomalacia, as it was done in that study. however, it does not seem to provide note: aoct-anatomical optical coherence tomography, copd-chronic obstructive pulmonary disease, di-deep inspiration, fev -forced expiratory volume in s, fot-forced oscillation technique, frc-functional residual capacity, hrct-high-resolution computed tomography, laba-long-acting b agonist, mch-methacholine, pc -the provocative concentration of methacholine causing a % decline in fev , raw-airway resistance, rv-residual volume, saba-short-acting b -agonist, sbnw-single breath nitogen washout, tlc-total lung capacity, vc-vital capacity, and v d -dead volume. a it was assumed that asm is arranged orthogonally in relation to the long axis of the airways. b it was assumed that the volume of the airways expands isotropically during an increase in lung volume from frc to tlc. c it was assumed that the changes in resistance are inversely proportional to the changes in airway luminal radius at the fourth power. the resolution to quantitatively evaluate the changes in airway luminal area during a di. whole-body plethysmography. the measurement of airway resistance (raw) by whole-body plethysmography (raw (pleth) ) is routinely performed in laboratories of lung physiology. raw (pleth) truly represents the resistance attributed to the viscous flow of air passing through the airways. the practical and theoretical principles involved were previously expatiated [ ] . briefly, the subject is enclosed in a hermetic box of known internal volume. the subject is then instructed to pant inside the box into a tube connected to an open shutter and a pneumotachograph. the shutter is then suddenly closed while the subject tries to keep panting. the airflow at the mouth and the pressures at the mouth and inside the box are monitored continuously. the variations of pressure inside the box (dpbox) when the shutter is open reflect corresponding but opposite changes in alveolar pressure (dpalv). using the law of boyle-mariotte (v p ¼ p v ), the dpbox are converted into changes in volume (dvbox: often called the shift volume). the dvbox happens to be the mirror image of the changes in thoracic gas volume (tgv) caused by rarefaction and compression of air during the panting, which are required to generate the driving pressure (dpalv) that draws air in-and-out of the lungs. when the shutter is closed, the dvbox can be related to the changes in alveolar pressure (dpalv), since dpalv are then the same as the changes in mouth pressure. once the relationships between both airflow/dvbox (when the shutter is open) and dpalv/dvbox (when the shutter is closed) are known, the dpalv required to accommodate any chosen airflow (usually l/s) can be determined. the ratio of dpalv/airflow is raw (pleth) . conveniently, the same panting maneuver and boyle-mariotte's law also allow the tgv from which the subject is panting to be measured. the panting can be performed at different lung volumes to measure the relationship between raw (pleth) and lung volume. from this relationship, the airway wall strain from frc to tlc can be roughly estimated ( table ). the limitations are discussed below. the greatest advantages of the whole-body plethysmography are that it is noninvasive and nonionizing. deflation. the interrupted deflation can be used to measure pulmonary resistance (r l ) and to estimate airway resistance (or conductance). the subject is instructed to take a di to tlc and then to expire slowly (sometimes at a controlled rate) while the flow is being interrupted at the mouth for a short period at specified intervals. the flow at the mouth and p l need to be measured simultaneously. p l is measured conventionally using an oesophageal balloon to estimate pleural pressure and to subtract it from airway opening pressure. during the measurement, the change in lung volume is being tracked by integrating flow. this is required to relate punctual changes in p l caused by the interruptions to the lung volumes at which they were measured. the increase in p l caused by the interruption is the driving pressure required to accommodate the airflow (and the lung tissue flow) just preceding the interruption. since the resistance attributed to tissue flow (sometimes called tissue viscance) is small [ ] , especially during deflation, the rise in p l caused by the interruption of flow is mainly ascribed to the frictional resistance to airflow within the airway tree. by dividing the increase in p l to the flow at the mouth, airway resistance can then be calculated at every lung volume at which the interruption took place. the change in resistance between frc and tlc can then be converted to estimate asm strain the same way it is done for raw (pleth) (discussed below) ( table ). the advantage of the interrupted deflation in comparison to the measurement of raw (pleth) is that the breathing maneuver performed by the subject is much simpler. technique. the acoustic reflection technique is an especially good tool to diagnose tracheal stenosis and malacia [ ] . the use of this technique to assess large airway caliber has also been validated in glass tube models and in humans [ , ] . changes in large airway caliber caused by changing lung volumes were also estimated ( table ). the acoustic reflection technique consists of a sound wave emitted by a loudspeaker that is traveling along the airways where it is reflected and perceived by a recording microphone located on the external device near the subject's mouth. the time gap between the emitted sound and the reflections is a measure of the distance between the microphone and the points within the airway where those reflections come from. the amplitude of the reflections is a proxy of the change in airway cross-sectional areas at those specified airway locations. the greatest advantages of the acoustic reflection technique are that it is noninvasive, nonionizing, and quick. it is also easy for the subject, as no complicated breathing maneuvers are required. . high-resolution computed tomography. high-resolution computed tomography (hrct) has first been used by wilson and coworkers in to assess airway caliber at different lung volumes [ ] . since then, hrct was used extensively to assess the bronchodilator effect of di ( table ). the breathing protocol used varied widely, but a scan is normally taken at frc and then at tlc and the differences in airway caliber at corresponding airway locations are measured. hrct is still considered by some the gold standard for imaging the airways. this is because it assesses directly the geometry of individual airways. it is also noninvasive and does not require sedation. the tools to analyze ct scan have also evolved to provide an accurate measure of airway wall thickness. another important asset is the fact that many airways of different sizes can be assessed simultaneously. magnetic resonance imaging (mri) is a technology that is currently emerging to image the geometry of airways in vivo in humans. the technology has evolved to offer a good temporal resolution (scanning time of . s), providing images without blurring, even during cough [ ] . although mri is primarily used in the field of pulmonary medicine to image ventilation defects [ ] [ ] [ ] [ ] [ ] , it can now be used to obtain descent three-dimensional reconstruction of the tracheobronchial tree [ ] . however, the resolution does not equate hrct [ ] . despite other limitations that will be discussed in sec. , mri still represents an appealing noninvasive and nonradiating alternative strategy to image airway caliber. further developments are awaited before mri can accurately assess the changes in airway caliber that occur during a breathing maneuver from frc to tlc (table ). probe. the pitot static probe is a device used to measure the velocity of a moving fluid. it has several applications in modern world. a miniature pitot static probe (few centimeters long and mm external diameter) can be inserted into the airway tree to assess airway caliber on the basis of physiological measurements. the probe is positioned at desired heights down the airway tree, which is determined prior the measurements using a bronchoscope. the probe has an opening at the distal end leading to a first catheter coming out at the mouth, which is used to measure impaction pressure (also called total pressure or ptot). the probe also has side holes that merge into a distinct catheter to measure lateral pressure (plat). the difference between impaction pressure and lateral pressure is the drop of pressure due to convective acceleration (i.e., the kinetic energy of the gas passing the cross-sectional area). assuming incompressible air flowing in a tube with a blunt velocity profile, bernoulli equation can then be used to estimate air velocity (ptot ¼ plat þ qv / , where q is air density and v is velocity). the flow at the pitot static probe can also be calculated from the mouth flow and correcting for difference in air pressure at that location (barometric pressure þ plat) using boyle's law. with this local flow and velocity, one can than calculate the cross-sectional area at the location of the pitot static probe ( is flow in l/s and v is velocity in m/s). calculating the cumulative expelled volume by integrating the flow at the mouth allows for changes in airway caliber to be related to changes in lung volume. theoretically, one maneuver would allow the measurement of airway caliber at every lung volume. the greatest advantage of the pitot static probe is thus to follow the kinetics of change in airway caliber. the insertion of an esophageal balloon as a proxy of pleural pressure (ppl) can also provide additional insights. for example, by relating airway caliber to transmural pressure (ptm, which is plat-ppl), airway compliance (caw) can be obtained. the pitot static probe is not often used to assess airway caliber at different lung volumes. in fact, only one study documented the utility of the pitot static probe for that purpose. in that study, the measurement was made during a maximal expiratory flow-volume maneuver (mefv), i.e., the subject was instructed to inhale to tlc and then provide a maximal expiratory effort to rv. several limitations are associated with this method. of special concern is the instability of the probe during the maneuver. the plugging of the end and side holes by mucus and secretions is also common. maximal reproducible efforts are also required, which can be demanding for the subject. these limitations, together with the other limitations discussed in sec. , may have dissuaded many investigators from using this method. technique. this technique consists of forcing the movement of air in or out the respiratory track using different devices and to then calculate impedance from the resulting flow and pressure that are measured at different locations in relation to the subject (reviewed in ref. [ ] ). the most common devices force air directly into the subject's mouth and the resulting flow and pressure within the mouth are used to calculate impedance (input impedance). the motion of air imposed by the device can also take different shapes, but sinusoidal forcing at one or several simultaneous frequencies are often used. during the measurement, the subject is usually instructed to keep breathing normally at tidal volume. the forced oscillation is thus simply superimposed on top of the natural motion of air resulting from breathing. the forced oscillation technique (fot) has gained tremendous momentum in human respiratory research. this is because it provides valuable readouts pertaining to the mechanics of the respiratory system, including respiratory system resistance (rrs) and reactance (xrs) at every tested frequencies. these measurements allow one to infer on phenomena as complex as recruitment-derecruitment and airway caliber heterogeneity. one of the greatest advantages of the fot is its fine time resolution. because of this fine time resolution, it is possible to measure the kinetics of events happening over a short time-scale, e.g., such as the extent and dynamics of airway dilatation during a di. its usefulness is also ascribed to its noninvasiveness, the ease with which it is operated and the very low level of cooperation that is required from the tested subjects. forced oscillation technique can be used to estimate asm strain during a di from frc to tlc because rrs and its inverse, respiratory system conductance (abbreviated grs), represent proper proxies of airway caliber when measured near the resonant frequency ( - hz) [ ] . this is especially true at zero flow, when the resistance is not affected by frictional airway resistance to airflow. many have measured rrs (or grs) to assess the changes in airway caliber during a di (see table ). . anatomic optical coherence tomography. anatomic optical coherence tomography (aoct) is an in vivo imaging technology that broadcasts live the interior of the airways [ ] . aoct uses a fiber-optic probe that passes through a bronchoscope and emits a beam of near-infrared light toward the airway wall. the resulting reflections are detected and analyzed using lowcoherence interferometry, which allows the measurement of the position of the airway wall in relation to the probe. by rotating the probe, one can obtained two-dimensional cross-sectional images of the lumen, and by moving the probe forward or backward, these serial two-dimensional images can provide a full d representation of the airway lumen. although not used extensively so far, aoct is likely to provide insightful information regarding the excursion of airway caliber during a di. i am aware of only one published study that has used aoct for that purpose [ ] (table ) . a new cousin of oct has also recently been developed, called orientation-resolved oct (or-oct) [ ] . or-oct uses the birefringent property of asm to capture its exact localization within the wall of large airways. asm in humans in vivo can be visualized with a sensational level of resolution by this method [ ] . or-oct should ultimately enable the direct measurement of asm strain during a di from frc to tlc. none of the aforementioned methods directly measure asm strain. they rather reported a change in the cross-sectional area of the lumen in individual airways, a change in luminal volume of the entire airway tree or a change in resistance to airflow in the airways or to the whole respiratory system. this obliges the transformation of the original data into radial airway wall strain. for the sake of this review, it was then assumed that the airway wall strain can be used as an appropriate proxy of asm longitudinal strain. this section describes the limitations associated with several methods, as well as the limitations associated with the transformations and assumptions that were made to convert the original data into asm strain. muscle. the strain on the airway wall reported by most methods during the di refers to the change in perimeter at the apical surface of the epithelium (i.e., the size of the lumen). this is true for imaging methods such as bronchography, hrct, mri, and aoct, but also for the methods relying of physiological measurements, such as v d and resistance (plethysmography, interrupted deflation, acoustic reflection technique, pitot static probe, and fot). it is important to understand that the change in luminal geometry does not represent precisely the change in perimeter occurring at the middle of asm. due to the combined thickness of the epithelium, the lamina propria, and the asm, the extent by which asm is stretched during a di is slightly overestimated when the luminal geometry (radius, diameter, perimeter, and cross-sectional area) is used to calculate the change in asm length (fig. ) . the size of this effect is more prominent in smaller airways. in fact, the size of this effect increases as the ratio of the perimeter measured at the middle of asm to the perimeter of the lumen increases. the thickening of the airway wall observed in some respiratory disorders also amplifies the size of this effect (fig. ). smooth muscle alignment. the reported strain (the one estimated in table ) represents more accurately the radial strain on the airway wall than the longitudinal strain on the asm. this is because the orientation of asm within the airway wall varies. here, i assumed that asm is arranged in an orthogonal fashion in relation to the long axis of the airways. this assumption is correct for the trachea and the main stem bronchi. however, asm is oriented differently in smaller airways. the angle of orientation of asm relative to the long axis of the airways is on average deg [ ] . however, this angle is obviously another parameter that is variable from one airway generation to another, as well as from one asm bundle to another. the implication here is that the strain reported is somewhat overestimated compared to the real strain on the long axis of the asm bundle. for example, when one assumes a radial airway wall strain (viz., an increase of airway perimeter) of % with no longitudinal airway strain (viz., no lengthening of the airway) and an asm bundle oriented deg off the long axis of the airway, the strain on the long axis of the asm bundle is . % (fig. ) . the magnitude by which asm strain is overestimated depends on how far off the angle of orientation deviates from deg. the reality, however, is that the airway is also strained on its longitudinal axis (this is discussed below). if both the radial strain and the longitudinal strain of the airway increase by %, the asm are then also strained by % regardless of the orientation of the asm bundle in relation to the long axis of the airway (fig. ). containing airway smooth muscle. although the bundles of asm completely encircle the small airways, it is not the case for the trachea and the main stem bronchi. in fact, asm occupies approximately / and / of the total airway wall circumference in the trachea and the main stem bronchi, respectively. the remaining circumference is made up of cartilage. since the cartilage is stiffer than the asm, the strain is not distributed homogeneously along the entire circumference of the airway. in reality, near % of the change in circumference is taken up by / to / of the airway wall; the portion occupied by asm and free of cartilage. asm strain during a di in the trachea and the main stem bronchi may thus be well beyond the one estimated based on the change in airway caliber. dimensions. the estimation of asm strain during a di from frc to tlc is sometimes accomplished by measuring a change in volume. this is the case for the methods measuring and comparing v d at different lung volumes. to convert a change in volume into radial airway wall strain (or $asm longitudinal strain), an additional step is required. this is because not only the caliber but also the length of the airways fluctuates during breathing maneuvers [ ] . the relative extent by which the length of the airways elongate and shorten during breathing maneuvers compared to the extent by which they dilate and narrow have been poorly studied. one can assume that the percentage change is equal in every directions; so that the luminal airway volume expands or shrinks isotropically without changing shape during both inflation and deflation. some experimental data support this assumption [ , [ ] [ ] [ ] . studies showing that dv d is linearly related to dlung volume also support this assumption [ , ] . however, other evidences suggest that it is not always the case [ ] . in fact, the accuracy of this assumption seems greatly affected by the starting lung volume and the magnitude of the lung volume excursion [ , , ] . the strain in one direction relative to the other directions is also likely to vary between airways from different individuals and between airways within the same individual. regardless, the degree by which the deformation of the airways deviates from isotropy obviously affects the precision with which asm strain can be estimated by these methods. fig. the changes in luminal geometry overestimate the stretch the asm undergoes during a deep inspiration (di). the schematic illustrates a normal (left) and an asthmatic (right) airways at frc and after a di to tlc. the dimensions are zoomed but at scale to an average airway of the ten generation. the springs represent lung recoil. they are stretched at tlc relative to frc. the arrow at approximately o'clock is the radius (in mm) of the airway lumen (r l ). the arrow at approximately o'clock is the radius up to the middle of the airway smooth muscle layer (r m ). the material composing the airway wall was considered inextensible. notice the thinning of the airway wall when the lungs are inflating to tlc. it this schematic it was assumed that the luminal geometry at frc was equal between normal and asthmatic. it was further assumed that the di was increasing luminal radius by % in both normal and asthmatic, so that the luminal geometry at tlc was also equal between normal and asthmatic. the schematic demonstrates that a % increase of luminal perimeter (p l ) only causes a . % increase of the perimeter at the middle of the asm layer (p m ). this effect is further amplified in asthmatic because of a thicker airway wall ( versus . %). the mathematics is developed in the middle. other abbreviations: a aw -area of the airway wall from the lumen to the middle of the asm layer, a l -luminal area, a m -area internal to the middle of the asm layer, and %d-change in percentage. due to the elongation of the airway tree during a di, the relative distance of a specific point within an airway to a recording device at the mouth increases. this is an issue for several methods. this vertical displacement is important to consider in order to allocate a change in caliber from frc to tlc at specific airway locations. otherwise, erroneous changes are caused by comparing a upper (and larger) part of the airway to a lower part due to lung and airway elongation at tlc. for the acoustic reflection technique, coronal radiographs can be acquired. the radiograph allows the identification of the carina and to subsequently measure its distance from the microphone. the other measured areas can then be allocated to specific airway locations. since the carina is moving at different locations during a breathing maneuver, several radiographs for monitoring its location at different lung volumes is ideally required. however, this is not always done [ ] . it is thus likely that a small variability arises due to a vertical displacement of the airways relative to the recording microphone. to limit this problem, the cross-sectional area is usually averaged over a few centimeter-long segment. the vertical displacement is also an issue for the pitot static probe. indeed, the distance of the probe from the mouth is always the same, as the catheter is fixed at the mouth. this means that the probe is actually moving in relation to the airway tree during the forced expiratory maneuver. this is not taken into account with the pitot static probe. the issue of vertical displacement is more easily overcome by hrct. this is because an image of the entire (or a major portion of the) lungs is obtained. landmarks can thus be identified to assess the vertical displacement of the lungs during the di. the strain can be applied in the three dimensions, as well as in two directions (stretch or compress). the focus of this review is on the expansion of airway caliber during a di causing a stretch to asm. however, the elongation of the airways during a di also implies that asm is strained in at least another dimension, namely on its transversal axis. the effect of transversal strain on asm contractility has not been studied sufficiently. one study demonstrated that cyclical lengthening of isolated airways mainly increases their contractile capacity [ ] . the third dimension (thinner versus thicker airway wall) is also important to consider. one may think that an increase in p l should thicken the airway wall, for the same reasons as it increases the length and the perimeter of the airways. however, the materials constituting the airway wall hardly change in volume during a di compared to the air-filled compartments (i.e., the lumen of the airways and the alveoli). increasing the length of an airway and its caliber during a di to tlc should thus exert a tensile stress on the airway wall, which tends to make it thinner. many ct studies actually reported that the airway wall is thinner at tlc, supporting that the airway wall is compressed in this dimension during a di [ , ] . this was also shown in cat lobes frozen at different volumes [ ] , as well as in isolated airways stretched to the letter a represents the airway perimeter and, in this example, is set to mm. the letter b is the airway longitudinal distance covered by the asm bundle going around the full circumference of the airway. finally, the letter c represents the length of the asm bundle and, in this example, is set at an angle deg off the long axis of the airway. using trigonometry, b and c can be determined. now imagine that this airway is stretched radially to increase its perimeter by %. on the flattened airway shown in (a), this would increase the height of the rectangle by % without changing its length (b). compared to (a), the length of a would increase by %, the length of b would remain unchanged and the length of c would increase by . %. the angle of c would also change to . deg. therefore, a radial stretch to the airway increasing its perimeter by % is expected to strain the asm bundle by only . %. now imagine that the airway length is also strained by %. on the flattened airway shown in (b), this would increase the length of the rectangle by % (c). compared to (a), the length of a, b, and c would all increase by %. in contrast to (b), the angle of c would remain unchanged. therefore, when both radial and longitudinal strains are applied simultaneously at the same magnitude on an airway, the strain on the asm is also of this magnitude. elongated lengths [ ] . overall, these suggest that the airway wall is stretched in two dimensions and compressed in one dimension during a di to tlc. the asm should thus be longer and wider but thinner at tlc. this is different from stretching an asm bundle in vitro, where it becomes longer but consequently less wide and less thick. thus, in contrast to in vivo, the asm in vitro is stretched in only one dimension and compressed in two directions. the effects of varying the orientation and the direction of strain on asm contractility have been largely overlooked. only few studies conducted with isolated cells have examined the effect of strain orientation on asm contractility [ , [ ] [ ] [ ] [ ] [ ] . the findings suggest that strain orientation has a major impact on the outcome. to the best of our knowledge, no study has yet investigated the impact of strain orientation and direction, as well as their different combinations, on the contractility of asm tissues. these studies are clearly warranted. airways. the effect of the upper airways (mouth, larynx, and pharynx) cannot always be discounted. whether the upper airways can be considered a mere extension of the lower airways can be justifiably questioned. some evidence suggests that the cross-sectional area of upper airways is also dependent on lung volume [ ] . however, the upper airways also have an intricate geometry. the concepts of fluid mechanics that apply to a single rigid cylindrical tube in which the flow regime is laminar might not be relevant. for example, the flow path during an expiration bends when it reaches the larger space of the mouth. the flow path also encounters many protruding structures and diverticula that can alter its flow regime such as the vocal cords, the uvula, the epiglottis, the tongue, and the alimentary and nasal canals. all these structure are likely to foster turbulent flow. the measurements of resistance by the fot, the interrupted deflation, and the whole-body plethysmography are affected by upper airways. in the case of raw (pleth) , attempts were made to subtract upper airway resistance from raw in order to obtain lower raw [ ] . in these experiments, the pressure difference between two lateral taps, one at the mouth and one inserted midstream in the tracheal lumen via a puncture - cm below the cricoid cartilage, was measured. this difference in pressure over the mouth flow was then used to estimate upper raw and to subtract it from raw (pleth) [ ] . the acoustic reflection technique is one convenient method to assess upper airways. this is because the time at which the signal is collected can be allocated to a specific distance from the mouth. the intensity of the signal is thus dictated only by the structural features that are in place at that distance. however, it does not provide information regarding the structural features altering the signal. therefore, a notch, a dent or a circular decrease in caliber are equally detected. capturing the excursion of small airway caliber during a di is a challenge. indeed, many methods described in sec. are restricted to the assessment of large airways. this is probably the main limitation of the acoustic reflection technique. according to the results obtained with rigid casts of the airway tree, the areas determined acoustically agreed well with the real areas for airway segments extending up to cm past the carina [ ] . therefore, the airways further down the main bronchi cannot be assessed. this obviously restrains the use of the acoustic reflection technique for the assessment of airway wall strain during di. also, only one measurement per distance can be obtained. so beyond the branching point, the signal emanates from the cumulative effect of the two main bronchi and their respective contribution cannot be distinguished. the pitot static probe is also limited to the assessment of the large airways. the lowest position measured in brackel and coworkers' study was at the entrance of the middle lobe [ ] . in this case, the penetration depth of the device is limited by its size in relation to airway caliber. the same constraints apply to aoct. high-resolution computed tomography is probably the best method to assess the small airways. yet, adequate resolutions are restricted to airway size over $ mm of internal diameter. the contribution of the small airways is certainly embodied in measurements such as v d , raw (pleth) , and fot. however, the extent by which the small airways contribute to the overall signal cannot be quantified. fot was initially suggested by some to distinguish the small from the large airways. the rationale lies in the traveling distances of the oscillations at different frequencies. while oscillations at low frequencies travel far down into the lungs, the traveling distance of high frequencies are shorter. therefore, while the low frequencies should probe the entire airway tree, the high frequencies should only probe the large airways. by looking at the frequency-dependence of rrs, or by using a simpler readout such a r - (which is the subtraction of rrs at hz from the rrs at hz), it was suggested that the degree of obstruction of the small airways can be deduced. although this line of reasoning is logical, this is no longer the prevailing belief. first, the penetration depth of the forced oscillations at different frequencies cannot be ascertained. it is thus not sure from how far deep into the lungs the signals used to calculate rrs at different frequencies emanate. second, and most importantly, computational models have clearly demonstrated that the frequencydependence of resistance is particularly sensitive to the pattern of constriction in peripheral airways [ ] [ ] [ ] . on one hand, a homogeneous pattern of small airway constriction increases rrs equally throughout the frequency range. on the other hand, a heterogeneous pattern of small airway constriction increases rrs way more at low than at high frequencies. therefore, the frequencydependence of rrs (or r [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) is now considered a readout that is more useful to quantify the degree of airway narrowing heterogeneity in the periphery than to assess the degree of small airway obstruction. near total lung capacity. the measurements made at tlc are sometimes difficult and sometimes just not possible. raw (pleth) , for example, cannot be measured at tlc but only near tlc. the measurement requires a certain amount of air to flow in-and-out of the lungs to calculate raw (pleth) . this panting maneuver near tlc is also difficult for the subject. the measurement of r l with the interrupted deflation also required a certain amount of air to evacuate the lungs in order to measure the change of p l caused by the interruption. therefore, the value at tlc cannot be determined. holding the breath at tlc during hrct can also be difficult, especially for older and/or sicker patients. concerning the pitot probe, the airway caliber at different positions were grouped in volume range (e.g., the caliber between % and % of fvc). the total strain experienced by asm may thus have been underestimated because many measurements were not at tlc. notwithstanding this possibility, the error associated with not assessing airway caliber at tlc compared with near-tlc would be small since the airways are stiffer in that range of lung volume. the variation in transmural pressure may disproportionately exceed the strain on asm. in fact, submaximal volumes and/or p l are generally sufficient to achieve maximal dilation [ , ] . other issues are also more common at or near tlc than other lung volumes and can affect the accuracy of the measurement. for example, glottic closure commonly occurs at extreme volumes (rv and tlc). therefore, the changes in airway caliber sometimes need to be assessed within these volumes (i.e., a little above rv and a little below tlc), which may slightly underestimate the full range of airway wall excursion that occurs during a di from frc to tlc. . bronchial tree geometry. the estimation of asm strain during a di from frc to tlc is often accomplished by measuring the changes in resistance at different lung volumes. this is the case for the whole-body plethysmography, the interrupted deflation, and the fot. to convert resistance into asm strain, some arithmetics are needed. the calculation is based on poiseuille's equation. this equation states that within a tube with a perfect cylindrical geometry in which flow is running in a laminar fashion, airway resistance is inversely proportional to the luminal radius at the fourth power. a change in airway resistance at different lung volumes can thus be converted into asm strain, as the percent change in radius is the same as the percent change in airway perimeter (viz., $asm length). as aforementioned, the airways also elongate during a di. according to poiseuille's equation, resistance to airflow is proportional to the length of the airway. so while the increase in caliber at tlc decreases resistance, the elongation increases resistance. as resistance is related to the radius at the fourth power, the effect of changing caliber largely predominates over the effect of changing the length of the airway. however, the estimation of asm strain based on measurements of resistance might be slightly underestimated because the increase in airway length is usually not taken into account. it is understood that the airway tree is way more complicated than a single cylindrical tube with identical size on both ends. it is also understood that the flow is not always laminar. the airway tree branches in different directions, contains numerous bifurcations, and many bronchi are bending and exhibit a tapered end. all these elements obviously disturb flow and increase the degree of turbulence. notwithstanding this complexity, the most accurate computational model of the human lungs is the singlecompartment model [ ] . this model is merely constituted of a single airway with a perfectly cylindrical geometry that runs into a single elastic compartment. the resistance that stems from the viscous flow of air within the airways in a real airway tree, as well as its response to different interventions, thus seem to be predicted quite precisely just by altering the caliber of a single conducting airway in the modeled lungs. it seems that, sometimes, the gestalt behaves as a simple element. there is thus no reason to treat it differently as such in order to extract information that would otherwise be impossible to infer. the presence of chokepoints (sometimes called stenosis) is also important to consider. the excursion of asm length at these chokepoints during breathing maneuvers can be greatly altered [ ] . chokepoints also obstruct the dynamics of fluid. consequently, they severely increase resistance without necessarily affecting the caliber along the entire airway. the presence of chokepoints is thus likely to insert inaccuracies in the estimation of asm strain in methods using measures of resistance. in addition, chokepoints affect the regional transmural pressures, especially during forced maneuvers. while the transmural pressure downstream of the chokepoint increases during expiration (as the luminal pressure increases in the airways and alveoli subtended by the choked airway), it decreases upstream of the chokepoint. the opposite occurs during inspiration. either way, the transmural pressures are altered. the strain asm undergoes is thus attended to be affected in these regions. also, these chokepoints are usually not present at every lung volume. the chokepoint may thus affect resistance at low lung volumes but not at high lung volumes, and thus affect the change measured between two lung volumes. more generally, the entire geometry of the airway tree (not only chokepoints) is likely to change at different lung volumes, which inevitably affects differently the resistance to airflow. . chest wall. the chest wall can also affect some measurements. this is the case for the fot. the estimated change in airway caliber calculated from the change in rrs from frc to tlc needs to be taken with caution. this is because rrs at volumes surrounding frc is compounded by the resistance of the chest wall (rcw), which is not the case at tlc [ ] . consequently, the delta of rrs from frc to tlc overestimates asm strain. this is consistent with brown and coworkers' study, in which the estimated asm strain by fot using rrs was greater than the one predicted based on delta v d assessed by the single-breath nitrogen washout [ ] . r l estimated with the esophageal balloon can remedy this overestimation by discarding the effect of the chest wall. . ionizing radiation. the exposure to ionizing radiation is obviously a major concern. it is probably the most important downside of hrct. repeated measures should absolutely be avoided. unfortunately, the investigators are left with a few snapshots at a few chosen lung volumes to quantify the excursion of airway caliber, which is far from ideal. radiation is also a concern with bronchography, although the doses used are lower. many factors influence the estimated strain asm undergoes during a di from frc to tlc and should thereby be considered as confounders. the confounders can be related to methodological procedures (an intervention or the breathing maneuvers during and preceding the measurement), which can either be controlled or not. alternatively, some confounders are inherent biological factors that cannot be controlled. this section describes the many procedural and biological confounders that are known to affect airway wall strain during a di. the magnitude by which the airways are strained during a di is dictated in great part by the swing in transmural pressure (i.e., the pressure across the airway wall). the swing in transmural pressure during a di depends on the extent by which the lung parenchyma is pulling the airway open, which, in turn, depends on the transpulmonary pressure (p l ). a greater swing in p l and/or lung volume during a di inflicts a greater excursion of airway caliber and, thereby, a greater stretch to asm. p l and the lung volume attained at tlc are variable between individuals. these biological factors thus affect asm strain during a di and should thus be considered as important confounders. as discussed above, many methods that are used to estimate asm strain from frc to tlc rely on tricky breathing maneuvers near tlc (e.g., raw (pleth) ) or a holding maneuver at tlc (e.g., hrct). the level of inspiration that is achieved/maintained by the subjects during such maneuvers relative to their maximum also contributes to variability [ ] . it is also important to mention that considerable regional differences in p l exist within the lungs. during the same breathing maneuvers, the airways in lower lobes are more strained than airways of the same generation in the apical lobes [ , , , ] . the estimated strain asm undergoes is thus highly influenced by the region of the lungs in which the airways are embedded. pressure and volume. the pressure-volume curve of the lungs is not linear, so as the curve defining the relationship between transmural pressure and airway radius. by changing the starting (frc) transpulmonary pressure or lung volume not only the swing in pressure and/or volume from frc to tlc may be affected [ ] but it also implies that the airways are now operating on a different part of the transmural pressure-radius curve. this is important as it indicates that the same swing in pressure now translates to different changes in radius and thus to different degrees of asm strain. this was predicted by computational modeling [ ] and clearly shown in vitro [ , ] . an increase in frc (i.e., hyperinflation) also impairs the ability to generate high p l [ ] . consequently, it reduces the pressure distending the airways during a di. in fact, hyperinflation is a strong predictor of the bronchodilator effect of di [ ] . more severe is the hyperinflation, smaller is the excursion of airway caliber from frc to tlc and less efficient is the bronchodilator effect of a di [ ] . lung volume and p l at frc also vary between individuals. these biological factors are thus important confounders that affect the strain asm undergoes during a di. all the methods used to estimate asm strain during a di rely on measurements made at frc. they are thus influenced by the starting lung pressure and volume. . hysteresis. the pressure-volume curve of the lungs during inflation and deflation do not overlap. in fact, when the two curves are combined they form a loop that rotates clockwise, meaning that the volume at any given pressure is greater during deflation. this is called hysteresis and is due to the resistive component of the lungs, which is chiefly perceived during inflation but dissipated and barely not perceived during deflation. this hysteretic behavior also applies independently to both the airways and the lung parenchyma. therefore, when tracking the transpulmonary pressure and the cross-sectional area of an airway during breathing, a loop is also formed. however, the direction of rotation depends of the relative hysteresis between the airway and the surrounding parenchyma. when the airway is more hysteretic than the parenchyma the loop rotates clockwise. inversely, when the parenchyma is more hysteretic than the airway the loop rotates anticlockwise. in healthy individuals, airway hysteresis dominates and is largely due to asm tone [ , ] . this was also shown in isolated airways [ , ] . hysteresis has important implications. it indicates that the caliber of an airway at a particular lung volume varies according to whether it follows a deflation from a higher lung volume or a inflation from a lower lung volume [ ] . after either a deflation or an inflation, the caliber can be larger or smaller depending on whether airway or parenchymal hysteresis predominates. for example, when airway hysteresis predominates, the caliber of the airways at frc after a di to tlc is greater than the caliber at frc prior to the di. in essence, this is the definition of the bronchodilator effect of a di. hysteresis also implies that the estimated strain asm undergoes during a di is not the same when the measurements are made from frc to tlc than when they are made from tlc to frc. an important procedural confounder is thus related to whether the change in airway caliber is measured during inflationary maneuvers versus deflationary maneuvers. the methods used to estimate asm strain described in sec. involve either types of maneuvers. for example, the measurements of r l at different lung volumes with the interrupted deflation are performed during deflation. the pitot static probe also takes the measurement during a forced deflationary maneuver. contrastingly, the change in crosssectional area of the airway lumen with the mri is measured during inflation. this is because the image has to be captured during the flow of hyperpolarized gas ( he or xe) into the conducting airways in order to achieve a better contrast between the airways and the parenchyma. this also implies that the image is not obtained at fixed volumes but rather during a dynamic change in lung volume. the caliber at which an airway settles after a perturbation, such as a di, also takes time due to hysteresis. the timing at which the measurements are made can thus affect the results and should thereby be considered as a confounder. likewise, some methods used to estimate asm strain require interferences with the normal pattern of breathing. hrct, for example, requires breath-holding to assess airway caliber at different lung volumes. the breathhold usually decreases the bronchodilator effect of a di [ , ] . however, whether the breath-hold overestimates or underestimates airway wall strain depends: -on the relative hysteresis between the airways and the parenchyma; -at which lung volume it is measured; and -whether the lungs were inflating or deflating before the breath-hold. regardless, the longer time that is allowed for the airways to settle at a new lung volume is likely to affect its caliber. consequently, the estimated strain excursion undergone by asm during such a change in lung volume will also be different compared to the one caused by the same change in volume with no breath-hold. all the other methods using measurements made at static (or quasi-static) lung volumes instead of dynamic lung volumes are likely to be affected by this procedural confounder. other procedures that clearly affect the estimation of asm strain include forced breathing maneuvers, such as forced expiratory volume in s (fev ) and coughing. the transitory peak of negative airway transmural pressure achieved during these maneuvers can transiently narrow the airway lumen to a level beyond that observed at the end of the maneuver [ , , , ] . this phenomenon is also known for causing dynamic collapses and expiratory flow limitation. one method used to estimate asm strain that can certainly be affected by forced maneuvers is the pitot static probe. the flow rate (or speed of inspiration) during the di is also important to consider. a di at a high flow rate is more effective to bronchodilate the airways than a di at slow flow rate [ , , ] . this was also demonstrated with isolated airways [ , ] and asm strips stretched at different rates [ ] . an increase in transmural pressure was proposed to account for the greater bronchodilator effect observed at high flow rate [ ] . with regards to the estimation of asm strain, both inspiratory and expiratory forced maneuvers should be considered as procedural confounders. beyond the different breathing maneuvers used during the measurements (inflation versus deflation, static versus dynamic, slow versus forced maneuvers), the sequence of events preceding the measurements should be considered. the most prominent example is the bronchoprotective effect of a di [ ] [ ] [ ] [ ] [ ] [ ] . so a di, or a series of dis, prior to a bronchoconstriction elicited by inhaled methacholine, facilitates bronchodilatation induced by a subsequent di postmethacholine [ ] . the time elapsed between the di premethacholine and the subsequent di under a bronchoconstricted state also affects its bronchodilator effect [ ] . the time spent without di under bronchoconstriction also affects the excursion of airway caliber during a di because it increases narrowing [ , ] . the bronchodilator effect of a di is also not the same after a sequence of dis than after a single di [ ] . all together, these results indicate that the strain asm undergoes during a di is affected by lung history. the geometry of the airways at frc can also be affected if the preceding measurements were performed at rv compared to if they were performed at tlc [ ] . the caliber of the airways at a given lung volume is greater when preceded by measurements done at a higher lung volume [ ] . as explained above, part of it is due to hysteresis. notably, history can affect the measurements in both directions, as it can either increase or decrease the estimated strain asm undergoes during a di. lung history thus represents a serious procedural confounder and should be controlled whenever possible. . posture. the lungs undergo important deformation during changes in posture. this is because it changes the direction of the gravitational forces, which then affects the local p l and the gradient of p l [ ] . a computational model predicted that a change of $ cmh o can occur at specific locations in the lungs during a change in posture from supine to prone [ ] . in turn, this can be expected to change lung volume by approximately twofold, depending on which part of the pressure-volume curve this change in pressure occurs. this is then predicted to strain asm by $ %, assuming that the perimeter of the airways changes proportionally to the changes in the cube root of lung volume. a change in posture also affects the starting (frc) lung volume, engendering the consequences stated above. for example, frc is decreased in supine posture [ ] . the methods used to estimate asm strain involved measurements at different postures. the measurements of airway resistance by fot and plethysmography are usually performed seated upright. contrastingly, hrct and mri are usually performed supine. the changes in gravitational forces due to these different postures are likely to affect asm strain differently in different locations of the lungs. the posture should thus be considered a procedural confounder. all the elements that constitute the airway wall can be organized differently, in addition to vary in absolute amount and relative proportion [ ] . these elements include the cellular and the extracellular components of the airway wall, as well as other structures such as the cartilages. any elements constituting the airway wall should thus be considered as biological confounders. this is because any change in the structural composition of an airway is likely to modify its mechanical properties and thereby affects the strain asm undergoes during a di from frc to tlc. many of these elements vary in healthy subjects according to airway size. small airways, for example, has a structural composition that makes them more compliant [ , , , , , ] . their percentage of dilation during a di is thus greater compared to larger airways [ , ] . interestingly, it was shown in mice that the specific compliance of the small airways are greater than the specific compliance of the lungs [ ] . the p l at which the airway dilation plateaus during an inflation maneuver also varies with airway size. in healthy individuals, the p l at which this plateau occurs increases progressively with increasing airway size [ , ] . this indicates that maximal airway dilation occurs rapidly in small airways during inflation, while the large airways continue to dilate until later on in the maneuver. this is concerning for the methods that use the change in the entire volume of the airway tree in order to estimate the longitudinal strain on asm. this is because these methods assume that all the airways expand to the same extent. as stated above, airways of different size have different compliance. their compliance also varies according to the pressure range across which they are studied [ , ] . in airway generations (trachea) through , the curve that describes the relationship between luminal area and p l in every generation is sigmoidal. in fact, most of the change in luminal area occurs within a narrowed range of p l of about cmh o [ ] . therefore, a limited window exists where changes in p l actually cause changes in airway caliber. this implies that no change in airway caliber occurs at either low and high p l . contrastingly, in the steeper part of the curve, large changes in caliber occur across a very small range of p l . similar to the mouse study stated above [ ] , the specific compliance of the small airways in dogs and sheep are greater than the specific compliance of the lungs in that steeper part of the curve [ , ] . collectively, these last observations suggest that the starting p l , the maximal p l attained during the di, and the curve describing the relationship between p l and the caliber of the airways under scrutiny need to be known in order to allocate an exact amount of strain on asm during a di. as mentioned above, the swing in transmural pressure during a di is a major determinant of asm strain. apart from the transpulmonary pressure, the transmural pressure across the wall of any airway is influenced by the stiffness of the surrounding lung parenchyma and the degree of interdependence between the airway and the parenchyma [ ] . a more compliant parenchyma may dampen the swing in distending pressure generated by lung inflation and thus limit dilation. a loss of connectivity between the airway and the parenchyma, due to alveolar detachments [ ] or geometric decoupling due to thicker adventitia [ ] , also limits the transmission of pressure across the airway wall. the stiffness of the surrounding parenchyma and the level of interdependence between the airways and the parenchyma are thus biological confounders that are important to consider. . age. aging influences the mechanical properties of the lungs and its constituents [ ] . age should thus be considered a biological confounder. for example, the extensibility (i.e., the ease by which it is elongated) of extraparenchymal airways, such as the trachea, decreases with age [ ] . in contrast, intraparenchymal airways and the parenchyma seem to be more distensible [ ] . indeed, loss of lung elastic recoil was reported with aging [ ] . the larger percentage change in caliber of small versus large airways during a change in lung volume also seems to vanish with age, probably due to an increase in lung compliance [ ] . age can thus affect asm strain during a di from frc to tlc due to various reasons. these phenomena may account for the attenuated bronchodilator effect of di with aging [ ] and the lack of bronchoprotective effect of di in infants [ ] . . health status. respiratory disorders can influence to various extent and by several ways the strain asm undergoes during a di. the driving pressure, the mechanical properties of the airways and the lung parenchyma, as well as the forces of interdependence can all be affected by diseases. in fact, changes in the structural composition of the airways, a process called remodeling, are common in many respiratory disorders and can have a major impact on the mechanical properties of the airway wall [ ] . airway distensibility is decreased in asthma [ , , , , , , ] , especially at high [ ] but also at low lung volumes [ ] . this seems to be mainly caused by airway remodeling [ , ] but also inflammation [ , ] . asthmatic patients also tend to exhibit airways of smaller caliber [ , , , ] . the sigmoidal curve that describes the relationship between p l and airway caliber is also shifted to the left in asthmatic patients, at least when a bronchodilator (salbutamol) is administered prior to measurements [ ] . this suggests that the changes in airway caliber due to breathing maneuvers occur at lower p l in asthma [ ] . this may explain why fluctuation of rrs during tidal breathing is increased in asthma [ ] . interestingly, this increased tidal fluctuation of rrs also negatively correlates with the bronchodilator effect of di [ ] . this is somewhat logical. by operating at a steeper part of the sigmoidal curve during tidal breathing, less of the total possible strain is available during a di to tlc. in contrast to asthmatic patients, the lungs of nonasthmatic individuals seem to breath in a range of p l below the one where airway caliber is greatly affected by small changes in p l (based on small tidal fluctuation of rrs [ ] ). however, for the same reason, the total possible strain the airways can undergo is still available and may occur during a di to tlc. the benefit is a greater stretch to asm and, thus, a commensurately greater bronchodilator effect of di. finally, a decline in lung elastic recoil was also reported in longstanding asthma [ ] [ ] [ ] . all these phenomena may account for the attenuated bronchodilator effect of di in asthma [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , as well as its attenuated bronchoprotector effect [ , , ] . airway distensibility also seems to be attenuated in copd patients [ , ] , especially in emphysema-predominant compared to airway-predominant subtypes of copd [ ] . in this case, the lower apparent distensibility seems mainly attributed to a loss of recoil due to parenchymal destruction [ , ] , as well as hyperinflation, which limits inspiratory capacity and the driving p l during the di [ ] . these phenomena may account for the attenuated bronchodilator effect of di in copd [ , , ] . in contradistinction, the distensibility of the airways is increased in patients with cystic fibrosis [ ] . lung volume dependence of tracheal cross-sectional area in these patients was calculated to . cm /l, compared to almost nil in healthy subjects. cystic fibrosis patients also demonstrate increased lung compliance [ ] , which may limit the swing in p l . therefore, the reported increase in tracheal distensibility may be attributable to flaccidity, as previously reported in older patients with cystic fibrosis, as well as postmortem on isolated tracheas from patients with cystic fibrosis [ ] . bronchiectatic bronchi, which are common in cystic fibrosis patients, are also prone to bronchomalacia. it is important to mention though that the incidence of bronchomalacia is high ( %) in patients with bronchiectasis with or without coexistent lung disorders [ ] . even nonrespiratory disorders are sometimes important to consider. allogeneic haematopoietic stem-cell transplantation, for example, increases the changes in airway caliber during a lung excursion from frc to tlc [ ] . this seems to be entirely related to an increase in lung parenchymal stiffness rather than an increase in airway distensibility. obesity is another example that can significantly impact asm strain during a di, mainly by changing lung volumes [ ] . this phenomenon may account for the attenuated bronchodilator effect of di in obesity [ ] [ ] [ ] . most respiratory disorders also affect the airways nonuniformly [ , ] . the amount of strain the asm experiences during a di is thus expected to vary according to this patchy pattern of affection. overall, diseases can exert a huge and variable impact on the strain asm undergoes during a di. they should thus be considered as biological confounders. among all the elements that constitute the airway wall, asm is one that is very special. this tissue is endowed with the ability to quickly adjust the mechanical properties of the airway wall. greater is its level of activation, stiffer are the airways. in turn, stiffer airways are intuitively expected to expand less in response to a di. however, this is not always the case. the change in airway caliber caused by a simulated di is sometimes greater when asm is activated by methacholine than when it is relaxed by atropine [ ] . this is because increasing the level of asm activation may also cause airway constriction, which affects asm strain during a di for at least two reasons. first, it modifies the length of asm before the di, which impacts its contractile capacity. more precisely, shortening the length of asm decreases its contractile capacity [ , ] . second, airway constriction also changes the positions over which the airways are operating on the pressure-radius curve [ ] . following airway narrowing, the di thus takes place on a more compliant part of the pressure-radius curve and thereby increases the strain for any given stress [ ] . therefore, while the maximal distention attained by the airway during a di is certainly limited by the level of asm activation, the strain excursion may not. these have important implications for in vitro studies that attempt to emulate in vivo situations. for example, it may be appropriate to impose an equivalent level of strain on asm during a simulated di with or without spasmogeninduced contraction. however, this simulation only recreates adequately the in vivo situation if the asm is also adjusted to a shorter starting length in the presence of a spasmogen. the intrinsic level of asm activation is tuned by many spasmogens and bronchodilators that are produced endogenously [ ] . the intrinsic level of asm activation should thus be considered a biological confounder. however, some interventions also affect the level of asm activation, which consequently changes the stiffness of the airway wall and the strain caused by a di. on one hand, many methods used to estimate asm strain during a di intentionally manipulate the level of asm activation with bronchoactive substances (bronchoconstrictor such as methacholine or bronchodilator such as salbutamol). some studies are actually designed to dissociate the active contribution of asm in determining airway wall stiffness from the passive structural components of the airway wall [ , , , ] . these studies investigated the response to dis before and after the activation or the inhibition of asm. on the other hand, many methods used to estimate asm strain during a di unintentionally manipulate the level of asm activation by utilizing topical or systemic anesthesia. this is the case for aoct [ ] . for these reasons, the level of asm activation should sometimes be considered a procedural confounder. some patients also take medications with either short or long duration of action. those are likely to affect the stress-strain relationship of the airway wall and thus the distension of the airways during a di. they should thus be considered as confounders. the duration of withholding from these medications prior to measurements is also an important confounder to take into account. apart from medications, some interventional procedures also have the potential to affect asm strain during a di. this is the case for bronchial thermoplasty, which is an alternative treatment for severe refractory asthma. it was demonstrated in dogs that bronchial thermoplasty increases airway caliber at any airway pressure from to cmh o [ ] . the change in airway caliber though from to cmh o was identical pre-versus postbronchial thermoplasty. based on these observations, geometrical considerations need to be taken into account to understand the impact of bronchial thermoplasty on asm strain. airway caliber changes proportionately to the square of airway perimeter (viz., $asm length). the same change in caliber induced by rising the pressure from to cmh o in an airway starting at a larger caliber implies that asm strain was attenuated postbronchial thermoplasty. therefore, the initial (frc) length of asm may be greater after bronchial thermoplasty, but the strain asm undergoes during the di may be attenuated. the goal of this review was to estimate the strain asm undergoes in vivo in humans during a di from frc to tlc. this transient stretch is important to quantify as it was proposed to contribute to the beneficial effect of di by decreasing the contractile capacity of asm. however, it remains unclear to what extent asm is strained during a di. despite this gap in knowledge, the effect of strain on asm contractility has been tested extensively in vitro. a variable range of strains at different frequencies and for different durations in a variety of asm preparations have been tested. we came to learn that the dynamic environment in which asm operates in vivo has the potential to greatly affect its contractile capacity. in general, the in vitro results indicate that the decline in contractile capacity is largely dictated by the amount of strain [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] , which is consistent with in vivo results in rabbits [ ] and humans [ , , , , , ] , as well as with isolated cells [ ] . together, these studies support the hypothesis that the beneficial effect of di on respiratory mechanics is related to the decrease in the contractility of asm elicited by the stretch. however, the in vitro results are also variable, sometimes contradictory, and it is still uncertain if the strain required to significantly decrease the contractile capacity of asm is physiologically attainable [ , ] . incorporating an appropriate range of strains in these in vitro studies is obviously essential in order to understand the integrated role of asm in respiratory mechanics. as seen in sec. of this review, many former methods and more recent technological advents can be used to estimate asm strain in humans in vivo during a di. on one hand, some methods assess the distensibility of the entire airway tree. these methods have the advantage to assess the effect of all the airways combined, but provide little insights on the localized response in any given airway and miss completely in reporting the spatial heterogeneity of the response. they are also unable to distinguish between radial versus longitudinal airway strain. among those methods, the measurement of v d , rrs near the resonance frequency with the fot and raw by whole-body plethysmography are the most relevant. the fot is especially appealing and increasingly used in clinical settings. combined with imaging technique and computational modeling, fot may eventually be able to document the changes in airway caliber during breathing maneuvers with a tremendous temporal and spatial resolution [ , ] . on the other hand, other methods assess individual airway distensibility. these methods have the advantage to measure directly the localized response, but usually fail to assess the small peripheral airways. the better being hrct, which can provide adequate resolution for airways down to $ mm of internal diameter. the other methods include bronchoscopy, fluoroscopy, acoustic reflection technique, mri, pitot static probe, and aoct. these methods were all very useful as they have clearly demonstrated that asm strain during a di differs according to airway size and location. as seen in sec. of this review, several limitations need to be considered as none of these methods assess asm strain directly. each of these limitations can putatively add inaccuracies that are sometimes not negligible. a few assumptions also need to be made in order to transform the original data into asm strain. the veracity of these assumptions can certainly be argued. owing to these limitations and assumptions, it is understood that the strains reported in this review are nothing more than estimations. as seen in sec. of this review, a single value of asm strain cannot be attributed to a di. this is because many procedural and biological confounders contribute to the variability between individuals and between airways within the same individual, as well as to the temporal variability of any given airway of a given individual [ ] . at this point, it seems more appropriate to provide a rough estimated range of strains that is physiologically relevant. despite the different setbacks of each method and the many confounders, the level of concordance between the methods for predicting the level of asm strain during a di is decent (table ) . this suggests that, although a greater level of temporal and spatial resolution can now be achieved, the new and refined technologies have simply confirmed the estimations made previously with antecedent methods. set apart the stiffer trachea and main stem bronchi, the longitudinal strain asm undergoes during a di from frc to tlc is estimated to reside within the range of - %. these values should assist investigators who attempt to impose a physiologically relevant level of strain to different asm tissues in in vitro settings in order to mimic a di. it is also important to realize that the value of strain that i was chasing in this review is the maximal attainable strain, i.e., the one achieves at tlc. involuntary dis, or should i call them sighs, which occur spontaneously every few minutes [ , , ] , do not reach tlc. provided that an in vitro study is interested to investigate the regulatory effect of these sighs on asm contractility, the range of acceptable strains is quite large. this is because beyond all the factors discussed in this review, the size of the breath during a sigh is obviously variable. the strain asm undergoes during sighs is thus commensurately variable. any intermediate values of strain occurring between frc to another higher lung volume anywhere from end-tidal inspiration to tlc can justifiably be used to simulate these spontaneous respiratory motions of the lungs. i think the one advice to take from this review is to stay within the reported range of strains and, more importantly, to never exceed the limit of what is physiologically attainable. to simulate more accurately particular in vivo situations, more data will be needed. indeed, owing to the number of biological confounders affecting the variability, more studies using different populations of patients and controlling for as many confounders as possible are required. these studies will allow us to adjust the strain in our in vitro settings according to the particular real-life situations that we are trying to model. in turn, applying a correct range of strains in our in vitro settings should: -ameliorate the validity of these in vitro simulations; -justify the incorporation of those in vitro results in 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fibrosis excessive collapsibility of bronchi in bronchiectasis: evaluation on volumetric expiratory high-resolution ct the effects of body mass index on lung volumes a deep breath bronchoconstricts obese asthmatics impaired response to deep inspiration in obesity deep inspiration avoidance and airway response to methacholine: influence of body mass index intrasubject variability in airway inflammation in biopsies in mild to moderate stable asthma heterogeneity of airway wall dimensions in humans: a critical determinant of lung function in asthmatics and nonasthmatics does the length dependency of airway smooth muscle force contribute to airway hyperresponsiveness? asthmatic airway hyperresponsiveness: the ants in the tree relationship between improved airflow limitation and changes in airway calibre induced by inhaled anticholinergic agents in copd effect of bronchial thermoplasty on airway distensibility bronchodilator effects of exercise hyperpnea and albuterol in mild-to-moderate asthma bronchomotor effect of bronchoconstriction-induced deep inspirations in asthmatics respiratory impedance measurements for assessment of lung mechanics: focus on asthma oscillometry and pulmonary mri measurements of ventilation heterogeneity in obstructive lung disease: relationship to quality of life and disease control spontaneous sigh rates during sedentary activity: watching television vs reading pattern of ventilation in young adults key: cord- - b wtim authors: borriello, giorgia; lucibelli, maria g; pesciaroli, michele; carullo, maria r; graziani, caterina; ammendola, serena; battistoni, andrea; ercolini, danilo; pasquali, paolo; galiero, giorgio title: diversity of salmonella spp. serovars isolated from the intestines of water buffalo calves with gastroenteritis date: - - journal: bmc vet res doi: . / - - - sha: doc_id: cord_uid: b wtim background: salmonellosis in water buffalo (bubalus bubalis) calves is a widespread disease characterized by severe gastrointestinal lesions, profuse diarrhea and severe dehydration, occasionally exhibiting a systemic course. several salmonella serovars seem to be able to infect water buffalo, but salmonella isolates collected from this animal species have been poorly characterized. in the present study, the prevalence of salmonella spp. in water buffalo calves affected by lethal gastroenteritis was assessed, and a polyphasic characterization of isolated strains of s. typhimurium was performed. results: the microbiological analysis of the intestinal contents obtained from water buffalo calves affected by lethal gastroenteritis exhibited a significant prevalence of salmonella spp. ( %), characterized by different serovars, most frequently typhimurium ( %), muenster ( %), and give ( %). the s. typhimurium isolates were all associated with enterocolitis characterized by severe damage of the intestine, and only sporadically isolated with another possible causative agent responsible for gastroenteritis, such as cryptosporidium spp., rotavirus or clostridium perfringens. other salmonella isolates were mostly isolated from minor intestinal lesions, and often ( % of cases) isolated with other microorganisms, mainly toxinogenic escherichia coli ( %), cryptosporidium spp. ( %) and rotavirus ( %). the s. typhimurium strains were characterized by phage typing and further genotyped by polymerase chain reaction (pcr) detection of virulence genes. the isolates exhibited nine different phage types and different genetic profiles. three monophasic s. typhimurium (b: , :i:-) isolates were also found and characterized, displaying three different phage types and three different virulotypes. the molecular characterization was extended to the s. muenster and s. give isolates collected, indicating the existence of different virulotypes also within these serovars. three representative strains of s. typhimurium were tested in vivo in a mouse model of mixed infection. the most pathogenic strain was characterized by a high number of virulence factors and the presence of the locus agfa, coding for a thin aggregative fimbria. conclusions: these results provide evidence that salmonella is frequently associated with gastroenteritis in water buffalo calves, particularly s. typhimurium. moreover, the variety in the number and distribution of different virulence markers among the collected s. typhimurium strains suggests that within this serovar there are different pathotypes potentially responsible for different clinical syndromes. salmonella spp. found in water buffalo (bubalus bubalis) herds are a matter of concern since they are responsible for serious economic losses in livestock and are a zoonotic agent responsible for foodborne illness [ ] . as for bovine calves, salmonella-induced diseases in water buffalo calves are characterized by severe gastrointestinal lesions, profuse diarrhea, and severe dehydration [ ] . acute salmonellosis generally induces diarrhea, mucous at first, later becoming bloody and fibrinous, often containing epithelial casts. ingestion is the main route of infection, although it can also occur through the mucosa of the upper respiratory tract and conjunctiva. the major source of infection in the herd is represented by asymptomatic older animals shedding heavy loads of bacteria through feces. other sources of infection are contaminated forages and water, as well as rodents, wild winged animals, insects and man [ , ] . the disease can also cause sudden death without symptoms. occasionally, the infection is systemic, affecting joints, lungs and/or the central nervous system (cns) [ ] . moreover, several salmonella serovars seem to be able to infect water buffalo, mainly affecting - week old calves, even though reports on salmonellosis in b. bubalis are scarce [ , ] . water buffalo calves are more frequently affected by gastroenteritis than bovine calves, with mortality rates as high as % in water buffalo species vs. % in bovine [ , ] . this difference might be due to a greater susceptibility of water buffalo to gastroenteric pathogens, although it also may reflect the lack of appropriate management practices for this animal species. therefore, water buffalo represents a suitable model to study causative agents of gastroenteritis. in water buffalo, s. enterica serovar typhimurium can induce a variety of clinical syndromes with different anatomopathological lesions [ , ] . the severity of the disease can depend on several factors, including host-pathogen interactions, which is highly influenced by the route of infection, the infectious dose, natural or acquired host resistance factors, and the possible presence of other pathogens. moreover, specific salmonella virulence factors, frequently located on salmonella pathogenicity islands (spis), prophage regions or virulence plasmids, play a key role in the pathogenesis of the gastroenteritis [ ] . the current study investigated the intestinal contents collected from water buffalo calves affected by gastroenteritis with lethal outcome to: (i) evaluate the prevalence of salmonella spp., and (ii) perform a polyphasic characterization of the collected isolates of s. typhimurium. salmonella spp. were isolated from % of the intestinal contents collected from water buffalo calves affected by gastroenteritis with lethal outcome. positive samples were detected in subjects bred in of farms (interherd prevalence, %). the s. enterica serovars most frequently isolated were typhimurium (n= ), muenster (n= ) and give (n= ). other recovered serovars were: derby (n= ), bovismorbificans (n= ), newport (n= ), monophasic s. typhimurium (b: , :i:-; n= ), blockley (n= ), meleagridis (n= ), umbilo (n= ), altona (n= ), anatum (n= ), bredeney (n= ), enterica (−;i; , ; n= ), gaminara (n= ), haardt (n= ), hadar (n= ), infantis (n= ), isangi (n= ), kottbus (n= ), london (n= ), muenchen (n= ), and s.ii: ;z; , (n= ). phage-typing of the s. typhimurium and monophasic typhimurium strains (table ) indicated a variable distribution of phage types among strains with nine different phage types of typhimurium strains, and three different phage types out of three monophasic typhimurium strains. this study reports a significant prevalence of salmonella spp. ( %) in diarrheic water buffalo calves, that are more relevant than those reported in previous studies ( and . %) [ , ] . moreover, in contrast with bovine species where salmonellosis results primarily associated with serovars dublin and typhimurium [ ] , the extremely variable distribution of the observed serovars confirms the absence of a serovar specifically adapted to water buffalo, as previously suggested [ ] . these data provide therefore evidence that salmonella, particularly s. typhimurium, can be potentially considered an important pathogen for this animal species. the definitive phage type (dt ), which has often been associated with multiple-antibiotic-resistant strains with ascertained zoonotic potential and, in many countries, has increased over the past two decades [ ] , does not seem to be widely spread in water buffalo. three monophasic s. typhimurium (b: , :i:-) isolates were also found that are s. typhimurium lacking phase two flagellar antigens that have a rapid emergence and dissemination in food animals, companion animals, and humans. more significantly, the public health risk posed by these emerging monophasic s. typhimurium strains is considered comparable to that of other epidemic s. typhimurium [ ] . the diagnostic investigation indicated that non-typhimurium salmonella isolates were detected with at least another potential pathogen in % of cases ( figure a ). in % of cases salmonella was linked with pathogenic escherichia coli that were characterized for the presence of virulence factors. other frequent associations were found with cryptosporidium spp. ( %) and rotavirus ( %) ( figure a) . remarkably, s. typhimurium was never associated with pathogenic e. coli, while it was isolated sporadically with clostridium perfringens (strain # ), rotavirus (strain # ), and cryptosporidium spp. (strain # ) ( figure b) . the presence of more pathogens in the same subject might suggest that, as for other animal species [ ] , diarrhea in water buffalo calves can be characterized by a multifactorial etiology. data from necroscopic examinations of tissues indicated that the lesions caused by s. typhimurium were characterized by severe damage of the intestine, ranging from congestive to necrotic-ulcerative enterocolitis. in particular, the strains isolated from animals exhibiting the most severe lesions were # , # , # , and # . among these strains, the two dt strains were also found, thus supporting the pathogenic role of this phage type. the other salmonella serovars were instead isolated from subjects exhibiting a variety of different lesions, mostly minor lesions confined to the jejunum, and often ( % of cases) associated with other pathogens. similarly, the monophasic s. typhimurium strains were detected either with rotavirus (strain # ) or st-positive e. coli (strains # and # ). these data confirm the pathogenic potential of the serovar typhimurium for water buffalo calves. on the other hand, the scarcity of observed lesions and the frequent presence of more than one microorganism in the same subject hamper a clear understanding of the potential pathogenic role of the non-typhimurium salmonella serovars included in this study. s. typhimurium and monophasic s. typhimurium strains were further characterized by the molecular detection of genes coding for virulence factors. the genetic characterization (table ) included five loci (avra, ssaq, mgtc, siid, and sopb) located on spi - , respectively [ ] , eight loci (gipa, gtgb, sope, sodc , gtge, gogb, ssph , and ssph ) of prophage origin [ ] [ ] [ ] [ ] [ ] , the gene spvc, located on a virulence plasmid [ ] , and nine genes (stfe, safc, csga, ipfd, bcfc, stbd, pefa, fima, and agfa) coding for bacterial fimbriae, involved in surface adhesion and gut colonization [ ] . as a positive control for the pcr assay, amplification of the chromosomal gene inva was carried out for each strain. all the s. typhimurium and monophasic typhimurium isolates displayed the presence of avra, ssaq, mgtc, siid, sopb, ssph , stfe, ipfd, bcfc, stbd, and fima genes, and the absence of the sope gene. other loci were variably distributed among the strains, with frequency values ranging from - % (table ) . on the basis of the presence or absence of the loci included in the study, the strains of s. typhimurium were subdivided into different genotypes (table ) ; however, the isolates with identical genotype displayed different phage types suggesting the presence of different strains. interestingly, the three monophasic s. typhimurium strains exhibited three different genotypes (table ) . the following loci: inva, ssph , stfe, ipfd, bcfc, stbd, fima, avra, ssaq, mgtc, siid, sopb were present in all the strains; the sope gene was not found in any of these strains. b nt = not typeable. the loci-genetic characterization was also extended to the s. muenster and s. give isolates to investigate their pathogenic potential because of their large presence in water buffalo calves. in addition they have already been reported to cause saepticemic salmonellosis in cattle and calves [ , ] . the molecular results (table ) indicated that the loci inva, safc, bcfc, fima and ssaq were present in all the strains, the genes gipa, gogb, ssph , sodc , gtge, spvc, stfe, ipfd and pefa were not found in any of these isolates, while the remaining loci were variably distributed, with frequency values ranging from - %. in particular, the prophage genes were scarcely present ( loci in the muenster serovar, locus in the give serovar), the plasmidic spvc locus was absent in all the analyzed isolates, while the fimbrial genes and the spi - genetic markers were discretely represented ( loci for the former genes in both serovars, and loci for the latter genes in the serovar muenster and give, respectively). moreover, the molecular profiles allowed to identify different genotypes out of the s. muenster isolates, and different genotypes out of the s. give isolates (table ) . our data confirm the high variability of the typhimurium serovar [ , ] , mostly related to virulence factors, and highlight the high discriminating potential of the genotyping technique performed. our data also suggest that monophasic typhimurium strains are likely to possess a similarly high degree of genetic variability, particularly linked to virulence markers. moreover, the presence of virulence markers in the isolated strains of monophasic s. typhimurium, s. muenster and s. give could further support their pathogenic potential. the products of the genes included in the virulotyping assay performed here are known to be important during different stages of infection (table ) . however, the distribution of these factors among the tested strains highlights the complexity and the variety of potential mechanisms used by salmonella to induce disease in the host. the avra, ssaq, mgtc, siid, and sopb genes are genetic markers for the presence of the spi - in all s. typhimurium strains tested, although their presence does not necessarily implicate the presence of the entire spi. spis are clusters of genes on the chromosome, likely to be horizontally acquired, and variably associated with enhanced invasion and intracellular survival within both phagocytic and non-phagocytic cells. in particular, spi- has been largely associated with the ability to produce enteritis [ ] . the s. typhimurium strains included in this study all displayed the presence of the investigated spi markers. interestingly, these loci appeared widely distributed also among the serovars muenster and give. the sope gene is known to favor the entry of salmonella into host cells and its presence has been correlated with disease in humans [ ] and with the epidemic potential of s. typhimurium strains in cattle [ ] . this gene was absent in all the s. typhimurium strains included in the present study, while was present in all the s. muenster strains analyzed. the pefa (plasmid encoded fimbria), agfa (aggregative fimbria a) and spvc (salmonella plasmid of virulence gene c) genes are all located on plasmids [ ] . five s. typhimurium isolates tested in the current study possessed both pefa and spvc, two isolates were positive for only spvc, and three isolates were positive for only agfa (table ) . these results confirm the presence of more than one virulence plasmid among s. typhimurium strains isolated from diarrheic water buffalo calves, and suggest horizontal exchange of virulence factors. however, the loci pefa and spvc were absent in all the monophasic s. typhimurium, s. muenster and s. give strains tested. prophage genes are known to account for most of the variability of closely-related s. typhimurium strains. moreover, lysogenic bacteriophages promote changes in the composition of genomic dna often altering the phenotype of the host [ , ] . the prophage virulence genes included in this study exhibited a variable distribution among the isolates tested, thus suggesting synergistic and/or redundant effects of these loci on the pathogenicity of salmonella, likely contributing to the actgcgaaagatgccacaga phenotypic variability of this pathogen. these loci were mostly present in s. typhimurium and monophasic s. typhimurium rather than in s. muenster and s. give isolates. fimbrial genes appeared widely distributed among all the serovars tested, particularly in s. typhimurium strains, with frequency values ≥ %, except for the plasmid-borne pefa and agfa genes (with frequency values of % and %, respectively). these data are consistent with the essential functions of adhesion factors for the attachment and internalization processes that occur during pathogenesis. to better characterize in vivo virulence, three strains representative of all s. typhimurium isolates were chosen to perform mixed infections in mice. animal experiments included the two strains exhibiting the highest and the lowest number of virulence factors (strains # and # , respectively), and strain # , carrying the same virulotype as strain # , but that does not harbor the agfa locus (table ). in the competition assay, strain # outcompeted both strains # and # (ci . ; p< . , and ci . ; p< . , respectively). these results were confirmed in a gastrointestinal mouse model of infection, which better resembles the clinical form of salmonellosis in livestock. using oral inoculation, in the competition assay, again strain # outcompeted both strains # and # (ci . ; p< . , and ci . ; p< . , respectively). our data indicate that among those strains included in the experiment, strain # was the most virulent in mice. these competition assays in mice suggest a key role of the agfa gene coding for a thin aggregative fimbria involved in the colonization of host intestinal epithelial cells by attachment to glycoprotein or glycolipid receptors on epithelial cell surfaces. indeed, the strain which was more virulent in in vivo experiments was characterized by a high number of virulence factors and by the presence of the agfa locus. moreover, it was isolated from one of the subjects with necrotic-ulcerative enterocolitis. the presence of this type of fimbria has been reported in clinical human and animal isolates of salmonella + + ----+ --- -+ -------- -+ -------- - freq. a the following loci: inva, safc, bcfc, fima and ssaq were present in all the strains; the genes gipa, gogb, ssph , sodc , gtge, spvc, stfe, ipfd and pefa were not found in any of these strains. [ , ] . the data presented here suggest that agfa might increase bacterial pathogenicity. nevertheless, we cannot reject the hypothesis that the mouse model chosen for in vivo experiments could have influenced the virulence phenotype of the tested strains originally isolated from water buffalo calves. therefore, future studies will be necessary to exclude the possibility that the phenotypic differences observed among the tested salmonellae are dependent on the animal model or on other virulence factors not included in this study. however, in vivo experiments carried out in mouse models represent a good preliminary source of information on the expression of traits associated with pathogenicity of salmonella in mammalian species. this study showed a significant ( %) prevalence of salmonella spp. in water buffalo calves affected by gastroenteritis with lethal outcome. however, our results did not indicate the existence of a salmonella serovar specifically adapted to water buffalo and highlighted that s. typhimurium is the most frequently found serovar. the molecular and phenotypic characterization of the s. typhimurium isolates provided evidence that within this serovar there are different pathotypes potentially responsible for different clinical syndromes, therefore requiring prophylaxis protocols including the use of specific vaccines for the effective control of salmonellosis in water buffalo calves and possible contamination of the food chain. this study was carried out in the campania region, southern italy, during - , using samples taken from water buffalo calves bred in different farms. the animals were aged between - weeks old and were all affected by gastroenteritis with lethal outcome. during necropsy, the intestinal lesions were evaluated and the intestinal content of the involved sections was collected and tested for the presence of salmonella spp. in addition, the presence of e. coli, eimeria spp., cryptosporidium spp., giardia spp., coronavirus, rotavirus, and c. perfringens were also determined to investigate their association with salmonella spp. the isolation of salmonella spp. was performed according to iso : [ ] . the isolated salmonella spp. were serotyped according to the kaufmann-white scheme [ ] . phage-typing of the isolated s. typhimurium strains was performed by the italian national reference centre for salmonellosis (istituto zooprofilattico sperimentale delle venezie). the presence of rotavirus and coronavirus was detected by polymerase chain reaction (pcr) amplification [ , ] . cryptosporidium spp. and giardia spp. antigens were detected by chromatographic immunoassay (oxoid, basingstoke, uk). the presence of eimeria spp. was examined by flotation technique using saturated saline [ ] . e. coli and c. perfringens were isolated according to the protocol reported by quinn et al. [ ] . e. coli hemolytic activity was evaluated by growing colonies on blood agar base, while virulence factors (lt-heat-labile toxin, st-heatstable toxin, stx -shiga toxin , stx -shiga-toxin , eaeintimin, cnf-cytotoxic necrotizing factor, and cdt-cytolethal distending toxin) were detected by molecular assays, as previously reported [ ] [ ] [ ] . bacterial dna was extracted from ml of overnight cultures using chelex resin (biorad, hercules, ca) and used as the template for the pcr detection of genes listed in table , as described previously [ ] [ ] [ ] [ ] [ ] [ ] ] . the primers used to amplify the genes ssph , ssph , ssaq, sopb, siid, stfe, safc, csga, ipfd, bcfc, stbd, and fima were designed using the primer software (version . . ; http:// frodo.wi.mit.edu/), and pcr was performed in a final volume of μl containing hotstar taq master mix (qiagen, valencia, ca) ×, . μm each primer and μl of extracted dna. the thermal profile included an initial denaturation step at °c for min, followed by cycles at °c for s, °c for s, and °c for min, and a final extension step at °c for min. amplification products were visualized under ultraviolet (uv) light after electrophoresis on % agarose gels and staining with sybrsafe (invitrogen, carlsbad, ca). groups of five age matched ( - weeks old) female balb/c mice used in this study were purchased from charles river (calco, italy). three strains (s. typhimurium # , s. typhimurium # , s. typhimurium # ), representative of the genotypically characterized s. typhimurium isolates, were selected for an in vivo analysis of virulence by using the competitive index (ci) resulting from mixed infections [ ] . in particular, two strains were selected that exhibited the highest and lowest number of virulence factors (strains # and # , respectively), and strain # , carrying the same virulotype as strain # , but without the locus agfa (table ) . bacteria were grown overnight at °c in brain heart infusion medium (oxoid, basingstoke, uk), washed, and diluted in sterile saline. cultures were alternatively combined in a mixture of equivalent numbers ( : ratio) of two of the three selected strains (input). mice were inoculated intraperitoneally (ip) with a dose of × bacteria or received mg of streptomycin orally ( μl of sterile solution or sterile saline) h prior of being intragastrically administered with × bacteria. the number of colony-forming units (cfu) contained in the inocula were confirmed by plating serial dilutions and counting colony growth. at (ip) or (os) days after infection, mice were sacrificed, spleens were aseptically removed, and bacteria were counted by plating serial dilutions (output). the ratio of two strains in the input and in the output was evaluated by picking and transferring colonies on selective plates. antibiotics used were streptomycin and sulfonamide, for which strain and strains or were naturally resistant. the ci was calculated using the formula: ci = output (strain a/strain b)/inoculum (strain a/strain b). statistical differences between outputs and inputs were determined by student's t test. all animal handling and sampling procedures were performed under the conditions of the local ethics committee meeting the requirements of italian legislation. fao regional office for europe inter-regional cooperative research network on buffalo veterinary microbiology and microbial disease infectious diseases pathophysiology of diarrhea in calves pathogenesis of bacterial infections in animals studies on enteritis in buffalo calves scientific opinion on monitoring and assessment of the public health risk of "salmonella typhimurium-like" strains virulotyping and antimicrobial resistance typing of salmonella enterica serovars relevant to human health in europe characterization of salmonella enterica serovar typhimurium strains of veterinary origin by molecular typing methods variability in occurrence of multiple prophage genes in salmonella typhimurium strains isolated in slovack republic transposition of the heat-stable toxin asta gene into a gifsy- -related prophage of salmonella enterica serovar abortusovis rapid identification of salmonella serovars in feces by specific detection of virulence genes, inva and spvc, by an enrichment broth culture-multiplex pcr combination assay human salmonella clinical isolates distinct from those of animal origin salmonella muenster infection in a dairy herd outbreak of salmonella give in the province of quebec frequency and polymorphism of sope in isolates of salmonella enterica belonging to the ten most prevalent serotypes in england and wales isolation of a temperate bacteriophage encoding the type iii effector protein sope from an epidemic salmonella typhimurium strain elucidation of antimicrobial susceptibility profiles and genotyping of salmonella enterica isolates from clinical cases of salmonellosis in new mexico in dna-based diagnostic tests for salmonella species targeting agfa, the structural gene for thin, aggregative fimbriae characterization of salmonella isolates from captive lizards annex d "detection of salmonella spp. in animal feces and in environmental samples from primary production stage who collaborating centre for reference and research on salmonella phylogenetic analysis of a highly conserved region of the polymerase gene from coronaviruses and development of a consensus polymerase chain reaction assay real-time reverse transcription-pcr for detection of rotavirus and adenovirus as causative agents of acute viral gastroenteritis in children internal parasites in laboratory procedures for veterinary technicians production of cytolethal distending toxins by pathogenic escherichia coli strains isolated from human and animal sources: establishment of the existence of a new cdt variant (type iv) single multiplex pcr assay to identify simultaneously the six categories of diarrheagenic escherichia coli associated with enteric infections shiga-toxin producing e. coli (stec) and necrotoxigenic e. coli (ntec) isolated from diarrhoeic mediterranean water buffalo calves (bubalus bubalis) use of mixed infections with salmonella strains to study virulence genes and their interactions in vivo submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors declare that they have no competing interests.authors' contributions gb carried out the molecular genetic studies and drafted the manuscript. mgl contributed to the molecular analysis and the isolation and phenotypic characterization of the strains. mp designed and interpreted the results of the in vivo assays. mrc carried out the isolation and phenotypic characterization of the strains. cg participated in the design of the in vivo assays and performed the statistical analysis. sa and ab carried out the in vivo assays and participated in the phenotypic characterization of the strains. de contributed to the design of the molecular assays, the interpretation of the genotyping results and critical preparation of part of the manuscript. pp participated in the conception, design, and coordination of the study. gg conceived the study, and participated in its design and coordination, and helped to draft the manuscript. all authors read and approved the final manuscript. key: cord- - ubh u r authors: nelson, oranmiyan w.; garrity, george m. title: genome sequences published outside of standards in genomic sciences, july - october date: - - journal: stand genomic sci doi: . /sigs. sha: doc_id: cord_uid: ubh u r the purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. while our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the sigs editorial office. aeromonas aquariorum, sequence accession bafl through bafl , and ap [ ] aerococcus viridans ll , sequence accession ajtg [ ] bacillus anthracis h , sequence accession cp . ( chromosome), cp . . (plasmid pxo ), and cp . (plasmid pxo ) [ ] bacillus atrophaeus c , sequence accession ajrj [ ] bacillus cereus nc , sequence accession ap (chromosome), ap (plasmid standards in genomic sciences pnccld), ap (plasmid pnc , kb), ap (plasmid pnc , kb), ap (plasmid pnc , kb), and ap (plasmid pnc , kb) [ ] bacillus siamensis kctc t , sequence accession ajvf [ ] bacillus sp. strain b , sequence accession ajst [ ] bacillus sp. strain , sequence accession afsu [ ] citreicella aestuarii strain , sequence accession ajkj [ ] clostridium beijerinckii strain g , sequence acceession akwa [ ] corynebacterium pseudotuberculosis strain / -a, sequence accession cp [ ] enterobacter sp. isolate ag , sequence accession akxm [ ] enterococcus faecalis d , sequence accession cp through cp [ ] enterococcus faecalis strain np- , sequence accession ab [ ] enterococcus hirae (streptococcus faecalis) atcc , sequence accession cp (chromosome), nc_ (plasmid ptg ) [ ] "geobacillus thermoglucosidans" tno- . , sequence accession ajjn [ ] lactococcus garvieae ipla , sequence accession akfo [ ] lactobacillus mucosae lm , sequence accession ahit [ ] lactobacillus rossiae dsm , sequence accession akzk [ ] paenibacillus polymyxa osy-df, sequence accession aipp [ ] pediococcus pentosaceus strain ie- , sequence accession cahu through cahu [ ] pelosinus fermentans a , sequence accession akvm [ ] pelosinus fermentans b , sequence accession akvj [ ] pelosinus fermentans jbw , sequence accession akvo [ ] pelosinus fermentans r , sequence accession akvn [ ] planococcus antarcticus dsm , sequence accession ajyb [ ] pseudomonas stutzeri strain jm , sequence accession cp [ ] rhodococcus sp. strain dk , sequence accession ajlq [ ] staphylococcus aureus strain lct-sa , sequence accession ajlp [ ] staphylococcus capitis qn , sequence accession ajtg [ ] staphylococcus equorum subsp. equorum mu , sequence accession cajl to cajl [ ] staphylococcus hominis zbw , sequence accession akgc [ ] staphylococcus saprophyticus subsp. saprophyticus m - , sequence accession ahkb [ ] streptococcus mutans gs- , sequence accession cp [ ] streptococcus pyogenes m , sequence accession ap [ ] streptococcus salivarius ps , sequence accession ajfw [ ] streptococcus thermophilus strain mn-zlw- , sequence accession cp [ ] ureibacillus thermosphaericus strain thermo-bf, sequence accession ajik [ ] phylum tenericutes mycoplasma leachii strain pg t , sequence accession cp . [ ] mycoplasma mycoides subsp. mycoides, sequence accession cp . [ ] mycoplasma wenyonii strain massachusetts, sequence accession cp [ ] phylum actinobacteria actinomyces massiliensis strain t , sequence accession akio [ ] bifidobacterium animalis subsp. lactis b , sequence accesion cp [ ] bifidobacterium animalis subsp. lactis bi- , sequence accesion cp [ ] bifidobacterium bifidum strain bgn , sequence accession cp [ ] brevibacterium massiliense strain t , sequence accession cajd [ ] corynebacterium bovis dsm , sequence accession aenj [ ] corynebacterium diphtheriae biovar intermedius nctc , sequence accession ajvh [ ] corynebacterium pseudotuberculosis strain / -a, sequence accession cp [ ] corynebacterium pseudotuberculosis strain / - sequence accession cp . [ ] corynebacterium pseudotuberculosis strain / -a, sequence accession cp [ ] microbacterium yannicii, sequence accession cajf through cajf [ ] micromonospora lupini lupac , sequence accession caie [ ] mycobacterium bolletii strain m , sequence accession ajly [ ] mycobacterium intracellulare clinical strain mott- y, sequence accession cp [ ] mycobacterium massiliense m , sequence accession ajsc [ ] mycobacterium massiliense strain go , sequence accession cp [ ] mycobacterium massiliense strain m , sequence accession ajma [ ] mycobacterium tuberculosis rgtb , sequence accession cp [ ] mycobacterium tuberculosis mtb , sequence accession cp [ ] parascardovia denticolens ipla , sequence accession akii [ ] saccharothrix espanaensis dsm t , sequence accession he [ ] streptomyces auratus strain agr , sequence accession ajgv [ ] "streptomyces cattleya" dsm t , sequence accession fq and fq [ ] streptomyces globisporus c- , sequence accession ajuo [ ] streptococcus mutans gs- , sequence accession cp [ ] streptomyces sp. strain aa , sequence accession alap [ ] streptomyces sulphureus l , sequence accession ajtq [ ] phylum spirochaetes borrelia crocidurae, sequence accession cp (chromosome), cp to cp (plasmids) [ ] treponema sp. strain jc , sequence accession jq [ ] phylum bacteroidetes flavobacterium sp. strain f , sequence accession akzq [ ] fusobacterium nucleatum subsp. fusiforme atcc t , sequence accession akxi [ ] "imtechella halotolerans" k t , sequence accession ajju [ ] standards in genomic sciences phage clp , sequence accession jn [ ] pseudomonas aeruginosa siphophage mp , sequence accession jx [ ] pseudomonas aeruginosa temperate phage mp , sequence accession eu [ ] pseudomonas aeruginosa temperate phage mp , sequence accession jq [ ] pseudomonas phage Φ-s , sequence accession jx [ ] siphophage mp , sequence accession jx [ ] staphylococcus aureus bacteriophage gh , sequence accession jq [ ] vibrio vulnificus bacteriophage ssp , sequence accession jq [ ] african bovine rotaviruses rva/cowwt/zaf/ / /g p, sequence accession s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn [ ] african bovine rotaviruses rva/cowwt/zaf/ / /g p, sequence accession s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn [ ] african bovine rotaviruses rva/cowwt/zaf/ / /g p, sequence accession s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (vp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn , s (nsp ) jn [ ] avian leukosis virus, sequence accession jx [ ] avian influenza virus h n , sequence accession jx through jx [ ] avian influenza virus h n , sequence accession jq through jq [ ] avian-like h n swine influenza, sequence accession jx through jx [ ] avian paramyxovirus, sequence accession jq [ ] avian tembusu-related virus strain wr, sequence accession jx [ ] bluetongue virus serotype , sequence accession jx to jx [ ] bluetongue virus serotype , sequence accession [ ] bombyx mori nucleopolyhedrovirus, sequence accession jq [ ] bovine viral diarrhea virus , sequence accession jf [ ] bovine foamy viruses, sequence accession jx [ ] canine noroviruses, sequence accession fj and fj [ ] chicken anemia virus, sequence accession jx [ ] chikungunya virus, sequence accession jx [ ] chinese virulent avian coronavirus gx-yl , sequence accession hq [ ] chinese virulent avian coronavirus gx-yl , sequence accession hq [ ] coxsackievirus b , sequence accession jx [ ] enterovirus c (hev-c ), sequence accession jx [ ] genotype hepatitis e virus strain, sequence accession jq [ ] h n avian influenza virus, sequence accession jq to jq [ ] h n subtype influenza virus fjg , sequence accession jf . , jn . through jn . [ ] . herpes simplex virus strain mckrae, sequence accession jx [ ] human coronavirus nl , sequence accession jx [ ] human g p rotavirus, sequence accession ab through ab [ ] ikoma lyssavirus, sequence accession jx [ ] korean sacbrood viruses amsbv-kor , sequence accession jq [ ] korean sacbrood viruses amsbv-kor , sequence accession jq [ ] mitochondrion of frankliniella occidentalis, sequence accession jn [ ] new circular dna virus from grapevine, sequence accession jq [ ] novel porcine epidemic diarrhea virus, sequence accession jx [ ] pararetrovirus, sequence accession jq [ ] parechovirus, sequence accession jx [ ] peste des petits ruminants virus, sequence accession jx [ ] polyomavirus, sequence accession jq [ ] porcine circovirus b strain cc , sequence accession jq [ ] porcine circovirus type (pcv ), sequence accession jx [ ] porcine epidemic diarrhea virus strain aj , sequence accession jx [ ] porcine [ ] waterfowl aviadenovirus goose adenovirus , sequence accession jf [ ] plant genomes plants cpdna of smilax china, sequence accession nc_ [ ] elodea canadensis, sequence accession jq [ ] ogura-type mitochondrial genome, sequence accession ab [ ] fungus aspergillus oryzae strain . , sequence accession akhy [ ] rhodosporidium toruloides mtcc , sequence accession ajmj [ ] animal genomes helicoverpa armigera, sequence accession hq [ ] plasmids plasmidincn plasmid prsb , sequence accession jn [ ] plasmidincn plasmid prsb , sequence accession jn [ ] plasmidincn plasmid prsb , sequence accession jn [ ] plasmidincn plasmid prsb , sequence accession jn [ ] complete genome sequence of the hyperthermophilic cellulolytic crenarchaeon "thermogladius cellulolyticus" complete genome sequence of methanomassiliicoccus luminyensis, the largest genome of a human-associated archaea species isolated from a deep-sea hydrothermal sulfide chimney on the juan de fuca ridge complete genome sequence of leptospirillum ferrooxidans strain c - , isolated from a fresh volcanic ash deposit on the island of miyake sequence analysis of a complete . mb prochlorococcus marinus med genome cloned in yeast genome sequence of acinetobacter sp. strain ha, isolated from the gut of the polyphagous insect pest helicoverpa armigera draft genome sequence of the hydrocarbon-degrading and emulsan-producing strain acinetobacter venetianus rag- t genome sequence and mutational analysis of plant-growth-promoting bacterium agrobacterium tumefaciens ccnwgs isolated from a zinc-lead mine tailing draft genome sequence of alcaligenes faecalis subsp. faecalis ncib (ccug ) genome sequence of pectin-degrading alishewanella agri, isolated from landfill soil genome sequence of pectin-degrading alishewanella aestuarii strain b t, isolated from tidal flat sediment genome sequence of thermotolerant bacillus methanolicus: features and regulation related to methylotrophy and production of l-lysine and l-glutamate from methanol draft genome sequence of the sulfur-oxidizing bacterium "candidatus sulfurovum sediminum" ar, which belongs to the epsilonproteobacteria genome sequence of bartonella birtlesii, a bacterium isolated from small rodents of the genus apodemus complete genome sequence of brucella abortus a , a new strain isolated from the fetal gastric fluid of dairy cattle complete genome sequence of brucella canis strain hsk a , isolated from the blood of an infected dog genome sequence of brucella melitensis s , an isolate of sequence type , prevalent in china genome sequences of brucella melitensis m and its two derivatives m w and m w, which evolved in vivo complete genome sequence of the endophytic bacterium burkholderia sp. strain kj draft genome sequence of the soil bacterium burkholderia terrae strain bs , which interacts with fungal surface structures revised genome sequence of burkholderia thailandensis msmb with improved annotation complete genome sequence of the opportunistic food-borne pathogen cronobacter sakazakii es genome sequence of the rice pathogen dickeya zeae strain zju genome sequence of the plant growth-promoting bacterium enterobacter cloacae gs genome sequence of enterococcus faecium clinical isolate lct-ef genome sequence of enterobacter radicincitans dsm t, a plant growth-promoting endophyte genome sequence of escherichia coli j , a reference strain for genetic studies draft genome sequence of escherichia coli lct-ec complete genome sequence of klebsiella oxytoca e , a new delhi metallo-β-lactamase- -producing nosocomial strain complete genome sequences of six strains of the genus methylobacterium genome sequence of methylobacterium sp. strain gxf , a xylem-associated bacterium isolated from vitis vinifera l. grapevine complete genome sequences of methylophaga sp. strain jam and methylophaga sp. strain jam genome sequence of radiation-resistant modestobacter marinus strain bc , a representative actinobacterium that thrives on calcareous stone surfaces genome sequence of mycobacterium massiliense m , isolated from a lymph node biopsy specimen genome sequence of a neisseria meningitidis capsule null locus strain from the clonal complex of sequence type genome sequence of novosphingobium sp strain rr - , a nopaline crown gall-associated bacterium isolated from vitis vinifera l. grapevine complete genome sequence of providencia stuartii clinical isolate mrsn whole-genome shotgun sequence of the sulfur-oxidizing chemoautotroph pseudaminobacter salicylatoxidans kct genome sequence of pseudomonas aeruginosa strain sjtd- , a bacterium capable of degrading long-chain alkanes and crude oil genome sequence of the lactate-utilizing pseudomonas aeruginosa strain xmg genome sequence of the rice pathogen pseudomonas fuscovaginae cb draft genome sequence of arctic marine bacterium pseudoalteromonas issachenkonii pamc draft genome sequence of high-siderophore-yielding pseudomonas sp. strain hys genome sequence of the polychlorinated-biphenyl degrader pseudomonas pseudoalcaligenes kf complete genome sequence of the naphthalene-degrading pseudomonas putida strain nd genome sequence of pseudomonas putida strain sjte- , a bacterium capable of degrading estrogens and persistent organic pollutants draft genome sequence of pseudomonas sp. strain m t , carried by bursaphelenchus xylophilus isolated from pinus pinaster genome sequence of the moderately halotolerant, arsenite-oxidizing bacterium pseudomonas stutzeri ts genome sequence of ralstonia sp. strain pba, a bacterium involved in the biodegradation of -aminobenzenesulfonate genome sequences for six rhodanobacter strains, isolated from soils and the terrestrial subsurface, with variable denitrification capabilities genome sequence of rickettsia conorii subsp. caspia, the agent of astrakhan fever genome sequence of rickettsia australis, the agent of queensland tick typhus draft genome sequence of rickettsia sp. strain meam , isolated from the whitefly bemisia tabaci genome sequence of rickettsia conorii subsp. israelensis, the agent of israeli spotted fever draft genome sequence of the antagonistic rhizosphere bacterium serratia plymuthica strain pri- c draft genome sequence of serratia marcescens strain lct-sm complete genome sequence of the broad-host-range strain sinorhizobium fredii usda genome sequence of sphingobium indicum b a, a hexachlorocyclohexane-degrading bacterium genome sequence of stenotrophomonas maltophilia pml , which displays baeyer-villiger monooxygenase activity draft genome sequences of eight salmonella enterica serotype newport strains from diverse hosts and locations whole-genome sequences and comparative genomics of salmonella enterica serovar typhi isolates from patients with fatal and nonfatal typhoid fever in papua new guinea draft genome sequence of serratia sp. strain m t , isolated from pinewood disease nematode bursaphelenchus xylophilus draft genome sequence of a psychrotolerant sulfur-oxidizing bacterium, sulfuricella denitrificans skb , and proteomic insights into cold adaptation genome sequence of xanthomonas campestris jx, an industrially productive strain for xanthan gum draft genome sequence of yersinia pestis strain , an isolate from the great gerbil plague focus in xinjiang genome sequence of a novel human pathogen, aeromonas aquariorum genome sequence of aerococcus viridans ll complete genome sequence of bacillus anthracis h , an isolate from a korean patient with anthrax draft genome sequence of the sponge-associated strain bacillus atrophaeus c , a potential producer of marine drugs complete genome sequence of bacillus cereus nc , which produces high levels of the emetic toxin cereulide draft genome sequence of the plant growth-promoting bacterium bacillus siamensis kctc t isolated from a cherry tree genome sequence of the plant growth-promoting rhizobacterium bacillus sp. strain draft genome sequence of citreicella aestuarii strain , a member of the roseobacter clade isolated without xenobiotic pressure from a petroleum-polluted beach draft genome sequence of butanol-acetone-producing clostridium beijerinckii strain g complete genome sequence of corynebacterium pseudotuberculosis strain / -a, isolated from a horse in north america draft genome sequences of enterobacter sp. isolate ag from the midgut of the malaria mosquito anopheles gambiae complete genome sequence of the porcine isolate enterococcus faecalis d complete genome sequence of bacteriophage bc- specifically infecting enterococcus faecalis strain np- genome sequence of enterococcus hirae (streptococcus faecalis) atcc , a model organism for the study of ion transport, bioenergetics, and copper homeostasis complete genome sequence of geobacillus thermoglucosidans tno- . , a thermophilic sporeformer associated with a dairy-processing environment genome sequence of lactococcus garvieae ipla , a bacteriocin-producing, tetracycline-resistant strain isolated from a raw-milk cheese genome sequence of lactobacillus mucosae lm , isolated from piglet feces draft genome sequence of lactobacillus rossiae dsm t draft genome sequence of paenibacillus polymyxa osy-df, which coproduces a lantibiotic, paenibacillin, and polymyxin e genome sequence of pediococcus pentosaceus strain ie- draft genome sequences for two metal-reducing pelosinus fermentans strains isolated from a cr(vi)-contaminated site and for type strain r draft genome sequence of pelosinus fermentans jbw , isolated during in situ stimulation for cr(vi) reduction draft genome sequences for two metal-reducing pelosinus fermentans strains isolated from a cr(vi)-contaminated site and for type strain r genome sequence of the antarctic psychrophile bacterium planococcus antarcticus dsm genome sequence of pseudomonas stutzeri strain jm (dsm ), a soil isolate and model organism for natural transformation draft genome sequence and comparative analysis of the superb aromatic-hydrocarbon degrader rhodococcus sp. strain dk whole-genome sequence of staphylococcus aureus strain lct-sa genome sequence of staphylococcus capitis qn , which causes infective endocarditis genome sequence of staphylococcus equorum subsp. equorum mu , isolated from a french smear-ripened cheese whole-genome sequence of staphylococcus hominis, an opportunistic pathogen draft genome sequence of staphylococcus saprophyticus subsp. saprophyticus m - , isolated from the gills of a korean rockfish, sebastes schlegeli hilgendorf, after high hydrostatic pressure processing complete genome sequence of streptococcus mutans gs- , a serotype c strain complete genome sequence of streptococcus pyogenes m , isolated from a patient with streptococcal toxic shock syndrome complete genome sequence of streptococcus salivarius ps , a strain isolated from human milk complete genome sequence of streptococcus thermophilus strain mn-zlw- draft genome sequence of ureibacillus thermosphaericus strain thermo-bf, isolated from ramsar hot springs in iran complete genome sequences of mycoplasma leachii strain pg t and the pathogenic mycoplasma mycoides subsp. mycoides small colony biotype strain gladysdale complete genome sequence of mycoplasma wenyonii strain massachusetts draft genome sequence of actinomyces massiliensis strain t complete genome sequences of probiotic strains bifidobacterium animalis subsp. lactis b and bi- complete genome sequence of the probiotic bacterium bifidobacterium bifidum strain bgn draft genome sequence of brevibacterium massiliense strain t draft genome sequence of corynebacterium bovis dsm , which causes clinical mastitis in dairy cows draft genome sequence of corynebacterium diphtheriae biovar intermedius nctc complete genome sequence of corynebacterium pseudotuberculosis strain / -a, isolated from a horse in north america complete genome sequences of corynebacterium pseudotuberculosis strains / - and / -a, isolated from sheep in scotland and australia, respectively genome sequence of microbacterium yannicii, a bacterium isolated from a cystic fibrosis patient genome sequence of micromonospora lupini lupac , isolated from root nodules of lupinus angustifolius draft genome sequence of mycobacterium bolletii strain m , a rapidly growing mycobacterium of contentious taxonomic status complete genome sequence of mycobacterium intracellulare clinical strain mott- y, belonging to the int genotype genome sequence of mycobacterium massiliense m , isolated from a lymph node biopsy specimen complete genome sequence of mycobacterium massiliense annotated genome sequence of mycobacterium massiliense strain m , belonging to the recently created taxon mycobacterium abscessus subsp. bolletii comb. nov whole-genome sefquences of two clinical isolates of mycobacterium tuberculosis from kerala, south india genome sequence of parascardovia denticolens ipla , isolated from human breast milk complete genome sequence of saccharothrix espanaensis dsm t and comparison to the other completely sequenced pseudonocardiaceae insights into fluorometabolite biosynthesis in streptomyces cattleya dsm through genome sequence and knockout mutants draft genome sequence of streptomyces globisporus c- , which produces an antitumor antibiotic consisting of a nine-membered enediyne with a chromoprotein complete genome sequence of streptococcus mutans gs- , a serotype c strain draft genome sequence of the marine streptomyces sp. strain aa , isolated from the yellow sea draft genome sequence of the marine actinomycete streptomyces sulphureus l , isolated from marine sediment complete genome sequence of borrelia crocidurae draft genome sequence of treponema sp. strain jc , a novel spirochete isolated from the bovine rumen draft genome sequence of flavobacterium sp. strain f , isolated from the rhizosphere of bell pepper (capsicum annuum l. cv. maccabi) draft genome sequence of fusobacterium nucleatum subsp. fusiforme atcc t genome sequence of the halotolerant bacterium imtechella halotolerans k t genome sequence of a novel actinophage pis isolated from a strain of saccharomonospora sp complete genome sequence of aeromonas hydrophila phage cc complete genome sequence of bacteriophage bc- specifically infecting enterococcus faecalis strain np- complete genome sequence of bacteriophage ssu specific for salmonella enterica serovar typhimurium rough strains genome sequence of blattabacterium sp. strain bgiga, endosymbiont of the blaberus giganteus cockroach complete genome sequence of caulobacter crescentus bacteriophage φcbk complete genome sequence of celeribacter bacteriophage p l complete genome sequence of croceibacter bacteriophage p s complete genome sequence of cronobacter sakazakii temperate bacteriophage phies complete genome sequence of marinomonas bacteriophage p complete genome sequence of phytopathogenic pectobacterium carotovorum subsp. carotovorum bacteriophage pp complete genome sequences of two persicivirga bacteriophages, p s and p l genome sequence of the phage clp , which infects the beer spoilage bacterium pediococcus damnosus complete genome sequence of pseudomonas aeruginosa siphophage mp complete genome sequences of two pseudomonas aeruginosa temperate phages, mp and mp , which lack the phage-host crispr interaction genome sequence of the broad-host-range pseudomonas phage Φ-s complete genome sequence of pseudomonas aeruginosa siphophage mp complete genome sequence of staphylococcus aureus bacteriophage gh complete genome sequence of vibrio vulnificus bacteriophage ssp whole genome sequence analyses of three african bovine rotaviruses reveal that they emerged through multiple reassortment events between rotaviruses from different mammalian species complete genome sequence of an avian leukosis virus isolate associated with hemangioma and myeloid leukosis in egg-type and meat-type chickens genome sequence of a novel reassortant h n avian influenza virus in southern china complete genome sequence of an h n avian influenza virus isolated from a parrot in southern china complete genome sequence of an avian-like h n swine influenza virus discovered in southern china complete genome sequence of a novel avian paramyxovirus complete genome sequence of avian tembusu-related virus strain wr isolated from white kaiya ducks in fujian complete genome sequence of bluetongue virus serotype : implications for serotyping complete genome sequence of bluetongue virus serotype of goat origin from india genome sequence of a bombyx mori nucleopolyhedrovirus strain with cubic occlusion bodies complete genome sequence of a bovine viral diarrhea virus from commercial fetal bovine serum complete genome sequences of two novel european clade bovine foamy viruses from germany and poland complete genome sequences of novel canine noroviruses in hong kong complete genome sequence analysis of a recent chicken anemia virus isolate and comparison with a chicken anemia virus isolate from human fecal samples in china complete genome sequence of a chikungunya virus isolated in guangdong complete genome sequences of two chinese virulent avian coronavirus infectious bronchitis virus variants complete genome sequence of a recombinant coxsackievirus b from a patient with a fatal case of hand, foot, and mouth disease in guangxi complete genome sequence of a novel human enterovirus c (hev-c ) identified in a child with community-acquired pneumonia complete genome sequence of the genotype hepatitis e virus strain prevalent in swine in jiangsu province, china, reveals a close relationship with that from the human population in this area complete genome sequence of an h n avian influenza virus isolated from a live bird market in southern china complete genome sequence of a novel h n subtype influenza virus fjg strain in china reveals a natural reassortant event characterization and complete genome sequence of human coronavirus nl isolated in china whole genome sequence analyses of three african bovine rotaviruses reveal that they emerged through multiple reassortment events between rotaviruses from different mammalian species complete genome sequence of ikoma lyssavirus analysis of the complete genome sequence of two korean sacbrood viruses in the honey bee, apis mellifera the complete mitochondrial genome sequence of the western flower thrips frankliniella occidentalis (thysanoptera: thripidae) contains triplicate putative control regions genome sequence of methylobacterium sp. strain gxf , a xylem-associated bacterium isolated from vitis vinifera l. grapevine complete genome sequence of porcine epidemic diarrhea virus strain aj isolated from a suckling piglet with acute diarrhea in china complete genome sequence of a novel pararetrovirus isolated from soybean complete genome sequence of a novel type of human parechovirus strain reveals natural recombination events complete genome sequence of a peste des petits ruminants virus recovered from wild bharal in tibet complete genome sequence of a polyomavirus isolated from horses complete genome sequence of a novel field strain of rearranged porcine circovirus type in southern china complete genome sequence of a novel field strain of rearranged porcine circovirus type in southern china complete genome sequence of porcine epidemic diarrhea virus strain aj isolated from a suckling piglet with acute diarrhea in china complete genome sequence of a novel porcine sapelovirus strain yc isolated from piglets with diarrhea complete genome sequence of porcine reproductive and respiratory syndrome virus strain qy reveals a novel subgroup emerging in china genome sequences of sat foot-and-mouth disease viruses from egypt and palestinian autonomous territories (gaza strip) complete genome sequence of a street rabies virus from mexico genome sequence of a waterfowl aviadenovirus, goose adenovirus jenny) xiang q-y. complete cpdna genome sequence of smilax china and phylogenetic placement of liliales -influences of gene partitions and taxon sampling complete chloroplast genome sequence of elodea canadensis and comparative analyses with other monocot plastid genomes a complete mitochondrial genome sequence of ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (raphanus sativus l.) draft genome sequence of aspergillus oryzae strain . genome sequence of the oleaginous red yeast rhodosporidium toruloides mtcc complete genome sequence of a monosense densovirus infecting the cotton bollworm, helicoverpa armigera the complete genome sequences of four new incn plasmids from wastewater treatment plant effluent provide new insights into incn plasmid diversity and evolution key: cord- - j yic authors: donato, celeste; vijaykrishna, dhanasekaran title: the broad host range and genetic diversity of mammalian and avian astroviruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: j yic astroviruses are a diverse family of viruses that infect a wide range of mammalian and avian hosts. here we describe the phylogenetic diversity and current classification methodology of astroviruses based on the orf b and orf genes, highlighting the propensity of astroviruses to undergo interspecies transmission and genetic recombination which greatly increase diversity and complicate attempts at a unified and comprehensive classification strategy. astroviruses (astvs) were first described in as small viruses that are - nm in diameter with icosahedral morphology. astroviruses are named due to the distinct five-pointed or six-pointed star-like appearance of some virions when visualized under an electron microscope (em); astrovirus is derived from the greek word astron meaning star [ ] [ ] [ ] . astroviruses were first described from human infants with diarrhea and were subsequently identified in the young of numerous mammalian and avian species [ , ] . human astroviurses (hastvs) have been recognized as one of the major causes of acute gastroenteritis in children, associated with - % of infections [ ] . transmission of hastv occurs via the fecal-oral route, person-to-person contact, or contaminated food or water. following an incubation period of - days, symptoms including diarrhea, vomiting, abdominal pain, and fever are often reported [ , ] . whilst primarily associated with asymptomatic or diarrheal disease in humans, there are several reports of central nervous system (cns) complications such as acute flaccid paralysis [ ] , meningitis, and encephalitis [ , ] . animal astroviurses, have been isolated from numerous mammalian and avian species. in animals, astrovirus infection may be asymptomatic or associated with enteric disease and a range of other symptoms indicative of the involvement of other organ systems including hepatitis and nephritis in avian species [ , ] , and neurological symptoms in cattle [ ] [ ] [ ] and mink [ ] . astroviruses are classified within the unassigned astroviridae family and are non-enveloped viruses characterized by a positive sense, single-stranded rna (ssrna) genome . - . kb long comprised of a -untranslated region (utr), three open reading frames (orfs)-orf a, orf b, and orf , a -utr, and a poly a tail [ ] . the orf a region encodes a non-structural polyprotein (serine protease), orf b encodes a polyprotein including the rna-dependent rna polymerase (rdrp), and orf encodes the viral capsid protein [ ] . a further orf, termed orfx, has been observed in classic hastvs and some mammalian astroviurses, overlapping the end of orf which may be translated through a leaking scanning mechanism [ ] . astroviruses exhibit several distinctive features in addition to a distinctive morphology. the viruses lack a rna-helicase domain encoded within the genome and utilize a ribosomal frameshifting mechanism to translate the rdrp, which distinguishes the astroviridae family from other non-enveloped ssrna virus families such as picornaviridae and caliciviridae [ , ] . the greatest diversity in the genome is within the orf region, which is characterized by a highly-conserved n-terminal domain (amino acids (aa) - ), a hypervariable domain (aa - ) which is believed to form the capsid spike and contribute to receptor binding, and a highly acidic c-terminal domain [ , ] . the wide host range of astroviruses and the high degree of genetic diversity present within the astroviridae family have complicated attempts at a unified classification method. astroviruses are classified within the unassigned astroviridae family, which was initially comprised of a single genus (astrovirus) based on virion morphology [ ] . the classification of the astrovirus genus within the family astroviridae was recognized by the international committee for taxonomy of viruses (ictv) in and the classification scheme has been modified numerous times over the intervening years [ ] . in , two genera were recognized; mamastrovirus (mastv) and avastrovirus (aastv), that were known to infect mammalian and avian species, respectively, and viruses were classified solely on the species of origin [ ] . however, the advent of sequence based characterization rendered this approach inadequate, revealing that viruses isolated from different species can be genetically similar (reflecting prevalent interspecies transmission of viruses) and also revealing a large range of diversity of viruses within a single host species. with this in mind, the classification system proposed by the ictv astroviridae study group in recommended classification based on the amino acid sequence of the orf genome region, recommending that different strains of the same astrovirus species should share > % identity in the capsid proteins [ ] . additionally, there are proposals to define distinct variants within a recognized astrovirus species, with a variant defined as sharing < - % nucleotide similarity to the reference or prototype strain of each species or if phylogenetic analysis is used, a distance of > . based on analysis of the capsid protein [ , ] . astroviruses within the mamastrovirus genus are derived from numerous mammalian species in addition to humans (hastv), including farmed species such as pigs (pastv), sheep (oastv), cattle (boastv), domesticated animals including cats (fastv), and dogs (caastv), rodents and small mammals including mink (miastv), bats (bastv), rats (rastv), mice, rabbit (rabastv), fox, marmot (hhmastv), porcupine, shrew, vole, and larger species including deer (ccastv), monkeys, water buffalo (bufastv), yak, camel (dcastv), and cheetah (chastv) (figure a,b) . viruses from the mamastrovirus genus have also been characterized from marine mammals including stellar sea lion (sslastv) and california sea lions (cslastv), minke whale, orca whale, and bottlenose dolphins (bdastv) (figure a ,b) [ ] . the current ictv classification reveals recognized species of mamastrovirus (mastv- - ) within two genogroups gi and gii; mamastrovirus (gi.a-human); mamastrovirus (gi.b-feline); mamastrovirus (gi.c-porcine); mamastrovirus (gi.d-california sea lion); mamastrovirus (gi.e-canine); mamastrovirus (gi.f-human); mamastrovirus (gi.g-bottlenose dolphin); mamastrovirus (gii.a-human); mamastrovirus (gii.b-human); mamastrovirus (gii.c-mink); mamastrovirus (gii.d-california sea lion), mamastrovirus (gii.e-bat); mamastrovirus (gii.f-ovine); and mamastroviruses - (gii.g to gii.l-bat species), and numerous other strains awaiting classification, some of which are considered as tentative new species (designated by a ∧ symbol in the phylogenetic trees) (figure a ) [ , ] . viruses from the avastrovirus genus have been characterized from numerous farmed avian species including turkeys (tastv), ducks (dastv), chicken (castv), guineafowl (gfastv), pigeon (piastv), goose, as well as wild aquatic and terrestrial birds including heron, doves, penguins, and many other species (figure a) . the three species originally recognized within the genus were avastrovirus gi.a comprised of turkey astrovirus (tastv- ), avastrovirus gi.b comprised of avian nephritis virus (anv- ), avian nephritis virus (anv- ), and avastrovirus gii.a comprised of turkey astrovirus (tastv- ) and duck astrovirus dastv/c-ngb [ ] . avastrovirus gi.a, avastrovirus gi.b, and avastrovirus gii.a were renamed avastrovirus (aastv- ), avastrovirus (aastv- ), and avastrovirus (aastv- ), respectively [ ] . viruses from the avastrovirus genus have been characterized from numerous farmed avian species including turkeys (tastv), ducks (dastv), chicken (castv), guineafowl (gfastv), pigeon (piastv), goose, as well as wild aquatic and terrestrial birds including heron, doves, penguins, and many other species (figure a) . the three species originally recognized within the genus were avastrovirus gi.a comprised of turkey astrovirus (tastv- ), avastrovirus gi.b comprised of avian nephritis virus (anv- ), avian nephritis virus (anv- ), and avastrovirus gii.a comprised of turkey astrovirus (tastv- ) and duck astrovirus dastv/c-ngb [ ] . avastrovirus gi.a, avastrovirus gi.b, and avastrovirus gii.a were renamed avastrovirus (aastv- ), avastrovirus (aastv- ), and avastrovirus (aastv- ), respectively [ ] . currently, classification into species is based on the phylogenetic analysis of the amino acid sequence of the full length orf region of the genome that encodes the capsid. however, the limited number of capsid sequences available compared to rdrp sequences makes consistent classification difficult, especially with some novel viruses incompletely sequenced. there are numerous unclassified astroviruses, particularly isolated from aquatic and terrestrial wild birds which, according to the ictv, are "related viruses which may be members of the avastrovirus genus but have not been approved as species" [ ] . astrovirus infection in humans has been primarily associated with diarrhea and vomiting, accounting for up to % of sporadic gastroenteritis cases in some regions [ ] . cns complications associated with astrovirus infection have been reported in recent years, including acute flaccid paralysis, with some fatalities reported in children with underlying immune disorders [ , ] . historically human astroviruses (hastv) were classified into five serotypes in [ ] . subsequent molecular characterization based on viral reactivity to polyclonal antibodies and nucleotide sequence analysis led to the recognition of eight serotypes (hastv- - ), now termed "classic" hastv [ , , ] . the relatively recent advent of next generation sequencing (ngs) and metagenomic analysis has led to the identification of numerous novel strains considered "non-classic" hastv [ ] . currently, hastvs are classified within the species mastv- (hastv- - ), mastv- (mlb - ), mastv- (va /hmo-a, va , va , bf ), and mastv- (va /hmo-c, va /hmo-b) [ ] (figure a ). the mastv- species is comprised of hastv- - , and surveillance has revealed that hastv- is the most commonly detected type in children, followed by hastv- - , whereas hastv- - have been rarely detected [ ] . hastv- and hastv- have been associated with infection of older children and longer duration of diarrhea (> days) [ , ] . a hastv- strain was also isolated from an infant with fatal meningoencephalitis [ ] . based upon the phylogenetic analysis of the orf region, different lineages within each hastv type have been proposed; hastv- (hastv- a-d) and hastv- (hastv- a-d) have been divided into four lineages, whereas hastv- (hastv- a-b) and hastv- (hastv- a-c) have been classified into two and three lineages, respectively [ ] . the first "non-classic" hastv strain characterized was mlb , the virus was detected in a stool sample from a year old australian child with acute diarrhea in ; the child had previously received a liver transplant [ ] . the majority of mlb strains characterized to date have been detected in india, kenya, and japan with limited detected in the usa, china, bhutan, egypt, brazil, and italy and prevalence has been reported in the range of . % to % [ ] . however, a seroepidemiologic study in the usa revealed that primary exposure to mlb occurs in childhood and that seropositivity reached % by adulthood suggesting the widespread circulation of the virus in the human population [ ] . mlb viruses were first identified in vellore, india [ ] with the majority of strains subsequently identified in japan, the gambia, and switzerland with limited detection in turkey, usa, kenya, china, and thailand and prevalence reported in the range of . % to . % [ ] . mlb has been associated with meningitis and other cns complications and has been detected in immunocompromised children [ ] . mlb viruses were first detected in india in [ ] , with subsequent detection in kenya and the gambia and the prevalence in stools ranges from . % to . % [ , ] . there is a dual naming system for some hastv species due to the simultaneous characterization of these viruses by different researchers; these viruses are termed va/hmo named for va-virginia and hmo-human-mink-ovine-like viruses, due to their genetic relatedness to previously characterized mink and sheep viruses [ ] . in , va /hmo-a strains were detected in children with non-polio acute flaccid paralysis in nigeria, pakistan [ ] , and india [ ] . the prevalence of va /hmo-a viruses in stools ranges from . % to . %, with strains also detected in egypt, japan, usa, kenya, and china [ ] . va has only been detected in nepal [ ] and the bf strain has only detected in burkina faso [ ] . the va /hmo-b viruses were first identified in vellore, india [ ] with sporadic detection in nigeria, pakistan, and nepal with prevalence ranging from . % to . % [ ] . va /hmo-c viruses were first detected in during an outbreak of diarrhea in children in virginia, usa [ ] . va /hmo-c viruses have been detected and associated with encephalitis in immunocompromized children and adults [ , , ] and acute respiratory disease [ ] . the prevalence of va /hmo-c viruses in stools ranges from . % to . % in diarrheic and non-diarrheic subjects worldwide, with limited detection in nepal, japan, tanzania, the gambia, france, and the u.k. [ , , ] . seroprevalence of va /hmo-c has been reported at % in adults [ ] . the divergent human astrovirus-like virus tentatively named bastrovirus was isolated from patients in the netherlands. the capsid region is homologous to the capsid of hastv whilst the rdrp region is more closely related to members of the hepeviridae family. this virus remains to be classified by the icvt, however the capsid regions clusters closest to the mlb - viruses suggesting an evolutionary relationship to these divergent human astroviruses [ ] (figure a ). revealing the geographic and host diversity of this virus, divergent bastrovirus strains have also been isolated from bat, pig, and rat species in vietnam, forming a distinct cluster of strains that also await classification (figure a ). mamastroviruses are capable of infecting a wide range of mammalian species including companion animals (cats and dogs), intensively farmed species (pigs and cattle), as well as terrestrial and aquatic wild mammalian species. unexpectedly there is no clear clustering of viruses separated by hosts of terrestrial or aquatic origins (figure a,b) . in addition to the classified mastv species, there are numerous divergent viruses isolated from diverse species which likely represent new species awaiting formal classification (figure a ) [ ] . not surprisingly, domesticated animals such as cats and dogs harbor astrovirus strains more closely related to hastv than the viruses harboured by many other animal species. feline astrovirus was first identified in , and feline strains form a small discrete cluster defined as the species mastv- , closely related to the human strains comprising the species mastv- [ ] (figure a ). based on evolutionary analysis, an interspecies transmission pathway has been hypothesised whereby porcine strains may have been transmitted to cats and subsequently to humans, possibly involving other intermediary species suggesting sustained interspecies transmission events [ ] . characterization of an outbreak of diarrhea in a group of captive cheetahs in a breeding facility identified an astrovirus strain most closely related to feline strains (figure a ), however it is not clear if this is a recent transmission from domesticated cats or if the virus is circulating independently in cheetahs [ ] . despite dogs also being a companion animal, canine astrovirus, first described in the s, are not as closely related to human strains as feline strains appear to be, based on phylogenetic analysis (figure a,b) . canine strains form a small, discrete cluster comprising species mastv- , within a lineage comprised of dolphin and sea lion clades representing species mastv- and - [ , ] , and feline, porcine, and human strains are more distantly related within the same lineage (figure a) . a large diverse lineage, largely comprised of unclassified viruses or tentatively classified viruses awaiting approval encompasses divergent marmot and rabbit strains (mastv- ), small, discrete clusters of porcine/ovine/bovine strains (mastv- ), divergent rat strains (mastv- ), and unclassified mouse and mink strains forming a small discrete cluster (figure a) . numerous porcine strains also cluster within this lineage; pastv comprise mamastrovirus and were first detected by em in pigs in the u.k. and the usa [ ] , and are now recognized to have worldwide distribution [ ] . there is a high prevalence of astrovirus detection in pigs, up to % in some studies, suggesting that pigs may be persistently infected with various strains of pastv [ ] . there are five recognized lineages (pastv - ) reflecting the diversity of strains, suggesting swine are highly permissive to astrovirus infection often without symptoms of disease (figure a,b) . this diversity likely reflects the varying origins of these viruses, in particular highlighting frequent interspecies transmission and recombination events [ ] [ ] [ ] . a study in the usa revealed the high level of co-infections in pigs ( . %), revealing the frequent opportunity for recombination, especially between viruses of different lineages [ ] . the close clustering between some porcine and humans strains, in particular mastv- and mastv- viruses, reflects the close contact between these species with the close sharing of environments and co-housing documented in some countries facilitating frequent interspecies transmission [ ] . wild boars showing no symptoms of enteric disease, housed in a captive breeding park with no contact with domesticated pigs, were found to harbor astrovirus strains with a high degree of genetic similarity to porcine strains comprising mastv- and suggests that the virus may be derived from commonly circulating porcine strains [ ] (figure a,b) . porcupine strains isolated in china also cluster with unclassified pastv- strains suggesting further interspecies transmission of these viruses (figure a ) [ ] . unexpectedly, limited astrovirus strains have been isolated from sheep. ovine astrovirus was first identified by em in [ ] , and oastv- clusters with bovine strains comprising mastv- and a second strain from a healthy sheep characterized in , oastv- , clusters with porcine and bovine strains comprising mastv- [ ] (figure a) . similarly, astroviruses are not associated with a significant burden of diarrheal disease in bovine species. the first bovine astrovirus was detected in england in [ ] and bovine astrovirus strains have been detection in association with neurological disease, including encephalitis (boastv-ch /neuros ) [ ] [ ] [ ] [ ] [ ] [ ] and diarrheal disease in calves in south korea [ ] and cattle and buffalo calves in china [ ] . two serotypes were previously recognized, boastv- and boastv- [ ] , however based on phylogenetic analysis there are multiple lineages of boastv strains circulating in farmed bovine populations, and the close clustering of bovine, porcine, and ovine strains in multiple lineages reflects the common interspecies transmission events that occur between farmed animals (figure a,b) . phylogenetic analysis also reveals that bovine-like astrovirus strains have been isolated from numerous wild species including water buffalo, yak, and european roe deer (ccastv- and ccastv- ) suffering from gastroenteritis [ ] . unclassified astroviruses from dromedary camels (dcastv) [ ] also cluster in a lineage comprised of porcine and bovine strains, further suggesting multiple interspecies transmission events (figure a) . substantial astrovirus strain diversity has been observed in small mammals, primarily rodents and bats, forming both species-specific clusters reflecting endemic transmission and co-clustering of strains with other host species reflecting widespread interspecies transmission (figure a,b) . novel murine astrovirus strains have been isolated from laboratory mice in the usa and japan [ , ] . divergent viruses have also been detected, such as those detected in rats (mastv- ), highlighting the need for more detailed detection and characterization of these viruses to better understand the role these animals play in astrovirus transmission between varied species. one of the mammalian species with the highest burden of symptomatic astrovirus infection is mink; infection is associated with pre-weaning diarrhea syndrome and the neurological condition "shaking mink syndrome" [ , ] . mink viruses cluster within multiple lineages suggesting that the species is permissive to infection with multiple, diverse lineages of viruses (figure a) . bat astroviruses were first detected in in hong kong [ ] and subsequently detected in bats in china [ , ] , north america [ ] , germany [ ] , hungary [ ] , and the czech republic [ ] from numerous bat species, with detection rates ranging from % to % in miniopterus magnater bats and from % to % of miniopterus pusillus bats sampled in hong kong [ ] . a diverse population of viruses appears to be highly prevalent in bats without causing disease [ ] (figures and ) . the majority of bat astroviruses are divergent from other characterized mammalian astroviruses, and display a high degree of genetic diversity forming numerous recognized and proposed species (figure a) . some bat sequences clustered with strains from other species including fox, cattle, and mice, suggesting that bats are highly permissive to infection with diverse astrovirus strains from multiple hosts and play a key role in astrovirus diversity and interspecies transmission (figure a,b) . astroviruses have been detected in aquatic mammalian species including californian sea lions, steller sea lion, bottlenose dolphin, killer whale, and minke whale [ ] . the strains from these aquatic species do not cluster together, instead forming multiple discrete clusters, suggesting several transmission events from terrestrial mammals to aquatic mammals (figure a) . minke whale and bdastv- strains are divergent to characterized astrovirus strains, with bdastv- strains comprising mastv- and a diverse group of sea lion viruses comprising the mastv- cluster with porcine and canine strains (figure a ). there are numerous divergent strains that are yet to be classified that may reflect interspecies transmission events. a single divergent feline strain along with a fox strain cluster within a diverse lineage comprised of human hmo strains (mastv- and - ), sea lion (mastv- ), unclassified sea lion, mink (mastv- ), and bat (mastv- ) strains. a himalayan marmot strain clusters with mastv- strains (figure a,b ). there appears to be a large diversity of mastv strains not captured by the currently available sequences, highlighting the need for more detailed sampling and characterization of animal strains. the isolation of astroviruses from avian species predates their isolation in humans, with disease in ducklings described in , however the virus was not formally recognized as an astrovirus until [ , ] . avian astroviruses have been documented to cause infection in poultry leading to economic losses in farms and affecting food production worldwide [ ] . avian astroviruses have been associated with a spectrum of disease ranging from subclinical infection in seemingly healthy adult birds to heavy flock losses. pleomorphic symptoms include enteritis in turkeys, chickens, and guineafowl, mild growth depression and nephritis in chickens, and hepatitis in ducklings [ ] . astrovirus infection has been implicated in pre-hatching mortality in ducklings and goslings [ ] . in addition to aastv- comprised of tastv- , aastv- comprised of anv and anv , and aastv- comprised of tastv- and duck astrovirus (dastv- ), there are numerous yet-to-be classified viruses, including turkey astrovirus (tastv- ), chicken astrovirus a (castv-a), chicken astrovirus b (castv-b) [ , ] , gfastv, and multiple viruses isolated from ducks including duck hepatitis virus (dhv- /dastv- ) [ ] , dhv- /dastv- -like viruses, dastv-cph (dastv- ) [ ] , dastv- , dastv-yp/dastv- -like, and diverse viruses isolated from wild birds [ ] . avian astrovirus have been detected in outbreaks of enteric disease in turkey poults, and avian astrovirus were first reported as an agent of gastroenteritis and mortality in young turkeys in , associated with a condition known as poult enteritis mortality syndrome (pems) [ , ] . subsequently there have been sporadic reports of astrovirus outbreaks in turkeys, associated with enteritis and growth depression [ ] . based on serological and genetic analysis, two types of tastv have been recognized (tastv- and tastv- ). tastv- comprises the avastrovirus species and was first described in the u.k. [ ] . tastv- has limited detection in other avian species with sporadic detection in chicken and ducks and forms a discrete cluster in both the capsid and rdrp phylogenetic analysis (figure a,b) . tastv- was identified in [ ] , and is likely to be classified within the species avastrovirus and is primarily associated with pems [ ] . tastv- is the predominant tastv lineage, with a wider global circulation and greater genetic diversity compared to tastv- , reflected by the co-circulation of multiple sub-lineages (figure a,b) . based on phylogenetic analysis of the rdrp gene there appears to be limited interspecies transmission of tastv- -like viruses detected in chicken and duck (figure b) . astroviruses infecting guineafowl are closely related to tastv- strains based on analysis of the rdrp region [ ] , however the capsid region is distinct to tastv- strains, forming a discrete cluster in a lineage of unassigned viruses suggesting they are closely related to castv-b strains (figure a) . these gfastvs were possibly derived from recombination and interspecies events followed by sustained transmission in the guineafowl population. this highlights the limitation of classifying viruses based on a single region of the genome. chicken astrovirus has been associated with runting-stunting syndrome (rss) in chickens characterized by poor weight gain, lower feed conversion, and mortality resulting in economic losses [ ] , and "white chicks" disease associated with increased mortality of embryos and chicks, weakness, and white plumage [ ] . currently two serotypes of castv have been described [ ] , and both serotypes form discrete clusters in the phylogenetic analysis of the capsid region within a lineage of unclassified viruses (likely to be classified within aastv- ). the castv-a viruses form a smaller, discrete lineage, clustering closest to dastv- strains (figure a) . castv-b strains form a large lineage clustering closest to gfastv strains. based on phylogenetic analysis of the small region of the rdrp gene commonly sequenced, it does not allow castv-a and -b strains to form discrete clusters as seen in the capsid analysis, possibly reflecting multiple recombination events in castv strains (figure b ). castv strains have limited detection in other avian species with sporadic detection in pigeon and duck (figure a,b) . avian nephritis virus was identified in association with intestinal nephritis in chickens and growth retardation. the first serotype of anv (anv- ) was isolated from a healthy broiler chick in [ ] and was initially regarded as a picornavirus, subsequently reclassified within the astroviridae family in [ ] . based on capsid phylogenetic analysis, anv- strains cluster within the aastv- lineage forming a small, discrete, relatively conserved cluster (figure a) . the second serotype (anv- ) was later described from chicks with stunted growth [ ] . based on capsid phylogenetic analysis, anv- strains cluster within the aastv- lineage, forming a larger, more diverse sub-lineage compared to anv- . anv- and anv- viruses have also been sporadically detected in ducks and turkeys (figure a) . a third serotype (anv- ) detected in chickens and turkeys with rss and locomotion impairment has been proposed based on sequencing of the orf a region, however complete genome sequences are unavailable for adequate comparisons to the recognized serotypes [ ] . the virus designated pigeon anv (p-anv) was detected during an outbreak of gastrointestinal illness in young pigeons in china [ ] . p-anv represents an interspecies transmission event from chickens to pigeon; based on phylogenetic analysis, p-anv strains share a high degree of genetic similarity to anv- strains and cluster closely with strains also circulating in china suggesting a localized transmission event, and these viruses should be considered as anv- viruses and do not require a distinct designation (figure a ) [ ] . based on phylogenetic analysis of the small region of the rdrp gene commonly sequenced, it does not allow anv and strains to form discrete clusters as characterized in the capsid analysis (figure b ). whilst this may reflect common recombination events between anv strains, the analysis of a small region does not adequately allow for distinct clustering. astrovirus infection in ducks has been associated with a highly contagious and fatal hepatitis, historically known as duck hepatitis virus type (dhv- ), which was described in the u.k. and subsequently serotype dhv- was isolated in the usa [ , , , ] . the th ictv report classified dhv- and dhv- as dastv- and dastv- , respectively [ ] . dastv- strains form a small, discrete cluster within the aastv- lineage based on phylogenetic analysis of the capsid region, clustering closest to tastv- strains (figure a) . dastv- viruses, currently unclassified, form a discrete, highly conserved cluster closest to castv-a strains (figure a) . based on phylogenetic analysis of the rdrp region, dastv- is closely related to other unclassified duck strains whilst dastv- strains are more closely related to tastv- strains (figure b ). further unclassified serotypes have been described; dastv- -cph, dastv/c-ngb, and dastv- [ ] . duck astrovirus dastv- /cph has been suggested to transmit horizontally and vertically [ ] and dastv- /cph viruses have also been detected in goslings [ ] . phylogenetic analysis of the rdrp region highlights the diversity of aastv strains circulating in duck species (figure b) . strains endemic to ducks have limited detection in other avian species including geese (figure a,b) . astroviruses have been detected in numerous wild aquatic species including teals, pintails, and shovelers (belonging to the order anseriformes), sanderlings (order charadriiformes), and herons and spoonbills (order pelecaniformes) [ , ] . fewer land dwelling wild birds have been found to harbor avian astroviruses including doves and pigeons (order colombiformes), european roller (order coraciiformes), and black-naped monarch (order passeriformes) [ , , , ] . these viruses are highly divergent and largely unclassified, suggesting these viruses are endemic to the wild bird population (figure a,b) . the migratory behavior of some of these species provides ideal conditions for virus dissemination and diversification as during migration there is a high degree of co-mingling and increased density of birds of different species that may originate from varied geographic regions [ ] . phylogenies indicate that the genetic diversity of mamastroviruses has been shaped by extensive interspecies transmission events that have occurred in the past between wild and domestic species and humans. however, the inference of astrovirus interspecies transmission events is hampered due to (a) sequencing of only a small portion of the genome; (b) inadequate sampling; or (c) that the event occurred early during the divergence of astrovirus lineages. a few exceptions where host-jumps are apparent have been in livestock where a greater level of sampling has occurred. this includes hosts with a greater level of interaction (e.g., mastv- in ovine and bovine) or host genetic similarly (e.g., mastv- in wild boars and domestic pigs). with the exception of hastv- strains associated with fatal meningoencephalitis, it is interesting to note that viruses from multiple species, all recognized to cause neurological symptoms, are closely related including human va /hmo-c viruses and mink and bovine viruses also associated with neurological symptoms, suggesting that these related viruses may have a distinct phenotype compared to other mastv strains (figure a,b) . astrovirus strains identified from fecal samples of multiple non-human primate species from wild, captive, and peri-urban environments in bangladesh and cambodia reveal multiple interspecies transmission events, with viruses closely related to the va/hmo lineage of human viruses, and non-human mammalian and avian astroviruses (figure a,b) [ ] . similarly, there appears to be evidence for a high degree of cross species transmission of avian astroviruses between farmed poultry species as described in the above sections. there also appears to be transmission between avian and mammalian species. the highly divergent strains isolated from european roller (er/szal /hun/ and er/bmtk /hun/ ) exhibited low identity to avian and mammalian astroviruses, cluster within the mastv lineage, and were likely derived from multiple recombination and interspecies transmission events [ ] . the carnivorous diet of this avian species may have facilitated the interspecies transmission event between a rodent or small mammal species. this is the only report of a mamastrovirus strain in an avian species; in contrast, there have been more reports of avastrovirus strains detected in mammalian species which may reflect greater sampling density of the mammalian population. a highly divergent group of mink strains detected in china represent a novel clade of astroviruses that were distantly related to previously described mink astrovirus and were closely related to chicken and turkey astroviruses (figure a,b) [ ] . these viruses are recombinant strains with the capsid region clustering with castv-b strains and the rdrp region clustering with human mastv strains and castv strains (figure a,b) . there have been two avastrovirus strains detected in humans; a strain clustering close to anv- strains detected in turkey and chicken was isolated from a child in the gambia, and a strain clustering with anv- strains detected in chicken was isolated from a child in kenya [ ] . serological studies have been used to screen human sera from poultry workers for antibodies to tastv- , with up to % of participants positive with the highest detection in abattoir workers and turkey growers [ ] , suggesting avian strains may be readily transmitted to humans under prolonged close contact. the important role that ecotones play in astrovirus cross-species transmission has been proposed [ ] . ecotones are ecological transition areas such as small and medium sized farms which rear multiple species. the co-rearing of poultry such as domestic ducks, chickens, turkey, and guineafowl can facilitate transmission between these species but also transmission to wild birds [ ] . farms and abattoirs have also been recognized as environments facilitating transmission between livestock species and to farm and abattoir workers [ ] . many other species have contact with livestock in a farming environment; in addition to wild species, companion animals such as cats and dogs and other peri-domestic animals have contact with livestock and their biological waste providing substantial opportunities for cross-species transmission. astroviruses also persist in bodies of water making the aquatic environment ideal for the transmission of viruses infecting avian species, aquatic mammalian species, and possible transmission between terrestrial and aquatic species. untreated or inadequately treated sewage and waste water from domestic and farmed areas can reach fresh and marine bodies of water transmitting human and animal viruses. astroviruses have been detected in the environment and the durability of the virus in this environment may greatly contribute to cross-species transmission within and between terrestrial and aquatic species, generating significant diversity [ ] . in addition to interspecies transmission which generates significant diversity in astrovirus species, both intra-species and inter-species recombination can rapidly generate novel, divergent viruses. full-and partial-genome sequence analysis has identified multiple strains that have undergone recombination events, which are predominately located within the orf b/orf junction region of the genome, which is a region with an rna secondary structure predicted to contain a stable hairpin structure [ , , [ ] [ ] [ ] [ ] . a virus with a recombination event within the orf a region has been identified [ ] . some recombinants appear to be highly stable and show widespread detection [ ] , whilst others are detected sporadically as single strains. numerous human recombinant strains have been reported between hastv strains, including strains with hastv- (orf b) and hastv- (orf ) parental viruses, hastv- (orf b) and hastv- (orf ) parental viruses, hastv- (orf b) and hastv- (orf ), and va (orf b) and mlb (orf ) parental viruses [ , [ ] [ ] [ ] . divergent species often represent recombination events between strains of different species. in , the study conducted by rivera et al. suggested the possibility of a recombination event between human and california sea lion astrovirus strains [ ] . a recombinant strain derived from porcine astrovirus and human hastv- strains was reported from piglets and children from various regions of colombia [ ] . anv strains that appear to be derived from recombination events between anv- (orf b) and anv- (orf ) have been described in the usa [ ] . the increased sampling density of numerous host species combined with the more prevalent use of ngs technologies and viral metagenomic studies will increase the detection of novel strains, further driving the need for a unified, complex, and encompassing classification system. the previously common practice of sequencing a relatively conserved rdrp amplicon of - bp renders many sequences available in genbank of little use in detailed phylogenetic analysis, as does the frequent missing metadata regarding species of isolation, country, and date of collection. sequencing small regions is not adequate to determine if a virus strain is a novel, divergent strain or a recombinant virus. recommendations should be made to encourage full genome sequencing where possible and the deposition of associated host and demographic information. although analysis of amino acid sequences of the capsid region is required for classification, it leads to confusion regarding appropriate phylogenetic analysis, with highly inconsistent publication of nucleotide and amino acid trees further complicating attempts to clarify diversity and classification. the topologies of amino acid trees differ to those of nucleotide trees, particularly for the analysis of mastv, and whilst amino acid trees are required for classification, nucleotide trees may be more appropriate for describing within species diversity (figures s and s ) . incorporating a standardized nomenclature to aid in classification has proven invaluable in the classification of numerous viruses, including rotavirus and influenza [ ] . adopting a nomenclature that records the appropriate metadata associated with sample collection including host, location, date of collection, and determined species and serotype as proposed by martella and colleagues would vastly improve the usability of strains for more complex analyses [ ] . the following are available online at www.mdpi.com/ - / / / /s , figure s : maximum-likelihood phylogenetic tree of mastv (a) capsid nucleotide and (b) capsid amino acid sequences, figure s : maximum-likelihood phylogenetic tree of mastv rdrp, figure s : maximum-likelihood phylogenetic tree of aastv (a) capsid nucleotide and (b) capsid amino acid sequences, figure s : maximum-likelihood phylogenetic tree of aastv rdrp. the authors declare no conflict of interest. nm particles in faeces in infantile gastroenteritis ultrastructure of human astrovirus serotype letter: viruses and gastroenteritis in infants detection and transmission of nm virus particles (astroviruses) in faeces of lambs with diarrhoea duck hepatitis: vaccination against two serological types epidemiology of astrovirus infection in children the changing epidemiology of astrovirus-associated gastroenteritis: a review molecular epidemiology of childhood astrovirus infection in child care centers multiple novel astrovirus species in human stool astrovirus mlb , a new gastroenteric virus 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and sewage treatment plants, evaluated by a competitive reverse transcription-pcr high evolutionary rate of human astrovirus nationwide surveillance study of human astrovirus infections in an italian paediatric population detection of all serotypes of human astrovirus by the polymerase chain reaction evidence of a recombinant wild-type human astrovirus strain from a kenyan child with gastroenteritis lineage diversification and recombination in type- human astroviruses novel astroviruses in children uniformity of rotavirus strain nomenclature proposed by the rotavirus classification working group (rcwg) key: cord- -bpgb tgo authors: ferreira, maria isabel m.; marchesi, julian r.; janssen, dick b. title: degradation of -fluorophenol by arthrobacter sp. strain if date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: bpgb tgo a gram-positive bacterial strain capable of aerobic biodegradation of -fluorophenol ( -fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. the organism, designated strain if , was identified as a member of the genus arthrobacter on the basis of s ribosomal rna gene sequence analysis. arthrobacter strain if was able to mineralize -fp up to concentrations of mm in batch culture. stoichiometric release of fluoride ions was observed, suggesting that there is no formation of halogenated dead-end products during -fp metabolism. the degradative pathway of -fp was investigated using enzyme assays and identification of intermediates by gas chromatography (gc), gc–mass spectrometry (ms), high-performance liquid chromatography, and liquid chromatography–ms. cell-free extracts of -fp-grown cells contained no activity for catechol , -dioxygenase or catechol , -dioxygenase, which indicates that the pathway does not proceed through a catechol intermediate. cells grown on -fp oxidized -fp, hydroquinone, and hydroxyquinol but not -fluorocatechol. during -fp metabolism, hydroquinone accumulated as a product. hydroquinone could be converted to hydroxyquinol, which was further transformed into maleylacetic acid and β-ketoadipic acid. these results indicate that the biodegradation of -fp starts with a -fp monooxygenase reaction that yields benzoquinone, which is reduced to hydroquinone and further metabolized via the β-ketoadipic acid pathway. during the past decades, widespread application of fluoroaromatic compounds as agrochemicals and pharmaceuticals has lead to an increased occurrence of environmental contaminants containing fluorine (key et al. ) . fluorinated compounds are rare in nature (harper and o'hagan ) . the stability of the carbon-fluorine bond ( kcal/mol in ch f, compared to kcal/mol for the carbon-chlorine bond in ch cl) makes most fluorinecontaining compounds much more resistant to biodegradation than their unsubstituted analogs (key et al. ) . furthermore, the van der waals radius of fluorine is small ( . Å, in between that of a hydrogen and an oxygen). yet, the electronegativity of fluorine causes strong polarization of c-f bonds, and fluorine substituents may be involved in biological interactions (howard et al. ) . regardless of the recalcitrance of most fluoroaromatics to biodegradation, several bacterial cultures have been described that aerobically degrade fluorobenzoic acids (oltmanns et al. ; engesser et al. ; harper and blakley ; schlomann et al. ). research on bacterial fluorophenol degradation has been limited to studies with whole cells, cell extracts, or purified enzymes from rhodococcus species that were obtained by enrichment with other aromatic compounds as a growth substrate (boersma et al. , bondar et al. bondar et al. , finkelstein et al. ) . the fluorobenzene-degrading organism rhizobiales f could grow on -fluorophenol ( -fp), but information on the pathway of -fp metabolism is lacking. cometabolic degradation of difluorophenols and trifluorophenols by several rhodococcus species is initiated by a phenol hydroxylase that catalyzes ortho-hydroxylation, resulting in the formation of the respective fluorocatechol, which is then cleaved by an intradiol dioxygenase to produce fluoromuconate (bondar et al. ). conversion of -fp by whole cells of the phenol-degrading organism rhodococcus opacus cp resulted in the formation of fluorocatechol, , , -trihydroxy- -fluorobenzene, and fluoromuconates (finkelstein et al. ) . yeasts and fungi that are able to cometabolically transform fluorinated phenols have also been described. whole cells of exophiala jeanselmei transformed -fp into -fluorocatechol and fluoromuconate . penicillium frequentans metabolized monofluorophenols in the presence of glucose or phenol. the metabolism of meta-or parafluorophenols yielded the corresponding catechol and -carboxymethylenebut- -en- -olide (hofrichter and schreibner ; hofrichter et al. ) . none of these organisms could utilize fluorophenols as a growth substrate. to our knowledge, studies on the metabolism of fluorophenols by a bacterial culture that is capable of using such as compound as a sole source of carbon and energy have not been reported. in the present paper, we describe the isolation and characterization of a bacterial strain growing on -fp as the sole source of carbon and energy. based on the identification of several intermediates, a metabolic route for the degradation of -fp by this strain is proposed. media and growth conditions nb medium contained g of nutrient broth (difco) per liter. mineral salts medium (mm) contained per liter . g na hpo h o, . g kh po , . g mgso h o, . g (nh ) so , ml trace metals solution (janssen et al. ) , and mg of yeast extract (difco laboratories). when required, the solid medium was obtained by adding g/l of difco agar. strain if was grown at °c on a rotary shaker ( rpm). cultures were grown in -ml flasks filled to % of their volume and were closed with teflon-lined screw caps. e. coli cells were grown in luria-bertani medium (lb) at °c on a rotary shaker. enrichment and isolation of -fp-degrading cultures a variety of soil samples, collected from different sites in the netherlands that are contaminated with halogenated aliphatic compounds (such as monochlorobenzene, hexachlor-obenzene, and trichloropropane), were used as the initial inoculum for the -fp enrichments. the soil samples were used to inoculate flasks containing ml of sterile minimal salts medium and mm of -fp, supplied in the liquid phase as the sole carbon and energy source. the cultures were incubated at room temperature on a rotary shaker ( rpm), and % of the suspension was transferred to a new flask containing fresh medium every days. during this time, growth (optical density at nm) and liberation of fluoride were monitored. samples of the enrichment culture were periodically spread onto minimal salts agar plates containing mm -fp and onto nb plates as soon as growth on -fp was established. pure cultures were obtained by repetitive streaking onto solid mm containing -fp and tested separately for growth on mm -fp liquid medium. growth and fluoride release were again monitored to verify -fp degradation. strains capable of -fp degradation as a sole source of carbon and energy were used for further experiments. strain if was deposited at centraalbureau voor schimmelcultures, utrecht, the netherlands, under accession number nccb . sequencing of the s rrna gene for cloning of the s ribosomal ribonucleic acid (rrna) gene, a single colony of strain if was directly used for polymerase chain reaction (pcr) amplification. the primers f ( ′-caggcctaac acatgcaagtc- ′) and r ( ′-gggcggwgtgta caaggc- ′; marchesi et al. ) were used for pcr amplification. the pcr reaction mixture ( μl) contained taq pcr buffer, . mm mgcl , pmol of each appropriate primer, mm of each deoxyribonucleotide triphosphate, u taq dna polymerase, and biomass of strain if . the pcr conditions were °c for min followed by min at °c, min at °c, . min at °c, and min at °c. the resulting fragments were cloned into the pcr -topo vector (invitrogen, carlsbad, ca) and transformed into e. coli top cells. the transformed cells were plated on lb plates containing . mg/ml of ampicillin, and the positive colonies were used for plasmid isolation and sequencing. phylogenetic analysis alignments of the s rrna gene were made using sequences downloaded from the ribosomal database project ii (rdp ii; cole et al. ) , after searching for nearest neighbors using the sequence match tool. further searches were conducted using blast and fasta of the european molecular biology laboratory (embl) database for s rrna gene sequences that are closely related to the s rrna gene of strain if but not available from the rdp ii. alignments were subsequently made using the profile alignment option in clustalx (thompson et al. ) , refined using bioedit ver . . . (hall ) , and subsequently parsed through gblock (castresana ) to remove ambiguously aligned sections and increase the robustness of the data. phylogenetic trees were determined using the neighbor-joining (nj) method. evolutionary distances for the global tree were calculated using the kimura- -parameter model with a transition/ transversion ratio of ( fig. ) . further trees were constructed in phylip version . a (j. felsenstein), using maximum parsimony and maximum likelihood methods. all nj trees were tested statistically by means of bootstrap analysis. preparation of cell extracts cells were grown in mm with mm -fp, harvested by centrifugation at , ×g for min, washed with ice-cold td buffer ( . m tris-hcl, ph . , and . m , -dithiothreitol), and stored at − °c until further use. the frozen cells were thawed, resuspended in td buffer, and incubated with lysozyme ( mg/ml) for h at °c. a french press was used to disrupt the cells, and the crude extracts were centrifuged at , ×g for min to separate the soluble from the particulate fraction. the supernatant was used for further experiments. protein concentrations were determined with the biorad protein assay kit. enzyme assays catechol , -dioxygenase and catechol , dioxygenase activities were measured spectrophotometrically at and nm, respectively. the reaction mixtures contained . mm catechol, td buffer, and cellfree extract ( . mg of protein) in a final volume of ml. -fluorocatechol , -dioxygenase was measured as catechol , -dioxygenase but with -fluorocatechol instead of catechol as the substrate. the -fp monooxygenase activity was measured spectrophotometrically by following the consumption of nadh at nm in a reaction mixture containing cellfree extract ( . mg protein), . mm -fp, . mm nadh, and buffer in a total volume of ml. the observed rates were corrected for substrate-independent nadh oxidation. hydroxymuconic semialdehyde dehydrogenase was measured at nm. reaction mixtures contained (in a final volume of ml) about . mm freshly prepared hydroxymuconic semialdehyde, td buffer, . mm nad, and cell-free extract ( . mg of protein). hydroxymuconic semialdehyde was obtained by incubation of catechol with the cell-free extract of pseudomonas putida mt- as described previously (mars et al. hydroquinone dioxygenase was assayed spectrophotometrically by monitoring the change in absorbance between and nm in a reaction mixture of ml final volume containing . mm hydroquinone, td buffer, and cell-free extract ( . mg of protein). hydroquinone hydroxylase and hydroxyquinol dioxygenase were assayed by using a fiber optic oxygen sensor. reaction mixtures contained mm of substrate, mm, and cell suspension ( . mg/ml protein) in a final volume of . ml. oxygen uptake measurements strain if was grown with glucose or -fp as the sole carbon source and harvested by centrifugation at , ×g for min at °c. cells were resuspended in mm, and o consumption was measured with a fiber optic oxygen sensor (mops- , prosense bv, hannover, germany). all reactions were performed in a stirred vessel at room temperature. the reaction mixtures contained mm of substrate ( -fp, hydroquinone, fluorocatechol, hydroxyquinol, or catechol), mm, and cell suspension ( . mg/ml protein) in a final volume of . ml. analytical methods for capillary gas chromatography (gc), ml samples were extracted with ml of diethyl ether. a model gas chromatograph (hewlett-packard) equipped with a flame ionization detector and a hp- column (agilent j- , m× . mm× . μm) were used for the analysis. gc-mass spectrometry (ms) analysis was carried out with a model mass selective detector (hewlett-packard) coupled to a hp series injector and a hp column (agilent z- ; m× . mm× . μm). high-performance liquid chromatography (hplc) was carried out using a chrompack c column ( cm× mm) connected to a jasco uv- detector, which monitored absorbance at and nm, and operated with jasco pu- pumps and a jasco as- sampler. the mobile phase was water/acetonitrile ( : ), mm potassium phosphate (ph= ), and mg/l sodium dodecyl sulfate, and the flow rate was ml/min. liquid chromatography (lc)-ms was carried out with a zmd micromass spectrometer, equipped with a xterra ms, symmetry shield c column ( . × mm), a waters photodiode array detector, and a waters separations module. samples of μl were analyzed, and compounds were isocratically eluted at a flow rate of ml/min with a solution of water/acetonitrile ( : ) and mm formic acid. concentrations of free fluoride in the culture supernatants were measured with a fluoride electrode (model - , thermo russell, scotland). fresh sodium fluoride standards were prepared for calibration curves. chemicals -fp (> %) was obtained from sigma-aldrich (steinheim, germany). all chemicals were of the highest purity grade available (sigma-aldrich; acros organics, geel, belgium). the compound -fluorocatechol was kindly provided by dr. erik de vries. purity of -fp, fluorocatechol, and hydroquinone was checked by hplc. nucleotide sequence accession numbers the s rrna sequence of strain if was deposited at genbank with the accession no. dq . isolation of a -fp-degrading bacterium to obtain a bacterial culture that is able to use -fp as carbon and energy source for growth, enrichments were performed and followed over time. two months of selective enrichment by repeated transfer of samples from a culture that displayed growth on -fp to fresh -fp containing media resulted in a microbial consortium that was capable of growth on -fp as a sole source of carbon and energy. samples of the consortium were repeatedly plated onto nb agar plates and mm agar plates containing -fp. this procedure resulted in the isolation of three pure strains named if , if , and if . the strains were restreaked on mm supplemented with -fp and inoculated in liquid cultures with mm -fp. fluoride liberation was observed for all three strains but not in control incubations to which no bacterial inoculum was added. all the three strains showed growth, as monitored at nm, and thus were able to use -fp as a sole carbon and energy source. after days of incubation in liquid culture supplied with mm -fp, strain if reached % fluoride release and an optical density (od) of . at nm. strain if released % of the fluorine and reached an od of . , while strain if reached an od of . and released % of the theoretical amount of fluoride ions. when a mixed culture of the three strains in liquid media was used, an od of . and % fluorine release were measured after days. apparently, the mixed culture contained, even after prolonged adaptation, organisms with varying efficiencies of -fp utilization. because strain if showed the highest degradation rates combined with stoichiometric release of fluoride, this organism was chosen for further studies. microbiological characterization strain if is a grampositive motile bacterium with a rhodococcus lifecycle. the optimal temperature for growth is °c. the s rrna gene sequence was determined. initial searches against the rdp ii and embl rrna databases resulted in very close associations with members of the genus arthrobacter, the closest of which were the quinaldine-degrading strain arthrobacter sp. ka - (overhage et al. ) and the -nitroguaiacol-degrading actinobacterium arthrobacter nitroguajacolicus sp. nov (kotouckova et al. ). the s rrna gene sequences of these organisms were greater than % identical to if 's. the phylogenetic analysis places the isolates in the phylum actinobacteria and the genus arthrobacter (fig. ) . the subtree that were obtained shows the detailed relationship of isolate if to other members of the genus arthrobacter. the topologies of all the trees that were obtained during statistical analysis were in agreement and clearly placed this isolate in the genus arthrobacter (data not shown). furthermore, the change in form during the growth cycle between rod and coccus is typical of the lifecycle of the arthrobacter genus. catabolic activities of arthrobacter sp. strain if to study the degradation potential of strain if , a range of organic compounds were tested as growth substrates. cells were inoculated into mm, and different organic substrates were added at a concentration of mm. after -h incubation growth, substrate disappearance and halide release were measured. growth and substrate removal were found when catechol, hydroquinone, hydroxyquinol, benzoate, phenol, -fluorocinnamic acid, and -nitrophenol were used as substrates. the organism did not grow on -fluorophenol, fluorophenol, -chlorophenol, -bromophenol, -iodophenol, fluoroacetate, trifluoroacetate, fluoroacetamide, trifluoroethanol, or on -bromoethanol. the fact that strain if is capable of growth on catechol, hydroquinone, and hydroxyquinol but not on -fluorocatechol indicates that -fluorocatechol is not the most likely intermediate in the -fp pathway, although toxic effects could also play a role. to test the range of -fp concentrations tolerated by strain if , experiments were conducted in sealed flasks with -fp at concentrations of to mm as a sole carbon and energy source. control assays without -fp showed no growth or release of fluoride, and sterile controls showed no abiotic loss of -fp. between and mm -fp, the substrate was completely consumed, stoichiometric release of fluoride was observed, and biomass increased linearly with the amount of -fp added (fig. a) . this indicates that the degradation of -fp by strain if does not give large amounts of fluorinated dead-end products. concentrations of -fp above mm caused a toxic effect on the growth of if (fig. b) . the use of mm -fp promoted growth, but a longer lag time was observed. at mm -fp, no growth occurred even after days of incubation. growth on -fp and formation of metabolites a batch culture of strain if supplied with mm -fp as the only source of carbon and energy was monitored in time. growth was accompanied by an increase in biomass, decrease in -fp, and formation of fluoride (fig. ) . after h, there was complete conversion of mm -fp, and mm fluoride was formed, indicating that there was no transient accumulation of fluorinated intermediates over the whole growth period. when cells grown on -fp were incubated in the presence of the iron chelator , ′-dipyridyl, no -fp degradation occurred, and no fluoride was released in the medium. this indicates that initial or further enzymes involved in -fp metabolism require ferrous ions for activity. to isolate intermediates of the degradation of -fp, samples from a batch culture containing mm -fp were taken at appropriate intervals and analyzed by gc, hplc, and lc-ms. metabolites that were detected were numbered in order of time of appearance in the culture. gc analysis indicated that at least four metabolites were formed and degraded over time (table ) . metabolite i, the earliest product that was observed, had the same retention time as an authentic hydroquinone standard and metabolite ii had the same retention time as a standard of hydroxyquinol. gc-ms analysis showed that the mass spectrum of metabolite i was indeed similar to that of the hydroquinone standard, with a molecular ion peak at and at m/z. metabolite ii gave a molecular ion peak at and m/z, which is typical for hydroxyquinol, and the spectrum coincided with that of a standard. metabolites v and vi were detected by gc but did not ionize in gc-ms under the conditions tested and were not identified. when the supernatant of if cells growing on mm -fp were subjected to hplc analysis, four peaks were detected (table ) . metabolite i had the same retention time as the hydroquinone standard, and metabolite ii coeluted with an authentic standard of hydroxyquinol. hydroquinone was detected in culture supernatants by hplc between . and h (fig. ) . lc-ms analysis of the extracted samples of the supernatant revealed the presence of a compound (vii) of which the negative ion spectrum shows a molecular ion peak at m/z and a peak at m/z, which are expected for the negative ionization of -oxoadipate. the above results suggest that -fp is initially converted to hydroquinone. the most likely enzyme involved in such a conversion is a -fp monooxygenase. enzyme activitiesto test inducibility of enzymes involved in -fp degradation, cells of arthrobacter sp. strain if were grown on glucose or -fp, washed, and tested for oxygen consumption in the presence of different substrates. washed suspensions of cells grown on -fp rapidly oxidized -fp, hydroquinone, and , , -benzenetriol (hydroxyquinol) without a lag. catechol and -fluorocatechol did not stimulate (table ). this result indicates that fluorocatechol and catechol are not likely intermediates and that hydroquinone and hydroxyquinol are. glucose-grown cells did not oxidize any of the aromatic substrates tested with the exception of hydroxyquinol. this observation implies that hydroxyquinol may be converted by a constitutive oxygenase, whereas most other enzymes seem inducible. cells grown with hydroxyquinol showed complete conversion of benzenetriol in the presence or absence of , ′dipyridyl, indicating that the putative hydroxyquinol oxygenase does not require ferrous ions to be active and that oxidation beyond this compound is not inhibited by the chelator. the formation of hydroquinone, which can be readily oxidized by -fp-grown cells, is in agreement with the involvement in -fp degradation of a -fp monooxygenase that is induced by -fp. to study in more detail the degradation pathway of -fp by strain if , cell-free extracts of if grown on -fp were investigated for the presence of several enzyme activities (table ) . when catechol , -and , -dioxygenases were tested for, no activity was found. furthermore, no activity was detectable for -fluorocatechol dioxygenase. consequently, the pathway does not proceed through catechol or a substituted catechol. when cell extracts were assayed for -fp monooxygenase, activity could be found in the presence of nadh but not in the presence of nadph as the reducing cosubstrate. this observation indicates that the cells contain an nadh-dependent -fp monooxygenase activity. hplc assays of incubations of cell extracts with -fp showed that substrate conversion was complete. the formation of the putative -fp monooxygenase was induced by -fp because no activity was found in extracts of cells grown in the presence of glucose. when hydroquinone was used as an assay substrate, its typical spectrophotometric peak at nm was not replaced by a peak absorbing at to nm, which would have pointed to formation of hydroxymuconic semialdehyde. thus, no hydroquinone dioxygenase was induced, or its product was rapidly further converted. the latter was ruled out by the observation that added hydroxymuconic semialdehyde was not converted by cell extracts of strain if , indicating that no hydroxymuconic semialdehyde dehydrogenase was induced during growth in the presence of -fp. these results make it unlikely that hydroxymuconic semialdehyde is an intermediate in the -fp pathway by strain if . when cell extracts were assayed for hydroxyquinol degradation, complete conversion of the substrate was observed with hplc. when the reaction was monitored spectrophotometrically, the peak at nm, typical of hydroxyquinol, was substituted by a peak at nm, typical of maleylacetate. this indicates the involvement of a hydroxyquinol oxygenase that converts hydroxyquinol into maleylacetate, which is further converted into -oxoadipate. this study reports the isolation and characterization of arthrobacter strain if , an organism capable of growth with -fp as a sole source of carbon and energy. the biodegradation of -fp by strain if was analyzed by gc, gc-ms, hplc, lc-ms, oxygen uptake experiments, and measurements of enzymatic activities. a metabolic pathway is proposed on the basis of the results of this study (steps and , ortho-cleavage) and by analogy with other systems ) . we suggest that the degradation of -fp in strain if starts with the conversion by a monooxygenase to benzoquinone, which is immediately reduced to hydroquinone. hydroquinone then undergoes a further hydroxylation to form hydroxyquinol. this benzenetriol is the ring fission substrate, which is transformed by ortho-cleavage and yields maleylacetate, which is further converted to oxoadipate. up to now, two main metabolic routes for the aerobic degradation of halogenated phenols have been described in the literature. in bacteria that degrade mono-and dichlorophenols, a degradation pathway is usually observed in which the substituted phenol is hydroxylated to the corresponding catechol, which is followed by orthocleavage of the aromatic ring (haggblom ; hollender et al. ; wieser et al. ). on the other hand, biodegradation pathways in which the substituted phenol is converted via hydroquinone to maleylacetate have been found, mainly in organisms that grow on polyhalophenols or -nitrophenol (kiyohara et al. ; xun et al. ; kadiyala and spain ; nordin et al. ; perry and zylstra ) . most studies have focused on the organisms and pathways that involve degradation via catechols. a clear indication for the first step in the -fp catabolic pathway of strain if was obtained by mass spectroscopic analysis of culture supernatants, which indicated the formation of hydroquinone as an early nonfluorinated intermediate. this result, in combination with the enzyme assays and oxygen uptake experiments, suggests the involvement of an inducible -fp monooxygenase that is nadh dependent, although the first expected product of this reaction, i.e., benzoquinone, was not detected. monooxygenation of an aromatic substrate carrying an electronwithdrawing halogen group or a nitro-substituent can result in simultaneous hydroxylation and dehalogenation or nitrite removal. this has, for example, been described for the conversion of tetrafluoro-p-hydroxybenzoate by parahydroxybenzoate hydroxylase (husain et al. ) and the oxidation of , - -trifluorophenol by a monooxygenase from ralstonia eutropha jmp (xun and webster ) . the quinone that is produced is chemically reduced by nadh to a hydroxyquinol, leading to a net stoichiometry of two nadh oxidized per halide or nitrite that is released (fig. ) . as in our case, no transient accumulation of a benzoquinone is usually observed, indicating that benzoquinone reduction is rapid. other examples of halophenol degradation with formation of hydroquinone derivatives are the degradation of trichlorophenol to , -dichlorohydroquinone by a strain of pseudomonas pickettii (kiyohara et al. ) , the degradation of pentachlorophenol by a sphingobium sp. through hydroxylation by a flavoprotein monooxygenase (xun and orser ) , and the degradation of -chlorophenol by an arthrobacter sp. (bae et al. ) . our results further indicate that hydroquinone is not the ring fission substrate because no enzymatic activities for hydroquinone dioxygenase or hydroxymuconic semialdehyde dehydrogenase were found. instead, upon conversion of hydroquinone by strain if , we observed the formation of a transient metabolite that was identified as hydroxyquinol, indicating that a second hydroxylation takes place before ring fission. the production of hydroxyquinol could be due to the action of a separate hydroquinone monooxygenase, or it could be caused by a second hydroxylation step by the -fp monooxygenase. conversion of hydroquinone to hydroxyquinol before ring fission was also suggested for strains arthrobacter that degrade -nitrophenol (perry and zylstra ) and -chlorophenol (nordin et al. ) . ring fission of hydroxyquinol would produce maleylacetate, which we did not detect as an intermediate in -fp degradation, but a transient metabolite was found with mass properties identical to those of oxoadipate, which is expected to be the next intermediate in hydroxyquinol degradation. most cometabolic transformations of fluorophenols described in the literature involve the initial action of a phenol hydroxylase that results in the formation of fluorocatechols, which are subsequently transformed into fluoromuconates through a catechol , -dioxygenase. the conversion usually proceeds with lactonization and elimination of fluoride (boersma et al. ; bondar et al. ; finkelstein et al. ; boersma et al. ; bondar et al. ). our results suggest that arthrobacter strain if , which grows on -fp, possesses a new pathway for the degradation of -fp that involves immediate defluorination by a monooxygenase. strain if avoids the accumulation of possible toxic metabolites such as -or -fluorocatechol by this initial dehalogenation. biodegradation of -chlorophenol via a hydroquinone pathway by arthrobacter ureafaciens cpr f nuclear magnetic resonance as a tool to investigate microbial degradation of fluorophenols to fluorocatechols and fluoromuconates f nmr metabolomics for the elucidation of microbial degradation pathways of fluorophenols f nmr study on the biodegradation of fluorophenols by various rhodococcus species preferential oxidative dehalogenation upon conversion of -halophenols by rhodococcus opacus g selection of conserved blocks from multiple alignments for their use in phylogenetic analysis the ribosomal database project (rdp-ii): sequences and tools for high-throughput rrna analysis adaptation of alcaligenes eutrophus b and pseudomonas sp. b to -fluorobenzoate as growth substrate identification of fluoropyrogallols as new intermediates in biotransformation of monofluorophenols in rhodococcus opacus cp microbial breakdown of halogenated aromatic pesticides and related compounds bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt the metabolism of p-fluorobenzoic acid by a pseudomonas sp fluorine-containing natural products utilization of aromatic compounds by the penicillium strain bi / unspecific degradation of halogenated phenols by the soil fungus penicillium frequentans bi / degradation of -chlorophenol via the meta cleavage pathway by comamonas testosteroni jh how good is fluorine as a hydrogen bond acceptor? fluoride elimination from substrates in hydroxylation reactions catalyzed by p-hydroxybenzoate hydroxylase degradation of halogenated aliphatic compounds by xanthobacter autotrophicus gj a two-component monooxygenase catalyzes both the hydroxylation of p-nitrophenol and the oxidative release of nitrite from -nitrocatechol in bacillus sphaericus js fluorinated organics in the biosphere isolation of pseudomonas pickettii strains that degrade , , -trichlorophenol and their dechlorination of chlorophenols arthrobacter nitroguajacolicus sp. nov., a novel -nitroguaiacol-degrading actinobacterium design and evaluation of useful bacterium-specific pcr primers that amplify genes coding for bacterial s rrna effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria novel -chlorophenol degradation gene cluster and degradation route via hydroxyquinol in arthrobacter chlorophenolicus a evidence for a new pathway in the bacterial degradation of -fluorobenzoate identification of large linear plasmids in arthrobacter spp. encoding the degradation of quinaldine to anthranilate cloning of a gene cluster involved in the catabolism of p-nitrophenol by arthrobacter sp. strain js and characterization of the p-nitrophenol monooxygenase enzymatic formation, stability, and spontaneous reactions of -fluoromuconolactone, a metabolite of the bacterial degradation of -fluorobenzoate the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools metabolism of -chlorophenol by azotobacter sp. gp : structure of the meta cleavage product of -chlorocatechol purification and properties of pentachlorophenol hydroxylase, a flavoprotein from flavobacterium sp. strain atcc a monooxygenase catalyzes sequential dechlorinations of , , -trichlorophenol by oxidative and hydrolytic reactions confirmation of oxidative dehalogenation of pentachlorophenol by a flavobacterium pentachlorophenol hydroxylase acknowledgments this work was supported in part by the european community human potential programme under contract hprtn-ct- - [biosap]. we thank erik de vries for fluorocatechol preparation and piet wietzes for technical assistance.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord- -ckms na authors: moser, kara a.; drábek, elliott f.; dwivedi, ankit; stucke, emily m.; crabtree, jonathan; dara, antoine; shah, zalak; adams, matthew; li, tao; rodrigues, priscila t.; koren, sergey; phillippy, adam m.; munro, james b.; ouattara, amed; sparklin, benjamin c.; dunning hotopp, julie c.; lyke, kirsten e.; sadzewicz, lisa; tallon, luke j.; spring, michele d.; jongsakul, krisada; lon, chanthap; saunders, david l.; ferreira, marcelo u.; nyunt, myaing m.; laufer, miriam k.; travassos, mark a.; sauerwein, robert w.; takala-harrison, shannon; fraser, claire m.; sim, b. kim lee; hoffman, stephen l.; plowe, christopher v.; silva, joana c. title: strains used in whole organism plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential date: - - journal: genome med doi: . /s - - - sha: doc_id: cord_uid: ckms na background: plasmodium falciparum (pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (chmi) in clinical trials. initial chmi studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. however, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and chmi strains, and how these strains are related to parasites in malaria endemic regions. methods: whole genome sequencing using long-read (pacific biosciences) and short-read (illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, nf , and for strains used in heterologous chmi ( g from brazil, nf .c from guinea, and nf .c from cambodia). the assemblies were used to characterize sequences in each strain relative to the reference d (a clone of nf ) genome. strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from south america, sub-saharan africa, and southeast asia. results: while few variants were detected between d and nf , we identified tens of thousands of variants between nf and the three heterologous strains. these variants include snps, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as pfap -l and pfap -g) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico cd (+) t cell epitope predictions. of all heterologous strains, nf .c had the highest number of unique predicted epitope sequences when compared to nf . comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, nf .c is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the greater mekong sub-region. conclusions: these results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous chmi. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the flattening levels of mortality and morbidity due to malaria in recent years [ ] , which follow a decade in which malaria mortality was cut in half, highlight the pressing need for new tools to control this disease. a highly efficacious vaccine against plasmodium falciparum, the deadliest malaria parasite, would be a critical development for control and elimination efforts. several variations of a highly promising pre-erythrocytic, whole organism malaria vaccine based on p. falciparum sporozoites (pfspz) are under development, all based on the same p. falciparum strain, nf [ ] , thought to be of west african origin, and which use different mechanisms for attenuation of pfspz. of these vaccine candidates, sanaria® pfspz vaccine, based on radiationattenuated sporozoites, has progressed furthest in clinical trial testing [ ] [ ] [ ] [ ] [ ] [ ] [ ] . other whole-organism vaccine candidates, including chemoattenuated (sanaria® pfspz-cvac), transgenic, and genetically attenuated sporozoites, are in earlier stages of development [ ] [ ] [ ] . pfspz vaccine showed % short-term protection against homologous controlled human malaria infection (chmi) in an initial phase clinical trial [ ] , and subsequent trials have confirmed that high levels of protection can be achieved against both short-term [ ] and longterm [ ] homologous chmi. however, depending on the immunization regimen, sterile protection can be significantly lower ( - %) against heterologous chmi using the g brazilian clone [ , ] , and against infection in malaria-endemic regions with intense seasonal malaria transmission ( % and % by proportional and time to event analysis, respectively) [ ] . heterologous chmi in chemoprophylaxis with sporozoites trials, in which immunization is by infected mosquito bite of individuals undergoing malaria chemoprophylaxis, have been conducted with nf .c from cambodia [ ] and nf .c from guinea [ ] , and have had lower efficacy than against homologous chmi [ , ] . one explanation for the lower efficacy seen against heterologous p. falciparum strains is the extensive genetic diversity in this parasite species, which is particularly high in genes encoding antigens [ ] and which combined with low vaccine efficacy against non-vaccine alleles [ ] [ ] [ ] reduces overall protective efficacy and complicates the design of broadly efficacious vaccines [ , ] . the lack of a detailed genomic characterization of the p. falciparum strains used in chmi studies and the unknown genetic basis of the parasite targets of pfspz vaccine-and pfspz cvac-induced protection have precluded a conclusive statement regarding the cause(s) of variable vaccine efficacy outcomes. the current pfspz vaccine strain, nf , was isolated from a patient in the netherlands who had never left the country and is considered a case of "airport malaria;" the exact origin of nf is unknown [ ] , but is thought to be from africa [ , ] . nf is also the isolate from which the p. falciparum d reference strain was cloned [ ] , and hence, despite having been separated in culture for over years, nf and d are assumed to be genetically identical, and d is often used in homologous chmi [ , ] . several issues hinder the interpretation of both homologous and heterologous chmi experiments conducted to date. it remains to be confirmed that d has remained genetically identical to nf genomewide, or that the two are at least identical immunogenically. indeed, nf and d have several reported phenotypic differences when grown in culture, including the variable ability to produce gametocytes [ ] . in addition, g , nf .c , and nf .c have not been rigorously compared to each other or to nf to confirm that they are adequate heterologous strains, even though they do appear to have distinct infectivity phenotypes when used as chmi strains [ , ] . while the entire sporozoite likely offers multiple immunological targets, no high-confidence correlates of protection currently exist. in part because of the difficulty of studying hepatic parasite forms and their gene expression profiles in humans, it remains unclear which parasite proteins are recognized by the human immune system during that stage, and elicit protection, upon immunization with pfspz vaccines. both humoral and cell-mediated responses have been associated with protection against homologous chmi [ , ] , although studies in rodents and non-human primates point to a requirement for cell-mediated immunity (specifically through tissue-resident cd + t cells) in long-term protection [ , , , ] . in silico identification of cd + t cell epitopes in all strains could highlight critical differences of immunological significance between strains. finally, heterologous chmi results cannot be a reliable indicator of efficacy against infection in field settings unless the chmi strains used are characteristic of the geographic region from which they originate. these issues could impact the use of homologous and heterologous chmi, and the choice of strains for these studies, to predict the efficacy of pfspz-based vaccines in the field [ ] . these knowledge gaps can be addressed through a rigorous description and comparison of the genome sequence of these strains. high-quality de novo assemblies allow characterization of genome composition and structure, as well as the identification of genetic differences between strains. however, the high at content and repetitive nature of the p. falciparum genome greatly complicates genome assembly methods [ ] . recently, longread sequencing technologies have been used to overcome some of these assembly challenges, as was shown with assemblies for d , g , and several other cultureadapted p. falciparum strains generated using pacific biosciences (pacbio) technology [ ] [ ] [ ] . however, nf .c and nf .c still lack whole-genome assemblies; in addition, while an assembly for g is available [ ] , it is important to characterize the specific g clone used in heterologous chmi, from sanaria's working bank, as strains can undergo genetic changes over time in culture [ ] . here, reference assemblies for nf , g , nf .c , and nf .c (hereafter referred to as pfspz strains) were generated using approaches to take advantage of the resolution power of long-read sequencing data and the low error rate of short-read sequencing platforms. these de novo assemblies allowed for the thorough genetic and genomic characterization of the pfspz strains and will aid in the interpretation of results from chmi studies. this study characterized and compared the genomes of four p. falciparum strains used in whole organism malaria vaccines and controlled human malaria infections using a combination of long-and short-read whole genome sequencing platforms (see below). in addition, these strains were compared to p. falciparum clinical isolates collected from patients in malaria-endemic regions globally, using short-read whole genome sequencing data. genetic material for the four pfspz strains were provided by sanaria, inc. clinical p. falciparum isolates from brazil, mali, malawi, myanmar, and thailand were collected between and from cross-sectional surveys of malaria burden, longitudinal studies of malaria incidence, and drug efficacy studies done in collaboration with the malaria research program within the center for vaccine development and global health at the university of maryland, baltimore, or were otherwise provided by collaborators (additional file ). all samples met the inclusion criteria of the initial study protocol with prior approval from the local ethical review board. parasite genomic sequencing and analyses were undertaken after approval of the university of maryland school of medicine institutional review board was received. these isolates were obtained by venous blood draws; almost all samples were processed using leukocyte depletion methods to improve the parasite-tohuman dna ratio before sequencing. the exceptions were samples from brazil and malawi, which were not leukocyte depleted upon collection. these samples underwent a selective whole genome amplification step before sequencing, modified from [ ] (the main modification being a dna dilution and filtration step using vacuum filtration prior to selective whole genome amplification [ ] ). additionally, samples for which whole genome short-read sequencing was previously generated were obtained from ncbi's short read archive to supplement the following malaria-endemic regions not represented in our data set and regions where pfspz trials are ongoing [ ] [ ] [ ] : peru, columbia, french guiana, guinea, cambodia, papua new guinea, burkina faso, kenya, and tanzania (additional file ). genetic material for whole genome sequencing of the pfspz strains was generated from a cryovial of each strain's cell bank with the following identifiers: nf working cell bank (wcb): san - ; g wcb: san - ; nf .c wcb: san - ; nf .c mother cell bank: san - . each cryovial was thawed and maintained in human o+ red blood cells (rbcs), from vitalant (blood system, inc.), phoenix, az, at % hematocrit (hct) in complete growth medium (rpmi with l-glutamine and mm hepes supplemented with % human o+ serum and hypoxanthine) in a six-well plate in % o , % co , and % n at °c. the cultures were then further expanded by adding fresh rbcs every - days and increased culture hematocrit (hct) to % hct using a standard method [ ] . the complete growth medium was replaced daily. when the pfspz strain culture volume reached - ml and a parasitemia of more than . %, the culture suspensions were collected and the parasitized rbcs were pelleted down by centrifugation at rpm for min. aliquots of . ml per cryovial of the parasitized rbcs were stored at − °c prior to extraction of genomic dna. genomic dna was extracted using the qiagan blood dna midi kit (valencia, ca, usa). pacific biosciences (pacbio) sequencing was done for each pfspz strain. total dna was prepared for pacbio sequencing using the dna template prep kit . (pacific biosciences, menlo park, ca). dna was fragmented with the covaris e , and the fragments were size selected to include those > kbp in length. libraries were prepared per the manufacturer's protocol. four smrt cells were sequenced per library, using p c chemistry and a -min movie on the pacbio rs ii (pacific biosystems, menlo park, ca). short-read sequencing was done for each pfspz strain and for our collection of clinical isolates using the illumina hiseq or platforms. prepared genomic dna, extracted from cultured parasites, leukocytedepleted samples, or from samples that underwent swga (see above), was used to construct dna libraries for sequencing on the illumina platform using the kapa library preparation kit (kapa biosystems, woburn, ma). dna was fragmented with the covaris e or e to~ bp. libraries were prepared using a modified version of the manufacturer's protocol. the dna was purified between enzymatic reactions and the size selection of the library was performed with ampure xt beads (beckman coulter genomics, danvers, ma). when necessary, a pcr amplification step was performed with primers containing an index sequence of six nucleotides in length. libraries were assessed for concentration and fragment size using the dna high sensitivity assay on the labchip gx (perkin elmer, waltham, ma). library concentrations were also assessed by qpcr using the kapa library quantification kit (complete, universal) (kapa biosystems, woburn, ma). the libraries were pooled and sequenced on a - -bp paired-end illumina hiseq or run (illumina, san diego, ca). canu (v . ) [ ] was used to correct and assemble the pacbio reads (cormaxevidenceerate = . for at-rich genomes, default parameters otherwise). organelle genomes were circularized using circlator (default settings, accessed october ) [ ] . to optimize downstream assembly correction processes and parameters, the percentage of total differences (both in bp and by proportion of the d genome not captured by the nf assembly) between the nf assembly and the d reference (plas-modbv ) was calculated after each round of correction. quiver (smrtanalysis v . ) [ ] was run iteratively with default parameters to reach a (stable) maximum reduction in percent differences between the two genomes and the assemblies were further corrected with illumina data using pilon (v . ) [ ] with the following parameters: --fixbases, --mindepth , --k , --minmq , and --minqual . the d annotation was mapped onto each assembly using gmap [ ] ( - - version) the following settings: -y -b -t -k --cross-species. assemblies were compared to the d reference (plas-modbv ) using mummer's nucmer [ ] , and the showsnps function was used to generate a list of snps and small (< bp) indels between assemblies. coding and noncoding variants were classified by comparing the show-snps output with the d gff file using custom scripts. for a subset of genes which are discussed specifically below (transcription factors, confirmed or suspected pre-erythrocytic genes, variants detected in nf relative to d , etc.), small variants were confirmed through manual inspection of extracted (using annotation coordinates) sequence alignments using clustal omega [ ] . structural variants, defined as indels, deletions, and tandem or repeat expansion and contractions each greater than bp in length were identified using the nucmer-based assemblytics tool [ ] (unique anchor length: kbp). translocations were identified by eye through inspection of mummerplots and confirmed through independent assembly runs using different assemblers and data generated with different sequencing technologies (see additional file : supplemental text). reconstructed exon sequences for var genes, encoding p. falciparum erythrocyte membrane protein (pfemp ) antigens, for each pfspz strain were recovered using the etha package [ ] . as a check for var exon sequences that were missed during the generation of the strain's assembly, a targeted read capture and assembly approach was done using a strain's illumina data, wherein var-like reads for each pfspz strain were identified by mapping reads against a database of known var exon sequences [ ] using bowtie [ ] . reads that mapped to a known exon sequence plus their mate pairs were then assembled with spades (v . . ) [ ] , and the assembled products were blasted against the pacbio reads to determine if they were exon sequences missed by the de novo assembly process, or if instead they were chimeras reconstructed by the targeted assembly process. to describe var sequences in the three heterologous chmi strains, exon sequences longer than . kb in length were kept for further characterization. domain composition was determined using vardom v [ ] . categorization of upstream promoter (ups) classification, and identification of domain cassette / vars, was done using hmmer [ ] , using profiles built from known sequences of upsa-e, dblα, and cidrα [ ] . (ups classification was not possible for a small number of sequences found within kb of the end of a contig, or for fragmented sequences). given the reported importance of cd + t cell responses towards immunity to whole sporozoites, mhc class i epitopes of length amino acids were predicted with netmhcpan (v . ) [ ] for each pfspz strain using protein sequences of pre-erythrocytic genes of interest. likely involvement in pre-erythrocytic immunity was inferred either from a literature review or experimentally, i.e., genes whose products were recognized by sera from protected vaccinees participating in whole organism malaria vaccine trials (both pfspz and pfspz-cvac) (n = ) [ , ] . (while the latter were detected through antibody responses, many have also been shown to have t cell epitopes, such as circumsporozoite protein and liver stage antigen ). hla types common to african countries where pfspz or pfspz-cvac trials are ongoing were used for epitope predictions based on frequencies in the allele frequency net database [ ] or from the literature [ , ] (additional file : table s ). shared epitopes between nf and the three heterologous pfspz strains were calculated by first identifying epitopes in each gene, and then removing duplicate epitope sequence entries (caused by recognition by multiple hla types). identical epitope sequences that were identified in two or more genes were treated as distinct epitope entries, and all unique "epitope-given-gene" combinations were included when calculating the number of shared epitopes between strains. to validate these in silico predictions, the predicted epitopes were compared to a published database of experimentally validated cd + t cell epitopes (filtered to remove epitope sequences longer than amino acids in length) [ ] . for the full collection of clinical isolates that had whole genome short-read sequencing data (generated either at igs or downloaded from sra), reads were aligned to the d reference genome (plasmodbv ) using bowtie (v . . ) [ ] . samples with less than million reads mapping to the reference were excluded, as samples with less than this amount had reduced coverage across the genome. bam files were processed according to gatk's best practices documentation [ ] [ ] [ ] . joint snp calling was done using haplotype caller (v . ). because clinical samples may be polyclonal (that is, more than one parasite strain may be present), diploid calls were initially allowed, followed by calling the major allele at positions with heterozygous calls. if the major allele was supported by > % of reads at a heterozygous position, the major allele was assigned as the allele at that position (otherwise, the genotype was coded as missing). additional hard filtering was done to remove potential false positives based on the following filter: dp < || qual < || fs > . || mq < . variants were further filtered to remove those for which the non-reference allele was not present in at least three samples (frequency less than~ . %), and those with more than % missing genotype values across all samples. a matrix of pairwise genetic distances was constructed from biallelic non-synonymous snps identified from the above pipeline (n = , ) across all samples (n = ) using a custom python script, and principal coordinate analyses (pcoas) were done to explore population structure using cmdscale in r. additional population structure analyses were done using admixture (v . ) [ ] on two separate data sets: south america and africa clinical isolates plus nf , nf .c , and g (n = ), and southeast asia and oceania plus nf .c (n = ). the data sets were additionally pruned for sites in linkage disequilibrium (window size of kbp, window step of kbp, r ≥ . ). the final south america/africa and southeast asia/oceania data set used for the admixture analysis consisted of , and snps, respectively. the number of populations, k, was tested for values between k = to k = and run with replicates for each k. for each population, the cross-validation (cv) error from the replicate with the highest log-likelihood value was plotted, and the k with the lowest cv value was chosen as the final k. to compare subpopulations identified in our southeast asia/oceania admixture analysis with previously described ancestral, resistant, and admixed subpopulations from cambodia [ ] , the above non-synonymous snp set was used before pruning for ld (n = , ) and was compared to a non-synonymous snp dataset (n = , ) from samples used by dwivedi et al. [ ] to describe eight cambodian subpopulations, in an analysis that included a subset of samples used by miotto et al. [ ] (who first characterized the population structure in cambodia). there were shared non-synonymous snps between the two datasets, of which were observed in nf .c . a pairwise genetic distance matrix (estimated as the proportion of base-pair differences between pairs of samples, not including missing genotypes) was generated from the shared snp set, and a dendrogram was built using ward minimum variance methods in r (ward.d option of the hclust function). to characterize genome-wide structural and genetic diversity of the pfspz strains, genome assemblies were generated de novo using whole genome long-read (pac-bio) and short-read (illumina) sequence data ("methods"; additional file : table s &table s ). taking advantage of the parent isolate-clone relationship between nf and d , we used nf as a test case to derive the assembly protocol, by adopting, at each step, approaches that minimized the difference to d (additional file : supplemental text & figure s ). the resulting pipeline generated very complete assemblies, with nuclear chromosomes represented by , , , and nuclear contigs, respectively, for nf , nf .c , g , and nf .c , with each chromosome in the d reference represented by one to three contigs ( fig. ) . several shorter contigs in nf ( , bps total), nf .c ( , bps total), and nf .c ( , bps total) could not be unambiguously assigned to an orthologous segment in the d reference genome; gene annotation showed that these contigs mostly contain members of multi-gene families and therefore are likely part of sub-telomeric regions. the cumulative lengths of the four assemblies ranged from . to . mbp (table ) , indicating variation in genome size among p. falciparum strains. in particular, the g assembly was several hundred thousand base-pairs smaller than the other three assemblies. to confirm that this was not an assembly error, we compared g to a previously published g pacbio-based assembly [ ] . the two assemblies were extremely close in overall genome structure, differing only by~ kbp in cumulative length, and also shared a very similar number of snp and small indel variants relative to d (additional file : table s ). many structural variants (defined as indels or tandem repeat contractions or expansions, greater than bp) were identified in each assembly by comparison to the d genome, impacting a cumulative length of . kbp in nf .c to . kbp in nf .c (additional file : table s ). many smaller variants fell into coding regions (including known pre-erythrocytic antigens), often representing variation in repeat units (additional file ). several larger structural variants (> kbp) exist in g , nf .c , and nf .c relative fig. pacbio assemblies for each pfspz strain reconstruct entire chromosomes in one to three continuous pieces. to determine the likely position of each non-reference contig on the d reference genome, mummer's show-tiling program was used with relaxed settings (-g -v -i ) to align contigs to d chromosomes (top). d nuclear chromosomes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] are shown in gray, arranged from smallest to largest, along with organelle genomes (m = mitochondrion, a = apicoplast). contigs from each pfspz assembly (nf : black, g : green, nf .c : orange, nf .c : hot pink) are shown aligned to their best d match. a small number of contigs could not be unambiguously mapped to the d reference genome (unmapped) to d . many of these regions contain members of multi-gene families, such as var genes (which encode pfemp proteins), and as expected the number of var genes varied between each assembly (additional file ). while pfemp proteins are most commonly studied in the context of blood-stage infections, several characteristics of these sequences may still be relevant for the interpretation of whole organism pre-erythrocytic vaccine trials. for example, nf .c and nf .c both had domain cassette sequences encoding dc -and dc containing pfemp s, which have been associated with severe malaria [ ] , while g did not. in addition, a recently characterized pfemp protein expressed on the surface of nf sporozoites (nf var sporo ) was shown to be involved in hepatocyte invasion (pf d _ ), and antibodies to this pfemp blocked invasion [ ] . no ortholog to nf var sporo was identified in the var repertoire of g , nf .c , or nf .c ; while there were var sequences in the three heterologous chmi strains that contained the general domain structure (nts-dbla-cidra-dbld-cidrb) of nf var sporo , none had its specific domain cassette (nts-dblα . -cidrα . -dblδ -cidrβ ) (additional file ). it remains to be determined whether a different, strain-specific, var gene fulfills a similar role in each of the heterologous pfspz strains. several other large structural variants impact regions housing non-multi-gene family members, although none is known to be involved in pre-erythrocytic immunity. examples include a -kbp-long tandem expansion of a region of chromosome in the g assembly (also present in the previously published assembly for g [ ] ) and a . -kbp-long repeat expansion of a region of chromosome in nf .c , both of which are supported by~ pacbio reads. the former is a segmental duplication containing a vacuolar iron transporter (pf d _ ), a putative citrate/oxoglutarate carrier protein (pf d _ ), a putative s ribosomal protein l (pf d _ ), gtp cyclohydrolase i (pf d _ ), and three conserved plasmodium proteins of unknown function (pf d _ , pf d _ , pf d _ ). the expanded region in nf .c represents a tandem expansion of a segment housing the gene encoding the multidrug resistance protein pfmdr (pf d _ ), resulting in a total of four copies of this gene in nf .c . other genes in this tandem expansion include those encoding an ironsulfur assembly protein (pf d _ ), a putative pre-mrna-splicing factor dub (pf d _ ), a putative zinc finger protein (pf d _ ), and a putative mitochondrial-processing peptidase subunit alpha protein (pf d _ ). in addition, the nf .c assembly contained a large translocation involving chromosomes ( d coordinates~ , to~ , ) and (start to coordinate~ , ) (additional file : figure s ). since large synteny breaks are uncommon within and even between plasmodium species, validation was done by generating oxford nanopore long-read data and building a canu-based pacbio-nanopore hybrid nf .c assembly; in addition, several new pacbioonly assemblies were made, with different assembly programs (additional file : supplemental text). all new assemblies supported a translocation event, although neither chromosome was resolved into a single supercontig. while an assembly artifact cannot be completely ruled out, the regions of chromosomes and where the translocation occurs are documented recombination hotspots that were identified specifically in isolates from cambodia, the site of origin of nf .c [ ] . several structural differences in genic regions were also identified between the nf assembly and the d genome (additional file ); if real, these structural variants would have important implications in the interpretation of trials using d as a homologous chmi strain. for example, a -bp tandem expansion was identified in the nf assembly on chromosome , which overlapped the region containing liver stage antigen (pflsa- , pf d _ ) . the structure of this gene in the nf strain was reported when pflsa- was first table the pfspz strains differ from the d in genome size and sequence. characteristics of the pacio assembly for each strain (first four columns), with the pf d reference genome shown for comparison (italics). single nucleotide polymorphisms (snps) and indels in each pfspz assembly as compared to d , both genome-wide (all) or restricted to the core genome cumulative length: total length of the contigs n : length of the contig which, along with all contigs larger than itself, contain % of the assembly (larger numbers indicate a more complete assembly) core genome as defined in [ ] characterized, with unique n-and c-terminal regions flanking a repetitive region consisting of several dozen repeats of a amino acid motif [ , ] ; the cds of pflsa- in the nf assembly was bp in length (matching the previously published sequence), but only bp long in the d reference. to determine if this was an assembly error in the nf assembly, the pflsa- locus from a recently published pacbio-based assembly of d [ ] was compared to that of nf . the two sequences were identical, likely indicative of incorrect collapsing of the repeat region of pflsa- in the d reference; nf and d pacbio-based assemblies had units of the -mer amino acid repeat, compared to only in the d reference sequence, a result further validated by the inconsistent depth of mapped illumina reads from nf between the pflsa repeat region and its flanking unique regions in the d reference (additional file : figure s ). several other potential differences between nf and d were ruled out as remaining errors in the d assembly, several of which are present in a list of d reference patches recently published [ ] (additional file ). very few small sequence variants were identified in nf compared to the d reference; nonsynonymous mutations were present in single-copy non-pseudogene-encoding loci (additional file ). short indels were detected in genes; many of these indels had a length that is not multiple of three and occurred in homopolymer runs, possibly representing remaining pacbio sequencing error. however, some may be real, as a small indel causing a frameshift in pf d _ , a putative protein-coding pseudogene that has previously been shown to accumulate premature stop codons in laboratory-adapted strains [ ] , and some may be of biologic importance, such as those seen in two histonerelated proteins (pf d _ and pf d _ ). it has been reported that some clones of d , unlike nf , are unable to consistently produce gametocytes in long-term culture [ ] ; no snps were observed within or directly upstream of pfap -g (pf d _ ) (additional file : table s ), which has been identified as a transcriptional regulator of sexual commitment in p. falciparum [ ] . however, g , nf .c , and nf .c had numerous non-synonymous mutations and indels within putative ap genes (additional file : table s ). a non-synonymous mutation from arginine to proline (r p) was observed in an ap -coincident c-terminal domain of pfap -l (pf d _ ), a gene associated with liver stage development [ ] , in all pfspz strains compared to d . interestingly, nf .c contained an insertion of almost bp in length relative to d in the ′ end of pfap -g; the insertion also carried a premature stop codon, leading to a considerably different c-terminal end for the transcription factor (additional file : figure s ). this alternate allele is also present in previously published assemblies for clones from southeast asia [ ] , including the culture-adapted strain dd , and variations of this insertion (without the in-frame stop codon) are also found in several nonhuman malaria plasmodium species (additional file : figure s ), suggesting an interesting evolutionary trajectory of this sequence. given that no absolute correlates of protection are known for whole organism p. falciparum vaccines, genetic differences were assessed both across the genome and in pre-erythrocytic genes of interest in the three heterologous chmi strains. as expected, the number of mutations between d and these three pfspz strains was much higher than observed for nf , with~ - k snps and as many indels in each pairwise comparison. indel length distributions showed distinct patterns in each strain (additional file : figure s ); the expected difference in the length distribution of small indels in coding versus non-coding regions across the genome suggests that most of the remaining indels correspond to true differences relative to d . snps were roughly randomly distributed among intergenic regions, silent and non-synonymous sites ( table , fig. ) , and corresponding to a pairwise snp density relative to d of . , . and . snps/kbp for g , nf .c and nf .c , respectively. increased diversity was observed in regions known to house variable members of multi-gene families such as vars, rifins, and stevors. nf .c had the highest number of unique snps genome-wide (snps not shared with other pfspz strains), with % more unique snps than nf .c and % more than g (additional file : figure s ) . a similar trend was seen when restricting the analyses to non-synonymous snps in the core genome ( . % and % more than nf .c and g , respectively). the lower number of unique snps in g may be due in part to the smaller genome size of this strain. snps were also common in a panel of preerythrocytic genes known or suspected to be implicated in immunity to liver-stage parasites (see "methods"; additional file : table s ). while the sequence of all these loci was identical between nf and d , there was a wide range in the number of sequence variants per locus between d and the other three pfspz strains, with some genes being more conserved than others. for example, the circumsporozoite protein, pfcsp, showed , , and non-synonymous mutations in g , nf .c , and nf .c , respectively, relative to d . however, pflsa- had over nonsynonymous mutations in all three heterologous strains relative to d (many in the repetitive, difficult-to-align, region of this gene), in addition to significant length differences in the internal repeat region (additional file : figure s ). the sequence variants mentioned above may impact the ability of the immune system primed with nf to recognize the other pfspz strains, impairing vaccine efficacy against heterologous chmi. data from murine and non-human primate models [ , , , ] demonstrate that cd + t cells are required for protective efficacy; therefore, the identification of shared and unique cd + t cell epitopes across the genome in all four pfspz strains may help interpret the differential efficacy seen in heterologous relative to homologous chmi. we predicted cd + t cell epitopes in genes whose product has been confirmed or suspected to be involved in preerythrocytic immunity (fig. ) . strong-binding mhc class i epitopes in the protein sequences from these loci were identified using in silico epitope predictions based on hla types common in sub-saharan africa populations (additional file : table s ). similar total numbers of epitopes (sum of unique epitopes, regardless of the hla-type, across genes) were identified in the three heterologous chmi strains, with each strain containing . k cd + t cell epitopes. nf had a slightly higher number of predicted epitopes compared to the other strains, possibly reflecting the slightly longer median sequence lengths in nf compared to the other strains (additional file : figure s ). while only a small number of cd + t cell epitopes, in a small number of antigens, have been experimentally validated [ ] , there was a strong overlap between these and the in silico-predicted epitopes. only a small number of fig. distribution of polymorphisms in pfspz pacbio assemblies. single nucleotide polymorphism (snp) densities (log snps/ kb) are shown for each assembly; the scale [ - ] refers to the range of the log-scaled snp density graphs-from to . inner tracks, from outside to inside, are nf (black), g (green), nf .c (orange), and nf .c (pink). the outermost tracks are the d reference genome nuclear chromosomes (chrm to chrm , in blue), followed by d genes on the forward and reverse strand (black tick marks). peaks in snp densities mostly correlate with subtelomeric regions and internal multi-gene family clusters validated epitope sequences failed to overlap with the predicted epitope set (fig. ) , at least one of which could be explained by differences in hla types used in experiments and in silico predictions. the majority of predicted epitopes were shared across all four strains, reflecting epitopes predicted in conserved regions of the genes used in this analysis. of the three heterologous chmi strains, nf .c had the highest number of unique epitopes relative to all other strains (n = , fig. ) or to nf (n = , additional file : table s ). both g and nf .c had a similar number of unique epitopes (n = and n = , respectively) and of epitopes not shared with nf (n = and n = , respectively). indels and repeat regions also sometimes affected the number of predicted epitopes in each antigen for each strain; for example, an insertion in g near amino acid residue in pflisp- (pf d _ ) contained additional predicted epitopes (additional file : figure s ). similar patterns in variation in epitope recognition and frequency were found in other pre-erythrocytic genes of interest, including pflsa- (pf d _ ), pfama- (pf d _ ), and pftrap (pf d _ ) (additional file : figure s ). some of these variations in epitope sequences are relevant for the interpretation of the outcome of pfspz vaccine trials. for example, while all four strains are identical in sequence composition in a b cell epitope potentially relevant for protection recently identified pfcsp [ ] , another b cell epitope that partially overlaps it [ ] contained an a g amino acid difference in g and nf .c relative to nf and nf .c . there was also variability in cd + t cell epitopes recognized in the th r region of the protein. specifically, the pfcsp encoded by the d / nf allele was predicted to bind to both hla-a and hla-c allele types, but the orthologous protein segments in nf .c and nf .c were only recognized by hla-a allele types; notably, and given the hla types studied, no epitope was detected at that position in pfcsp encoded in g (fig. ) . expansion of the analyses to additional hla types revealed an allele (hla- : ) that is predicted to bind to the th r region of the g -encoded pfcsp; however, hla- : is much more frequent in european populations ( - %) than in african populations ( - %) [ ] . therefore, if cd + t cell epitopes in the th r region of g are important for protection, which is currently unknown, the level of protection against chmi with g observed in volunteers of european descent may not be informative of pfspz vaccine efficacy in africa. fig. comparison of predicted cd + t cell epitopes from pre-erythrocytic antigen amino acid sequences. cd + t cell epitopes were predicted in silico for confirmed or suspected pre-erythrocytic antigens (see additional file : table s for a complete list of genes included in this analysis). the plot shows the number of shared or unique epitopes, as compared between different pfspz strain groupings. the height of the bar is the number of epitopes that fell into each intersection category, and the horizontal tracks below the bars show the pfspz strains that are included in that intersection. for example, the first bar represents the number of shared epitopes between nf , g , and nf .c . at the bottom left, colored tracks represent the total number of epitopes predicted across all genes (> k for each strain). as the vast majority of predicted epitopes were shared among all four strains, that group was removed from the bar plot to achieve better visual definition for the other comparison the four pfspz strains have been adapted and kept in culture for extended periods of time. to determine if they are still representative of the malaria-endemic regions from which they were collected, we compared these strains to over recent ( - ) clinical isolates from south america, africa, southeast asia, and oceania (additional file ), using principal coordinates analysis (pcoa) based on snp calls generated from illumina whole genome sequencing data. the results confirmed the existence of global geographic differences in genetic variation previously reported [ , ] , including clustering by continent, as well as a separation of east from west africa and of the amazonian region from that west of the andes (fig. ) . the pfspz strains clustered with others from their respective geographic regions, both at the genome-wide level and when restricting the data set to snps in the panel of pre-erythrocytic antigens, despite the long-term culturing of some of these strains (fig. ). an admixture analysis of south american and african clinical isolates confirmed that nf and nf .c both have the genomic background characteristic of west africa, while g is clearly a south american strain (additional file : figure s ). nf .c was isolated in the early s [ ] , at a time when resistance to chloroquine and sulfadoxinepyrimethamine resistance was entrenched and resistance to mefloquine was emerging [ , ] , and carries signals from this period of drug pressure. four copies of pfmdr- were identified in nf .c (additional file : table s ); however, two of these copies appeared to have premature stop codons introduced by snps and/or indels, leaving potentially only two functional copies in the genome. while nf .c also had numerous point mutations relative to d in genes such as pfcrt (conveying chloroquine resistance), and pfdhps and pfdhr (conveying sulfadoxine-pyrimethamine resistance), nf .c was isolated before the widespread deployment of artemisinin-based combination therapies (acts) and had the wild-type allele in the locus that encodes the kelch protein in chromosome (pfk ) on chromosome , with no mutations known to convey artemisinin resistance detected in the propeller region (additional file : table s ). the emergence in southeast asia of resistance to antimalarial drugs, including artemisinins and drugs used in artemisinin-based combination treatments (acts), is thought to underlie the complex and dynamic parasite population structure in the region [ ] . several relatively homogeneous subpopulations, whose origin is likely linked to the emergence and rapid spread of drug resistance mutations, exist in parallel with a sensitive subpopulation that reflects the ancestral population in the region (referred to as kh ), and another subpopulation fig. predicted cd + t cell epitopes in the p. falciparum circumsporozoite protein (pfcsp). protein domain information based on the d reference sequence of pfcsp is found in the first track. the second track are previously experimentally validated (exp. val.) epitopes (from [ ] , after removing duplicate epitope sequences and epitopes > amino acids in length) and the following tracks are epitopes predicted in the pfcsp sequences of nf , g , nf .c , and nf .c , respectively. each box is a sequence that was identified as an epitope, and colors represent the hla type that identified the epitope. the experimentally validated epitopes do not have hla types reflected and are simply jittered across two rows of admixed genomic background (referred to as kha), possibly the source of the drug-resistant subpopulations or the result of a secondary mix of resistant subpopulations [ , , , ] . this has been accompanied by reports of individual k mutations conferring artemisinin resistance occurring independently on multiple genomic backgrounds [ ] . to determine the subpopulation to which nf .c belongs, an admixture analysis was conducted using isolates from southeast asia and oceania, including nf .c . eleven total populations were detected, of which seven contained cambodian isolates (fig. ) . both admixture and hierarchical clustering analyses suggest that nf .c is representative of the previously described admixed kha subpopulation [ , ] (fig. ) , implying that nf .c is representative of a long-standing admixed population of parasites in cambodia rather than one of several subpopulations thought to have arisen recently in response to pressure from acts, an important observation if this strain is ever considered for use in a vaccination product. whole organism sporozoite vaccines have provided variable levels of protection in initial clinical trials; the radiation-attenuated pfspz vaccine has been shown to protect > % of subjects against homologous chmi at weeks after the last dose in clinical trials in the usa [ , ] and germany [ ] . however, efficacy has been lower against heterologous chmi [ , ] , and in field studies in a region of intense transmission, in mali, at weeks [ ] . interestingly, for the exact same immunization regimen, protective efficacy by proportional analysis was greater in the field trial in mali ( %) than it was against heterologous chmi with pf g in the usa at weeks after last dose of vaccine ( %) [ , ] . while evidence shows that whole organism-based vaccine efficacy can be improved by adjusting the vaccine dose and schedule [ ] , further optimization of such vaccines will be facilitated by a thorough understanding of the genotypic and immunologic differences among the pfspz strains and between them and parasites in malaria endemic regions. a recent study examined whole genome short-read sequencing data to characterize nf .c and nf .c through snp calls, and identified a number of nonsynonymous mutations at a few loci potentially important for the efficacy of chemoprophylaxis with sporozoites, the foundation for pfspz-cvac [ ] . the analyses described here, using high-quality de novo genome assemblies, expand the analysis to hard-to-call regions, for the genome-wide dataset, coordinate separated south american and african isolates from southeast asian and papua new guinean isolates ( . % of variation explained), coordinate two separated african isolates from south american isolates ( . %), and coordinate three separated southeast asian isolates from papua new guinea (png) isolates ( . %). similar trends were found for the first two coordinates seen for the pre-erythrocytic gene data set ( . and . %, respectively), but coordinate three separated isolates from all three regions ( . %). in both datasets, nf (black cross) and nf .c (orange cross) cluster with west african isolates (isolates labeled in red and dark orange colors), g (bright green cross) cluster with isolates from south america (greens and browns), and nf .c (pink cross) clusters with isolates from southeast asia (purples and blues) such as those containing gene families, repeats, and other low complexity sequences. the added sensitivity enabled the thorough genomic characterization of these and additional vaccine-related strains, and revealed a considerably higher number of sequence variants than can be called using short read data alone, as well as indels and structural variants between assemblies. for example, the insertion close to the ′ end of pfap -g detected in nf .c and shared by dd has not, to the best of our knowledge, been reported before, despite the multiple studies highlighting the importance of this gene in sexual commitment in p. falciparum strains, including dd [ ] . long-read sequencing also confirmed that differences observed between the nf and d assemblies in a major liver stage antigen, pflsa- , represent one of a small number of errors lingering in the reference d genome, which is being continually updated and improved [ ] . confirmation that nf and d are identical at this locus is critical when d has been used as a homologous chmi in whole sporozoite, nf -based vaccine studies. furthermore, the comprehensive sequence characterization of variant surface antigen-encoding loci, such as pfemp -encoding genes, will enable the use of the pfspz strains to study the role of these protein families in virulence, naturally acquired immunity and vaccine-induced protection [ ] . the comprehensive genetic and genomic studies reported herein were designed to provide insight into the outcome of homologous and heterologous chmi studies and to determine whether the chmi strains can be used as a proxy for strains present in the field. comparison of genome assemblies confirmed that nf and d have remained genetically very similar over time and that d is an appropriate homologous chmi strain. as expected, g , nf .c , and nf .c were genetically very distinct from nf and d , with thousands of differences across the genome including dozens in known pre-erythrocytic antigens. the identification of sequence variants (both snps and indels) within transcriptional regulators, such as the ap family, may assist in the study of different growth phenotypes in these strains. nf .c and nf .c merozoites enter the bloodstream several days earlier than those of nf [ ] , suggesting that nf may develop more slowly in hepatocytes than do the other two strains. therefore, each sample is a column, and the height of the different colors in each column corresponds to the proportion of the genome assigned to each k population by the model. bottom: hierarchical clustering of the southeast asian isolates used in the admixture analysis (branch and leaves colored by their assigned subpopulation) and previously characterized cambodian isolates (n = , black; [ ] ) place nf .c (star) with samples from the previously identified kha admixed population (shown in gray dashed box). the y-axis represents distance between clusters mutations in genes associated with liver-stage development (as was observed with pfap -l) may be of interest to explore further. finally, comparison of the pfspz strains to whole genome sequencing data from clinical isolates shows that, at the whole genome level, they are indeed representative of their geographical regions of origin. we note, however, that potential transcriptional differences between pfspz and field strains, which could be caused by a small number of variants, remain to be explored. these results can assist in the interpretation of chmi studies in multiple ways. first, of the three heterologous strains, nf .c is the most divergent from nf , containing the highest numbers of unique snps and epitope sequences relative to the vaccine strain, which was expected from their respective geographic origins. however, results were less consistent for nf .c and g . given its south american origin, g was expected to have more unique variants relative to nf than nf .c did, but this was not always the case (for example, nf .c had a slightly higher number of unique epitopes relative to nf , compared to g ). these results show that the practice of equating geographic distance to genetic differentiation is not always valid and that the interpretation of chmi studies should rest upon thorough genome-wide comparisons. lastly, since, of all pfspz strains, nf .c is the most genetically distinct from nf , if proteome-wide genetic divergence is the primary determinant of differences in protection against different parasites, the extent to which nf based immunization protects against chmi with nf .c is important in understanding the ability of pfspz vaccine and other whole-organism malaria vaccines to protect against diverse parasites present worldwide. these conclusions are drawn from genome-wide analyses and from subsets of genes for which a role in whole-sporozoite-induced protection is suspected but not experimentally established. conclusive statements regarding cross-protection will require the additional knowledge of the genetic basis of whole-organism vaccine protection. without more information on the epitope targets of protective immunity induced by pfspz vaccines, it is difficult to rationally design multi-strain pfspz vaccines. however, these data can potentially be used for the rational design of multi-strain sporozoite-based vaccines once knowledge of those critical epitope sequences is available. characterization of a variety of p. falciparum strains may facilitate the development of region-specific or multi-strain vaccines with greater protective efficacy. support for a genomics-guided approach to guide such next-generation vaccines can be found in other whole organism parasitic vaccines. field trials testing the efficacy of first-generation whole killed-parasite vaccines against leishmania had highly variable results [ ] . while most studies failed to show protection, indicating that killed, whole-cell vaccines for leishmaniasis may not produce the necessary protective response, a trial demonstrating significant protection utilized a multi-strain vaccine, with strains collected from the immediate area of the trial [ ] , highlighting the importance of understanding the distribution of genetic diversity in pathogen populations. in addition, a highly efficacious nonattenuated, three-strain, whole organism vaccine exists against theileria parva, a protozoan parasite that causes east coast fever in cattle. this vaccine, named muguga cocktail, consists of a mix of three live strains of t. parva that are administered in an infection-andtreatment method, similar to the approach utilized by pfspz-cvac. it has been shown recently that two of the strains are genetically very similar, possibly clones of the same isolates [ ] . despite this, the vaccine remains highly efficacious and in high demand [ ] . in addition, the third vaccine strain in the muguga cocktail is quite distinct from the other two, with~ snps/kb [ ] , or about twice the snp density seen between nf and other pfspz strains. these observations suggest that an efficacious multi-strain vaccine against a highly variable parasite species does not need to contain a large number of strains, but that the inclusion of highly divergent strains may be warranted. these results also speak to the promise of multi-strain vaccines against highly diverse pathogens, including apicomplexans with large genomes and complex life cycles. next-generation whole genome sequencing technology has opened many avenues for infectious disease research and holds great promise for informing vaccine design. while most malaria vaccine development has occurred before the implementation of regular use of whole genome sequencing, the tools now available allow the precise characterization and informed selection of vaccine strains early in the development process. the results presented here will greatly assist these future research efforts, as well as aiding in the interpretation of clinical trials using the pfspz strains for vaccination and chmi purposes. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. additional file . samples used for analyses in the manuscript "strains used in whole organism plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential". excel spreadsheet listing all samples used in this analysis; if a sample was sequenced as part of a previously published study, the reference is listed (and are also included in the main text of the manuscript, see methods and references sections), as well as accession ids. additional file . supplemental text, tables, and figures . word document containing supplemental information as cited in the main manuscript. additional file . description of structural variants. excel spreadsheet describing structural variants identified in each of the four pfspz strain assemblies. additional file . description of var exon sequences. excel spreadsheet describing var exon sequences recovered from each of the four pfspz strains. additional file . description of variants found in the nf assembly. excel spreadsheet listing snp and indel differences found in the nf assembly and d . informed consent included the generation of parasite genomic data. the respective field studies were approved by the following institutional review boards (irbs): institute of biomedical sciences, university of são paulo ( / /cepsh), for collection in acre, brazil; malawi national health sciences research committee, and the irb at the university of maryland université des sciences, des techniques, et des technologies de bamako, mali, and the irb at the university of maryland baltimore (fwa# , irb# ), for collections in myanmar; human subject protection branch (hspb) of the walter reed army institute of research, silver spring, md (fwa# , irb# ), and institute for the development 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collection of clinical isolates in malawi. we would also like to thank robin bromley for assistance with deposition of oxford nanopore data to sra. were also supported by the howard hughes medical institute. the parasites provided by sanaria were produced in part by funding from two sbir grants from national institute of allergy and infectious diseases, nih, r ai and r ai - a . collection of clinical isolates that were sequenced for this study were funded by u ai , grants from cnpq ( / - ), grants from the armed forces health surveillance center/global emerging infections surveillance and response system (wr and wr ), the nih international centers of excellence in malaria research program (u ai ) (brazil) , and the howard hughes medical institute. s.k. and a.m.p. were supported by the intramural research program of the national human genome research institute, nih ( ziahg ). p.t.r. was supported by a scholarship from the conselho nacional de desenvolvimento científico e tecnológico (cnpq), which also provided a senior researcher scholarship to m.u.f. the datasets generated during the current study are available on ncbi. biosample ids for raw sequencing data and assemblies for the four pfspz strains are samn (nf ), samn ( g ), samn (nf .c ), and samn (nf .c ). oxford nanopore data was obtained only for nf .c and has accession # srr . assemblies and annotation files can also be accessed at: https://doi.org/ . /m . figshare. .v . see additional file for biosample ids for the clinical isolates used in these analyses (sequenced as part of this study or previously published work).authors' contributions kam designed the study, performed analyses, and wrote the paper. jcs, cvp, and slh designed the study, contributed samples and funding, and wrote the paper. key: cord- - vl cvyq authors: mostow, s. r.; tyrrell, d. a. j. title: the behaviour in vitro of attenuated recombinant influenza viruses date: journal: arch gesamte virusforsch doi: . /bf sha: doc_id: cord_uid: vl cvyq influenza strains produced by recombination and tested as possible live vaccine candidates were studied in organ cultures of trachea. two strains which proved to be too virulent in human volunteers regularly caused damage to the ciliated epithelium and viruses grew to high titre. two strains which proved to be attenuated for volunteers did not cause appreciable damage, although they replicated to low titre in the epithelium. similar results were obtained with influenza a virus attenuated by passage in the presence of horse sera. the method may be of value for detecting virulent live influenza vaccine candidates without risking severe illness in volunteers. attenuated influenza viruses have been produced recently by genetic recombination of avirulent laboratory-adapted strains and virulent parents (beare and hall, ; mccaho~ and schild, ) . this method, which is the most rapid means of producing attenuated influenza strains to date, results in a spectrum of both attenuated and virulent clones as judged by their effects in human volunteers. unfortunately, virulence cannot be predicted by laboratory tests such as yield in eggs, mouse virulence, and growth at high temperatures and volunteer tests are slow and difficult to quantitate. this method might occasionally result in the production of new phenotypes with characteristics not seen in either parent and a recombinant more virulent than the virulent parent strain could be produced (kilbovr~s,, ) . for these reasons we wished to find a new in vitro marker of influenza virus virulence. this paper describes a method using the ciliated epithelium of human foetal tracheal organ culture. all the viruses tested were provided by dr. a. s. beare. the parent strains were a/pr / (hon ) of unknown passage history which had been passed once in organ cultures of human embryo trachea and once in leukosis free eggs, and a hong kong variant known as a/england/ / (h n )which had been isolated in leukosis-free eggs from an influenza patient. two cloned recombinants of • pr were also provided for testing. these had been prepared from the above parents by dr. d. mccahon and dr. g. c. schild of the national institute for medical research by methods previously described (mecai~ox and scmld, ) . finally, two strains of a/england/ / (h n ) were also tested. these were an unmodified inhibitor sensitive strain (is ) and an inhibitor resistant strain (ir ) which had received several passages at ~ c. the method is a modification of those used by others (chea•y and tayloi~-robinson, ; h.~r~ett and hoo]?e~i ; hoor~r and tyri~elru, ) . tracheau from human embryos were provided by the royal marsden hospital tissue bank. foetal age varied to weeks. after transportation on ice in parker's medium to the laboratory, the tracheas were dissected free of connective tissue. complete rings were prepared by cutting transversely between the tracheal cartilages in a petri dish supported on a plastic platform containing a light source. the rings ( -- per embryo) were carefully transferred to sterile screw-top tubes (flow laboratories) containing i ml of l- medium (leibowtz, ) to which had been added i per cent l-glutamine (final concentration), i units of penicillin, and ~g streptomycin. ferrets were given a lethal dose of sodium pentothal. the skin was dissected free over the abdomen and pulled upwards and over the head to provide a sterile environment over the trachea. the trachea was dissected free of connective tissue, removed, placed in medium, and handled as above. after positioning on the side of the tube opposite the medium, the percentage of each ring beating was estimated using transmitted light and the low power objective of an ordinary microscope. differences in the vigour and speed with which the cilia beat were discernible, but these were ignored. readings were recorded by the same observer (srm) each day. the ferret rings, because of their large size, were read with an inverted microscope. the tissue was rolled for -- hours before inoculation with virus to remove free cells and mucus. after an additional medium change, eids of virus in . ml was introduced into the tube which was then rolled continuously at ~ c. the virus was allowed to adsorb for hours and then the tissue was washed three times with medium. at hours, and thereafter every hours, the tubes were harvested and fresh medium added. the entire ml of medium from each tube was removed aseeptically every other day for the first two weeks and mixed with an equal volume of nutrient broth, pooled, and stored at -- ~ c. serial -fold dilutions of virus were injected into the allantoic cavity of groups of three -day embryonated chicken eggs. after incubation at ~ c for to hours, the allantoic fluid was harvested and tested for haemagglutinin (ha) with . per cent chicken red blood cells (rbc). titres were expressed as per cent egg infectious doses per millilitre (eids /ml). volunteers were studied in isolation by standard methods as already described (beare and hall, ). the number of days of symptoms and signs above a baseline were combined to provide a symptom score. the use of l- medium and rolling the screw-topped tubes prolonged the survival of the ciliated epithelium beyond that obtained using other standard media, and, in the conditions finally used, the human embryo trachea regularly survived for periods of -- and occasionally days. the rings tend to adhere to one side of the tube and remain in this position if undisturbed. to prevent the accumulation of debris in the centre of the x'ing, thus obscuring the cilia, the tubes were shaken gently each day. occasionally it was necessary to dislodge a ring because flbroblasts appeared at the point of attachment after -- days and obscured the cilia. finally, after preparation of the rings, there was a definite improvement of the ciliary activity over the first -- hours. therefore the reading on the day of inoculation was used as a baseline. it was frequently impossible to test all the viruses in the trachea from the same embryo on the same day, and for this report the data have been pooled and are based on the observation of -- rings infected with each virus. several uninoeulated cultures were included in each test. as shown in figure , the ciliary activity in cultures infected with this strain was the same as that in uninoculated tubes and in fact the cilia in some inoculated tubes were more active than in controls. the virus grew to low titre, as shown in figure . . . . virus strain a/eng.~ ~ (h n ) this strain, by contrast, regularly destroyed ciliated epithelium. the earliest effects, noticeable by the third day after inoculation, were the appearance of mucus and many desquamated cells within the ring. the cilia of some of these shed cells continued to beat for a few hours. at this time the ciliated epithelium looked intact, but by -- days it was markedly damaged and by days ciliary activity had ceased. histological sections showed that the epithelium was denuded of ciliated cells. in a few tracheas the process was delayed by two or three days. the growth of virus preceded the destruction of the ciliated epithelium and the titre was the highest of any virus tested (fig. ) . the growth of virus continued at a low level after the cilia were destroyed and was detected at titres between and eids /ml for up to days. this virus behaved similarly to the a/pr parent and caused very little ciliary damage. however, it grew to slightly higher titres in the ciliated epithelium than did the parent. . . . clone (ply x ) this recombinant was of intermediate pathogenicity for cilia, but more closely resembled the a/eng. parent. in other words some rings would resist growth and * destruction for a few days longer than average, but widespread damage occurred by -- days, and viral growth was quite similar to the a/eng. parent as shown in figure . thus, both by observation of ciliary activity and by titrating the virus produced, the viruses fall into the order pr , e, , a/eng./ / from the least to the most virulent for cultured cells. as shown in table , this corresponds exactly to the order of virulence found in experiments in human volunteers and recently reported by be~me and hall ( ) . as shown in figure , it was not possible to differentiate the a/pr parent from the other viruses tested by the effect on cilia. however, this virus grew to low titre as it has in human tissue (table ) . although the a/eng. parent destroyed the ciliated epithelium of human tissue, it has little effect on ferret trachea. in one experiment in which cultures were held for a long time, the eiliated epithelium was destroyed after about three weeks. furthermore, the a/eng. virus multiplied to high titre, range from . to . eid /ml at days. the recombinant viruses did not cause ciliary damage, but at days the growth in ferret epithelium was similar to that in human tissue. in other words, clone c attained titres (range at days . to . eids /ml) similar to the a/pr parent, and clone attained titres (range at days . to - eid /ml) similar to the a/eng. parent. since the differences in the behaviour of the viruses might have been related to the fact that they were prepared by recombination, rather than to the attenua- even when optimally constituted, the usual doses of inactivated influenza virus vaccines are variably effective in preventing disease (sehoenbaum, mostow, dowdl]~, co] ~an and kaye, ) . live attenuated vaccines are a possible alternative but before they can be introduced it is necessary to be able to obtain rapidly and reproducibly from wild virus, strains which grow to high titre in eggs and which are a attenuated for man. it is generally believed that virulence is determined by multiple genes and kilbourne ( ) suggested that recombining a virulent strain with an avirulent one would give progeny with intermediate virulence and might therefore be used as a means of simultaneous "instant" attenuation and adaptation to eggs. this has been shown to be possible but two of four reeombinants were inadequately attenuated (beare and hall, ) and it would therefore be useful to be able to distinguish attenuated reeombinants in the laboratory. the method of evaluation in organ culture seems to give repeatable and consistent results as was noticed in a preliminary paper (mostow and tyrrwll, ) . this paper reports further results and a number of important points of technique. there appears also to be some variability in the susceptibility of different tracheas. the data were pooled for this report, but nevertheless each individual experiment ranked the viruses in the same order; however, we think it is important to include the virulent and attenuated parent in each test as controls. using the technique described, there was a clear correlation between free growth in organ culture, damage to cells and the ability to produce disease in man. we do not know whether this correlation would hold with tracheas from other species, if not, there could be difficulty using the test because of lack of an adequate supply of suitable human tracheal cultures (shaaff, boswell and mufson, ) . our results with the parental strains of influenza were not particularly surprising. the basic pathology of the disease is damage of the tracheal cells by the virus and since the laboratory adapted strain, a/pr / , does not cause disease in humans, we did not expect it to damage the eiliated epithelium from man. nevertheless, the cultures were infected whereas the volunteers often were not. conversely, recently isolated influenza a strains cause clinical influenza and would be expected to destroy eiliated epitholium. it has been shown that influenza viruses which have squired ts markers as a result of recombination with mutagen-treated strains are attenuated for man (c~anock, personal communication) . it is now necessary to determine whether these strains are a]so avirulent in our tests. likewise recombinants made with other attenuated strains should be tested to establish the validity of the test. if it can be established it would suggest that the only really important factor in virulence for man is whether the virus multiplies in tracheal cells. attenuation of human influenza a viruses recombinant influenza a viruses as live vaccines for man large quantity production of chicken embryo trachea organ cultures for use of virus and mycoplasma studies test tube organ cultures of ciliated epithelium for the isolation of respiratory viruses future influenza vaccines and the use of genetic recombinants the growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere schild : segregation of antigenic and biological characteristics during influenza virus recombination tyrrell: detection of attenuation of recombinant influenza viruses in vitro mufso~r viral and mycoplasmal infections in minor respiratory illnesses and non-streptococcal pharyngitis. a search for coronaviruses using foetal trachea organ cultures studies with inactivated influenza vaccines purified by zonal ultracentrifugation key: cord- -utg k qs authors: yinda, claude kwe; vanhulle, emiel; conceição-neto, nádia; beller, leen; deboutte, ward; shi, chenyan; ghogomu, stephen mbigha; maes, piet; van ranst, marc; matthijnssens, jelle title: gut virome analysis of cameroonians reveals high diversity of enteric viruses, including potential interspecies transmitted viruses date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: utg k qs diarrhea remains one of the most common causes of deaths in children. a limited number of studies have investigated the prevalence of enteric pathogens in cameroon, and as in many other african countries, the cause of many diarrheal episodes remains unexplained. a proportion of these unknown cases of diarrhea are likely caused by yet-unidentified viral agents, some of which could be the result of (recent) interspecies transmission from animal reservoirs, like bats. using viral metagenomics, we screened fecal samples of humans (almost all with gastroenteritis symptoms) between and years of age with different degrees of bat contact. we identified viruses belonging to families that are known to cause gastroenteritis such as adenoviridae, astroviridae, caliciviridae, picornaviridae, and reoviridae. interestingly, a mammalian orthoreovirus, picobirnaviruses, a smacovirus, and a pecovirus were also found. although there was no evidence of interspecies transmission of the most common human gastroenteritis-related viruses (astroviridae, caliciviridae, and reoviridae), the phylogenies of the identified orthoreovirus, picobirnavirus, and smacovirus indicate a genetic relatedness of these viruses identified in stools of humans and those of bats and/or other animals. these findings points out the possibility of interspecies transmission or simply a shared host of these viruses (bacterial, fungal, parasitic, …) present in both animals (bats) and humans. further screening of bat viruses in humans or vice versa will elucidate the epidemiological potential threats of animal viruses to human health. furthermore, this study showed a huge diversity of highly divergent novel phages, thereby expanding the existing phageome considerably. importance despite the availability of diagnostic tools for different enteric viral pathogens, a large fraction of human cases of gastroenteritis remains unexplained. this could be due to pathogens not tested for or novel divergent viruses of potential animal origin. fecal virome analyses of cameroonians showed a very diverse group of viruses, some of which are genetically related to those identified in animals. this is the first attempt to describe the gut virome of humans from cameroon. therefore, the data represent a baseline for future studies on enteric viral pathogens in this area and contribute to our knowledge of the world’s virome. the studies also highlight the fact that more viruses may be associated with diarrhea than the typical known ones. hence, it provides meaningful epidemiological information on diarrhea-related viruses in this area. d iarrhea is the second most common cause of death worldwide and accounts for about to % of the . million yearly deaths in children under the age of ( , ) . most of these deaths occur in southeast asia and sub-saharan africa ( , ) . the chances of infection with enteric viruses are higher in developing countries than developed countries, probably due to suboptimal sanitation and hygienic conditions and low quality of drinking water, especially in rural areas ( ) . in cameroon, a limited number of studies have investigated the prevalence of enteric pathogens as the cause of gastroenteritis in humans. these studies mainly focused on the epidemiology of a limited number of pathogens such as rotavirus, norovirus, and enteroviruses, revealing significant differences in the prevalence of these viruses in different settings and time periods ( , , ) . in parts of cameroon, a high prevalence of several enteric viruses such as enterovirus, norovirus, rotavirus, and adenovirus was found in children and adults ( ) . generally in africa, many episodes of gastroenteritis remain unexplained as no etiological agent is determined ( , ) . a proportion of the unexplained gastroenteritis cases are likely due to other known viruses, for which no tests were performed. however, a part of these gastroenteritis cases could also be caused by novel viral agents. transmission of these enteric viruses is predominantly fecal-oral, and humans are constantly exposed to these viruses through various routes ( ) . one of these routes is zoonosis from reservoirs in wild or domestic animals, either by insect vectors or by exposure to animal droppings or tissues. one rich but, until recently, underappreciated reservoir of emergent viruses is bats. of the ϳ , known terrestrial species of mammals, about % are bats ( ) . several viruses pathogenic to humans are believed to have originated in bats over the last several years, including severe acute respiratory syndrome (sars)-and middle east respiratory syndrome (mers)-related coronaviruses, as well as filoviruses, such as ebola and marburg viruses, or henipaviruses, such as nipah and hendra viruses ( ) ( ) ( ) ( ) ( ) ( ) . in the southwest region of cameroon, bats are hunted and eaten. such close interactions provide ample opportunity for zoonotic events to occur ( ) . previously, we identified a plethora of known and novel eukaryotic viruses in cameroonian fruit bats using a viral metagenomics approach, including viruses known to cause gastroenteritis in humans (sapovirus, sapelovirus, and rotaviruses a and h) and those not yet associated with gastroenteritis (bastrovirus and picobirna-like viruses) ( ) ( ) ( ) ( ) . in the current study, we metagenomically screened human fecal samples collected in the same region (where bats are hunted and eaten), to assess (i) if any viruses of animal origin could be identified and (ii) which known human gastrointestinal viruses were present. these fecal samples were collected from children less than a year old to adults of more than years who had gastroenteritis and/or were in contact with bats. additionally, since the gut virome typically contains both eukaryotic and prokaryotic viruses (phages), of which the latter usually represents the largest fraction of the gut virome, we also analyzed the phageome of these samples. sample characterization. a total of human fecal samples ( from kumba and from lysoka) were collected from two hospitals in the southwest region of cameroon, for viral metagenomics screening. from these fecal samples, a total of pools were constituted in categories based on age, bat contact status, and location (see table s in the supplemental material). illumina sequencing of all the human pools generated in total approximately million raw paired-end (pe) reads (between . and . million reads per pool). after trimming, % of the reads ( million) were retained and % of these retained trimmed reads ( million) were annotated using diamond. of these, % ( million) could be attributed as viral. ngs viral read distribution/abundances. in each of the categories of pools, phages make up at least % of the total number of viral reads while the maximum proportion of eukaryotic viral reads is %. a similar annotation profile was observed for pools of patients in different age groups, different locations, and different bat contact statuses (fig. s ). further analysis of eukaryotic viral reads revealed that at least % of the reads mapped to viruses of the families astroviridae, reoviridae, and anelloviridae (fig. a) . other viruses were also present, particularly those that are known to cause gastroenteritis belonging to the families adenoviridae, caliciviridae (sapovirus and norovirus), and picornaviridae (of which about % were enteroviruses [fig. b and fig. s ] ). also, reads from viruses known to cause other human diseases (parvoviridae) or other animal diseases (circoviridae) or not associated with any diseases at all (picobirnaviridae) were present in variable numbers in the different groups ( fig. b to e) . the rest of the viral families were either plant-or insect-associated viruses. notably, in age groups a to d, the percentage of pools in which picobirnaviridae viruses were present increased with age with low percentages in age groups a and b (fig. c) . also, the percentages of pools positive for anelloviruses differed with respect to age, with higher percentages in young children and the elderly. further, there were no observable trends in the percentage of eukaryotic viral presence with respect to bat contact status or location ( fig. d and e) . figure f shows a heat map of the percentage of pools in which eukaryotic viral families were present in human and bat pools, while fig. s presence in human and bats at the genus level ( ) . astroviridae (mamastrovirus), calciviridae (sapovirus), picornaviridae (parechovirus), and reoviridae (rotavirus), viral families known to cause gastroenteritis in humans, were identified in both bat and human pools from the same region. also, mammalian viruses not yet established to cause gastroenteritis (picobirnaviridae, circoviridae, and parvoviridae [bocaparvovirus]) were also common in both bats and humans from the same regions ( fig. f and fig. s ). phylogeny of eukaryotic viruses. in this study, we focused on viruses from which near-complete genomes were obtained, particularly those that are known to cause viral gastroenteritis (belonging to the astroviridae, caliciviridae [norovirus and sapovirus] , picornaviridae [enterovirus, parechovirus, cosavirus] , parvoviridae, reoviridae, and adenoviridae [human mastadenovirus]). furthermore, we also looked at other viruses not fully proven to cause gastroenteritis in humans but which have only sporadically been associated with gastroenteritis, like picobirnaviridae and small circular singlestranded dna viruses. phylogenetic analysis was done for each of the selected viruses using the protein or nucleotide sequences of suitable conserved regions and representative members of their viral family, genus, or species. reoviridae. reoviridae is a large viral family of segmented dsrna viruses with a wide host range. they are further divided into two subfamilies and genera. genomes of viruses belonging to the reoviridae contain to segments ( ) . in total, reoviridae reads were found in pools, and (nearly) complete genomes of viruses of the family reoviridae were obtained from pool hp . samples in this pool were from two diarrheic children (less than years), originating from kumba and without contact with bats. mammalian orthoreovirus. mammalian orthoreoviruses (morvs) contain segments, l to l , m to m , and s to s , coding for to proteins ( , ) . a morv strain was identified represented by , reads ( . % of all viral reads of the pool). phylogenetic analysis based on the nucleotide sequences of each of the segments of this morv ( fig. and fig. s ) showed topological incongruence with four distinctive patterns. based on segments l and s , this strain clustered with bat strains wiv and wiv from china with % and % nucleotide (nt) identity, respectively ( fig. a and b ). for the l and s segments, the human strain clustered with the ndelle murine strain, also from cameroon, with % and % nt identity, respectively (fig. c and d) . on the other hand, segment s of the cameroonian morv strain clustered with a human strain and a civet morv strain from china ( % and % nt identity, respectively [fig. e] ). the rest of the segments (l , m to m , and s ) did not cluster together clearly with any of the abovementioned strains (fig. s ) . rotavirus a. rotavirus a (rva) contains segments coding for or proteins: vp to vp , vp , vp , and nsp to nsp ( , ) . we identified a near-complete rva sequence which made up % ( . million) of the eukaryotic viral reads of that pool. the nsp segment was not identified in the sample. the vp gene of this strain was genetically most related to rva/human-tc/usa/wa/ /g p a [ ] and rva/human-tc/usa/rotarix/ /g p [ ] (nt identity of and %, respectively) while the vp gene was % identical to the same strains. the phylogenetic trees of the remaining segments shared the same clustering pattern (fig. a and b and fig. s ). according to the rotavirus classification scheme, this strain is a typical wa-like g p [ ] named rva/ human-wt/cmr/cmrhp / /g p [ ] . cmrhp was distantly related to bat rva strains identified from the same regions (only to % nt identity). picornaviridae. the picornaviridae represent a large family of small, cytoplasmic, nonenveloped icosahedral ssrna viruses consisting of species, grouped into genera. they have a genome of . to . kb in size and are most often composed of a single orf encoding a polyprotein flanked by a = and = utr ( ) . the members of the family picornaviridae can cause gastroenteritis, meningitis, encephalitis, paralysis (nonpolio and polio-type), myocarditis, hepatitis, upper respiratory tract infections, and diabetes ( , ) . out of the pools, contained picornaviridae reads, making the picornaviridae the eukaryotic viral family of which reads could be identified in the highest number of pools. enterovirus. the genus enterovirus (ev) consists of species: enterovirus a to l and rhinovirus a to c. ev a, b, c, and d are found in humans; e and f in cattle; g in pigs; h, j, and l in monkeys; k in rodents; and species i in dromedary camels (http://www .picornaviridae.com). in this study, eighteen (nearly) complete genomes of evs were obtained. the strains were named ev/human/cmrhpxx/cmr/ , here referred to as ev-cmrhpxx. all eighteen genomes were found in pools of age groups a and b (Ͻ and to years, respectively). eight of these were identified in age group a, three (ev-cmrhp , a, and b) of which were pools consisting of samples of infants who had indirect contact with bats while the rest (ev-cmrhp , , a, b, and ) were those that had no contact with bats. the ten other strains were identified in pools belonging to age group b, three of which had direct contact with bats (ev-cmrhp a, b, and ), indirect contact , a, b, and ) and two with no contact . based on the phylogenetic analysis of the vp nucleotide sequences, the evs found in this study were quite divergent from each other, belonging to three different species of enterovirus, a, b, and c (fig. a ). most of the strains belonged to the enterovirus c clade (ev-cmrhp , , , a, b, , , , a, a, and ) , while ev-cmrhp b, , and clustered within the enterovirus b genotype, and evcmrhp a, b, b, and in the genogroup enterovirus a. some pools had multiple strains of ev present, and some of these clustered together (cmrhp a and b: vaccine type pv- ), whereas other pools contained distinct ev species (ev-cmrhp a and b; a and b). the presence of vaccine strains in pool hp probably indicates recent vaccination events of the infants in this pool. apart from ev-cmrhp (which clustered with c _cmr), all the ev strains identified here were distantly related to those previously identified in the far north region of cameroon ( ) . furthermore, none of the human strains from cameroon were related to any of the animal ev strains (from chimp or gorilla). a summary of the detailed classification of these evs using an online typing tool ( ) is shown in table . parechovirus. the genus parechovirus is comprised of two species, parechovirus a (human parechovirus [hpev] ) and parechovirus b (ljungan virus, isolated from bank voles) ( ) . hpev is subdivided into types (hpev to - ) . hpev is associated with mild gastrointestinal or respiratory illness; however, severe disease conditions, such as meningitis/encephalitis, acute flaccid paralysis, and neonatal sepsis, may occur ( ) ( ) ( ) . here, three (nearly) complete hpevs were identified in pools hp , hp , and hp with sequence lengths of , bp, , bp, and , bp, respectively, collected from children less than years old (age group a). in terms of bat contact status, they were in pools of those either in indirect contact with bats (hp and hp ) or without contact (hp ). they were all distantly related to each other, with hpev-cmrhp and hpev-cmrhp having the highest identity ( % and % nt and aa identity, respectively). phylogenetically, hpevs in hp and in hp fell into a clade of type hpevs (fig. b ). the hpev in hp clustered together with hpev /harris strain with % nt identity, while cmrhp clustered closely with japanese and norwegian strains a - and no- ( to % nt identity) . furthermore, hpev-cmrhp clustered distantly with type hpevs from china and bangladesh with only to % nt identity. considering the % identity demarcation for hpev types ( , ) , this strain potentially represents a novel type. cosavirus. the genus cosavirus consists of five species (cosavirus a, b, and d to f), which have been associated with gastroenteritis in children ( ) . six near-complete [ ] strains. red, cameroonian human rva strain identified in this study; blue, cameroonian bat rva strains. trees were constructed human cosavirus (hcosv) genomes were identified: from children less than years old (hp ), from those between and Ͻ years old (hp a and hp b, hp ), and from pools of individuals between and Ͻ years old (hp , hp ). some of these pools had direct or indirect contact with bats (hp , hp , and hp ), while others had no contact with bats (hp and hp ). phylogenetic analysis (fig. c ) showed that cosaviruses from hp b, hp , and hp formed a clade with two other strains from australia and nigeria (hcosv/e /aus and hcosv/ng /nga) in species hcosv e. meanwhile the strains in hp a, hp , and hp clustered with hcosv in species a, d, and b, respectively. therefore, it seems that humans in cameroon host a diverse range of cosaviruses. cardiovirus. the genus cardiovirus consists of three species, cardiovirus a to c. species b includes saffold virus (safv) infecting humans. it has been found in cases with acute flaccid paralysis, respiratory tract infections, and diarrhea in china ( ) ( ) ( ) . here, we found a near-complete genome of a safv in one pool (hp ) belonging to the age group between and Ͻ years old who had indirect contact with bats. the vp phylogenetic trees were based on the nucleotide sequences of the vp -p a region for the species hepatovirus a and the vp region for the rest of the genera. all the trees were constructed using the gtrϩgϩi nucleotide substitution model using raxml, with the automre flag, which enables a posteriori bootstrapping analysis. only bootstrap values greater than % are shown. bars indicate nucleotide substitutions per site. red, novel strains from this study; blue, human cameroonian enterovirus strains from other studies; green, animal enterovirus strains from cameroon. segment of the identified safv was to % and to % identical (on nt level) to safv strains in types and , respectively. phylogenetic analysis based on the vp region confirmed the clustering of the novel strain between types and with more phylogenetic relatedness to type ( fig. d) . hence, this novel safv strain may be a distant member of type or represent a new type. hepatovirus a. hepatitis a virus (hav), now hepatovirus a, belongs to the genus hepatovirus, which consists of nine species (hepatovirus a to i). the hepatovirus a species is comprised of a single serotype, hav, subdivided into human and simian viruses ( ) . it causes acute hepatitis throughout the world ( ) . there were three (nearly) complete hav genomes in pools hp , hp , and hp , all of which were pools from those in direct (hp ) or indirect (hp and hp ) contact with bats. these strains were either from infants less than years old (hp ) or from children between and Ͻ years old (hp and hp ). based on the vp -p a region, the nt identity between these strains was to %. strains in hp and hp were % identical to brab , isolated from a patient from the netherlands in , who was staying in a hippie community with visitors from all over the world and under primitive living conditions ( ) . on the other hand, the hav strain in hp was closely related to strain g b -vp from france ( % nt identity). therefore, all strains identified here are genotype iia (fig. e) , increasing the number of completely sequenced genotype ii strains to five (the other two strains are ba/ita/ and cf /berne). astroviridae. astroviridae is a family of nonenveloped, spherical viruses with a linear ssrna(ϩ) genome of . to kb, containing three overlapping orfs. the family is divided into two genera: genus mamastrovirus (mastvs) and genus avastrovirus (aastvs). the genera are further divided into and species, respectively ( ) . fourteen out of the sixty-three human pools contained astroviridae reads, and we were able to obtain eight near-complete genomes of mastvs (hp , , , , , , , and ) . additionally, these pools were either from children less than years old (hp , hp , and hp ), age group to Ͻ (hp , hp , and hp ), or between and Ͻ (hp and hp ). phylogenetic analysis of the rdrp and capsid regions ( fig. a and b) depicted clustering of the novel mastvs in species (cmrhp , , , d, , and ) , (cmrhp ), and (cmrhp ). in the mastvs clade, there seems to be topological inconsistency in the different phylogenetic trees. strain astv _yuc (af ) clustered with the novel strains cmrhp , , and d in the capsid tree, while in the rdrp tree it clustered with the chinese strain v -guangzhou, suggesting a recombination event between these strains in the past. bat astrovirus identified in cameroon ( ) phylogenetic trees based on the nucleotide sequences of the rdrp (a) and capsid (b) genes of the astvs identified in this study and representative strains from genbank. trees were constructed using the gtrϩgϩi nucleotide substitution model using raxml, with the automre flag, which enables a (continued on next page) formed a clade (in the rdrp tree) with other bat astroviruses from guangxi but was distantly related to the human astvs from the same region. caliciviridae. caliciviridae are a family of nonenveloped viruses with a linear ss-rna(ϩ) genome of . to . kb, containing two or three orfs. the family contains five genera ( , ) . in total, caliciviridae reads were found in pools belonging to either the norovirus or sapovirus genus. norovirus. this genus consists of a single species, norwalk virus (nv), divided into genogroups. genogroups i, ii, and iv infect humans, whereas genogroup iii infects bovine species and genogroup v has been isolated from mice ( ) . three nearcomplete nvs were present in the pools that contained caliciviridae reads (hp , hp , and hp ), from people who had indirect (hp and hp ) or no (hp ) contact with bats, and from age group a (hp ), b (hp ), or c (hp ). the phylogenetic tree (fig. a) showed that the four nvs belonged to two genogroups: i (nv_cmrhp , genotype i. ) and ii (nv_cmrhp and nv_cmrhp , genotypes ii. and ii. , respectively) . the novel strain nv_cmrhp was more than % similar to strain c / cmr_gi. (a partial sequence [jf ]) isolated from the littoral region of cameroon in , whereas strains of genogroup ii from the same study (ii. , ii. , ii. ) were distantly related to those identified here (ii. and ii. ) ( ) . strains from this previous study were not included in the phylogenetic analysis because only to nt of the capsid region was available in databases. sapovirus. the genus sapovirus (sav) consists of a single species, sapporo virus. it has been detected in humans, pigs, minks, dogs, sea lions, bats, chimpanzees, rodents, and carnivores ( , ) . three near-complete sav genomes were present in pools hp (age group b), hp (age group a), and hp (age group d) from people who were in indirect contact, were not in contact, and were in direct contact with bats, respectively. phylogenetic analysis (fig. b) showed that sav from hp could be classified as a giv genotype, and the savs hp , hp , hp , hp , and hp belonged to genotype gii. the phylogenetic tree showed that the bat savs found in cameroon (in blue) ( ) clustered together and formed a clade with other bat savs from china and hong kong but divergent from these human savs, indicating no evidence of interspecies transmission of savs in this region. picobirnaviridae. picobirnaviruses (pbvs) belong to the family picobirnaviridae, genus picobirnavirus, and are small bisegmented dsrna viruses with a total genome size of about kb. segment one encodes a polyprotein, containing the capsid protein, and segment two encodes the rdrp. based on the rdrp gene, pbvs are classified into two genogroups. although pbv is genetically highly diverse and has been found in stool samples of a broad range of mammals, its true host(s) remain(s) enigmatic. the disease association is unclear, but pbv infection has been associated with gastroenteritis in both animals and humans ( , ) . up to out of the pools contained reads annotated as picobirnaviridae with most of the positive pools from individuals in age groups above . we could obtain (near-complete) rdrp sequences of pbvs from these pools. phylogenetic analysis based on rdrp (fig. ) revealed the clustering of the novel strains in four different clades: in genogroup i ( strains) , in genogroup ii ( strains), and in clades ( strains) of uncharacterized picobirna-like viruses that use an alternative mitochondrial invertebrate genetic code (lysoka picobirna-like virus cmrhp and cmrhp b and kumba picobirna-like virus cmrhp a). interestingly, a wolf pbv strain from portugal (ans ) from genogroup i clustered together with human strains from cameroon with an aa identity of % with strains cmrhp a and cmrhp . likewise, in genogroup ii, strains cmrhp a, cmrhp b, and cmrhp c clustered closely ( to % aa identity) with a portuguese feline strain (agz ). intriguingly, the cameroonian human picobirna-like viruses cmrhp and cmrhp b were % identical to a cameroonian bat strain picobirna-like virus, p - , suggesting a possible interspecies transmission. however, their true host has not yet been determined. it could be that the true hosts of pbvs are found in both humans and bats and that this therefore explains their presence in both. small circular, rep-encoding, ssdna (cress-dna) genomes. (i) smacovirus. smacovirus (scv) is a relatively recently described virus with a small circular dna genome with a size of about , bp. it belongs to the smacovirus group and is an unclassified eukaryotic virus of unknown origin ( ) . in this study, we identified two scv sequences, one complete genome (huscv-cmrhp ) and a near-complete genome (huscv-cmrhp ). they were identified in pools of patients belonging to age group b, coming from lysoka and having direct (hp ) or indirect (hp ) contact with bats. these strains shared % amino acid identity. their replicase genes were % and % identical to chimpanzee (kp ) and human (huscv , kt ) strains from the united states, respectively. based on the capsid region, these novel cameroonian strains were to % identical to the chimp strain and only % identical to the human strain huscv . the close genetic relatedness of human strains to a strain found in a chimpanzee sample suggest that these viruses infect a host shared between chimps and humans, and if indeed smacovirus infects mammals, this could be a case of interspecies transmission ( ) . phylogenies of the replicase (fig. a ) and the capsid genes ( fig. b) indeed showed a cluster of these cameroonian strains with a human genogroup i and pecovirus (c and d) of the replicase and capsid genes, respectively. the trees were constructed using the lgϩgϩi substitution model using raxml, with the automre flag, which enables a posteriori bootstrapping analysis. only bootstrap values greater than % are shown. bars indicate amino acid substitutions per site. red, cameroonian human strains; blue, previously known human smacoviruses or pecovirus. and a chimpanzee strain from the united states. however, the topological inconsistency in the replicase and capsid trees may suggest a recombination event between these strains in the (distant) past. (ii) pecovirus. pecoviruses (peruvian stool-associated circo-like viruses [pecvs] ) are cress-dna genomes that were first identified in the feces of a patient during an outbreak of acute gastroenteritis in the netherlands and later in samples of peruvian children ( , ) . subsequently, they were identified in other humans, pigs, a dromedary camel, and a seal ( ) ( ) ( ) ( ) . here we identified a genome sequence (hupecv-cmrhp ) of , bases made up of two orfs that code for a capsid ( aa) and a replicase protein ( aa). unlike other human pecvs, the cameroonian strain shared the same canonical nonamer (nantattac) atop the predicted stem-loop structure with seal, dromedary, and porcine pecv strains. the rep showed to % aa sequence identity to all other rep genes, and a rep-based phylogenetic analysis (fig. c) showed that hupecv-cmrhp clustered together with pecovirus genomes from a seal and human strains. based on the cap protein (fig. d) , the cameroonian strain was only to % identical to all other pecoviruses and clustered only distantly from the seal strain, a porcine strain, and the human strains. this demonstrates the existence of a high level of genetic diversity within this group of circular dna genomes, pointing to the possible existence of multiple species in this clade. furthermore, we identified incomplete sequences related to sewage-associated circular dna molecules recovered from a sewage treatment oxidation pond in new zealand ( ), with only % aa identity on the rep protein, further expanding the great diversity of cress-dna genomes in the cameroonian population. bacteriophages. bacteriophages are viruses that infect and replicate within bacteria. their presence therefore reflects the gut microbiota of the patients. because most of the obtained viral reads were classified as bacteriophages, we further investigated the bacteriophage composition of the human samples. with virsorter ( ), a tool developed to identify highly divergent dsdna phages from metagenomics data, , of the contigs in our data set were identified as phages. from these, the tool diamond ( ) annotated , as bacterial, as metazoan, and only (ϳ %) as viral, while , contigs remained unannotated. from the contigs annotated as viral by diamond, most were phages belonging to the myoviridae ( contigs), podoviridae ( contigs), siphoviridae ( contigs), and microviridae ( contigs) families. to get insight into the differences in the bacteriophage communities, we compared the virsorter-identified bacteriophage richness between the different age groups (fig. s a) , locations (fig. s b, ) and bat contact status (fig. s c ), all of which showed no significant differences. to identify the potential bacterial hosts of these phages, we searched for bacterial crispr spacer sequences in the phage contigs, to identify its potential host. the search revealed that the most likely hosts of these phages are bacteria of the families bacteroidaceae, bifidobacteriaceae, enterobacteriaceae, enterococcaceae, erysipelotrichaceae, eubacteriaceae, lactobacillaceae, odoribacteraceae, streptococcaceae, and veillonellaceae (table s ) . network analysis of human and bat phageomes. in order to visualize the genetic relatedness between the human and bat gut phageome, a recently developed bioinformatics tool (vcontact) was used. it groups phages based on their genome sequences into viral clusters which correlate rather well with viral genera as defined by the international committee of taxonomy of viruses (ictv) ( ) . a total of , protein clusters were predicted using the prokaryotic and archaeal refseq combined with the proteins predicted from the phage contigs identified from the human and bats pools using virsorter. using a network analysis approach (fig. ) , viral genome clusters were predicted of which contained reference phages together with bat or human phage contigs, whereas the rest contained only bat, only human, or bat-human clusters. figure shows that both cameroonian human and bat phage contigs identified in our studies are spread across the known phage sequence space. however, several of the phage contigs constituted completely new clusters (indicated by filled gray ovals), completely unconnected to phages in the reference database. also, the genetic diversity of several previously known phage subclusters was significantly expanded (as indicated by open ovals) while some clusters (in brown ovals) were made up of only bat and human phages identified in this study. recently, we thoroughly investigated the gut virome of fruit bats from cameroon ( ) ( ) ( ) ( ) ) and showed the presence of many novel and divergent eukaryotic viral families, including viruses known to cause gastroenteritis in humans. the aim of the current study was to investigate the gut virome of humans (n ϭ ) from cameroon and to further determine if bat viruses are possible causative agents of gastrointestinal infections in humans. twenty-four percent of the million generated trimmed reads were assigned as viral. most of these reads were bacteriophages, which is in accordance with previous studies ( ) . the eukaryotic viral reads include those that belong to viral families that are commonly associated with gastroenteritis in humans (adenoviridae, astroviridae, caliciviridae, picornaviridae, and reoviridae), viruses that are uncommon causes of gastroenteritis (orthoreovirus), or those that have been identified in humans but not associated with disease (anellovirus, smacovirus, and picrobirna-like viruses). virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ 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_ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ halocynthia~phage~jm- bacillus~phage~notthecreek bacillus~phage~belinda listeria~phage~lp- - virsorter_node_ _length_ _cov_ _ -cat_ bacillus~phage~w.ph. enterococcus~phage~eflk virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _id_ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ 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_length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _id_ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ microviridae~bog _ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ synechococcus~phage~s-cbs virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _id_ -cat_ synechococcus~phage~s-rip virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ riemerella~phage~rap virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ pseudomonas~phage~af virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _id_ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ enterobacter~phage~tyrion virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ clostridium~phage~phicd bordetella~virus~bpp virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _id_ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ cyanophage~natl a- virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ escherichia~phage~tl- b virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ synechococcus~phage~s-cbs virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ pseudomonas~phage~phipsa virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ xanthomonas~phage~xp virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _id_ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -circular-cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ _gc _gc -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ virsorter_node_ _length_ _cov_ _ -cat_ family were identified most frequently (fig. b) . this is partly because it is one of the largest viral families and is made up of at least genera, many of which are transmitted through the fecal-oral or respiratory route ( ) . most of these infections were in pools of individuals less than years of age (fig. c) . this finding is consistent with previous findings from cameroon, where a high prevalence of ev in children was reported using pcr-based approaches ( ) . furthermore, most of the evs here were of genotype c, also supporting a recent study that identified a high rate of ev cs in the northern regions of cameroon ( ) . therefore, the high prevalence of ev c is probably national. however, the absence of genetic relatedness between the cameroonian human ev strains and animal strains (from chimp and gorilla [ ] ) does not indicate interspecies transmission of evs from animals. additionally, we report for the first time (nearly) complete genomes of picornaviruses of the genera parechovirus, cardiovirus, hepatovirus a, and cosavirus from cameroonian patients. this broadens the range of picornaviruses found in the cameroonian population, indicating that picornaviruses might be playing a vital role in gastroenteric viral infection in the cameroonian population, especially given that most of these were from samples of sick children. rotavirus a (rva), a common viral gastroenteritis-causing agent, was identified only in a limited number of pools. this was previously observed in cameroon, and possible reasons for the low prevalence could include the acute nature of rotavirus infections or seasonal changes in rotavirus infections ( ) . of note, rotavirus vaccination was introduced in cameroon in april , coinciding with the period of sample collection of this study (february to september ); however, the vaccination campaign had not started in the sampling locations within this period, and therefore, the result represents a prevaccination rotavirus prevalence status. the identified rotavirus strain showed % nt differences with the vaccine strain, further suggesting that this was a wild-type rva strain, rather than a vaccine-derived strain. uncommon human gastroenteric virus: mammalian orthoreovirus (morv). this first morv strain from cameroon showed topological incongruence in its phylogeny, thereby pointing to possible reassortment events in the past. the phylogenetic clustering of some segments to strains from animals (rodents and bats) could be an indication of a zoonotic event or could also be due to the absence of related strains in databases from an unknown host. given that this strain was from a pool of samples from two children suffering from severe diarrhea, it is not unlikely that this strain might have contributed to the disease. therefore, morv might be playing a greater role in diarrheal diseases in this region than was previously known. hence, extensive epidemiological studies in different regions and in different hosts are required to fully delineate the prevalence, genomics, and interspecies transmissibility of morv. viruses not (yet) associated with gastroenteritis: picobirnaviridae, smacovirus, and anelloviridae. apart from the above-mentioned gastroenteritis-related viruses, several other viruses with unelucidated gastroenteric roles were also identified in this study. first, we observed fewer reads of picobirnaviruses (pbvs) in pools from children than in adults. previous studies also detected a relatively low percentage of children with pbvs ( , ) . this therefore adds up to the notion that pbvs are likely to be absent in infants and young children and only start to increase with age and potentially a changing diet, though this needs to be further proven ( , ) . interestingly, the genetic relatedness of a human picobirna-like virus with one that was found in a bat pool from the same region suggests an interspecies transmission. however, these picobirna-like sequences are translated using an alternative mitochondrial codon, indicating that their hosts may not be mammals. a principal component analysis of the codon usage bias of different known mitochondrial genome sequences, mitoviruses, and pbvs seems to suggest that they may have the same lifestyle as mitoviruses known to infect fungal mitochondria ( ) . however, the recent identification of a bacterial ribosomal binding site in pbv genomes suggests prokaryotes as a potential host ( ) . given that the mitochondria have descended from ancient eubacterial endosymbionts ( ) , this may explain the clustering of these pbvs with mitoviruses. therefore, the question about the true host of pbvs remains controversial. second, for the first time, two strains of african smacovirus (scv) were identified in cameroonian samples. their genetic relatedness to a chimpanzee strain (isolated from a captive chimp in a zoo in san francisco) and a strain from a child from the united states ( , ) indicates either an interspecies transmission event or the presence of a shared viral host in both humans and chimps. although the role of smacovirus in gastroenteritis has not been elucidated, their presence in cases of unexplained diarrhea in french patients seems to indicate a potential role in gastroenteritis ( ) ; hence, these could be instances of interspecies transmissions. the percentages of pools positive for anelloviruses were higher in age categories of children and the old and lower for the middle-aged groups. given the well-established notion that infants and the elderly have reduced immunity ( , ) , this could be in line with previous studies that suggest a link between the burden of anelloviruses and host immune competence ( ) ( ) ( ) . despite their ubiquity, anelloviridae have an undefined implication in hosts' health and are thought to be probably asymptomatic (harmless) or even beneficial. however, they have been associated with hepatitis, pulmonary diseases, hematologic disorders, myopathy, and lupus, but it is not clear if their presence is the cause or the result of disease progression ( ) ( ) ( ) ( ) . human viruses and interspecies transmission from bats. in bats from the same area, we were able to identify gastroenteritis-related and nonrelated viruses. here, the corresponding viruses identified from the families astroviridae (astrovirus), caliciviridae (sapovirus), and reoviridae (rva) are genetically diverse from those identified in bats from the same region, indicating no evidence of recent interspecies transmissions between bats and humans ( ) . however, genetic relatedness of human morv to animal strains showed the possibility of zoonosis between humans and not only bats but animals in general. additionally, the presence of some cameroonian strains of scv and pbv in bats or other animals would indicate interspecies transmissions if their infectivity in these animals is fully elucidated. human and bat phageome. in this study, we detected a huge phage community with a great diversity beyond the range of known bacteriophages in reference databases, potentially representing the gut microbiome diversity in the patients ( , ) . overall, this further supports the idea that the full phageome richness is still to be completely elucidated ( ) . furthermore, network analysis indicates the presence of completely novel phage groups and that phage genera in the gut microbiota might be shared between humans and bats. conclusion. several diverse viruses were discovered in the gut virome of cameroonians. some of these were already known to be the causative agent of gastroenteritis, whereas others are likely to be the cause of gastroenteric problems in the patients. further screening of patients for these viruses will be needed to establish their prevalence in the population, allowing for more appropriate measures and treatment and prevention of viral gastroenteritis. also, to be able to completely elucidate the role of the novel viruses like pecovirus and smacovirus, more studies are required. further attention should also be given to newly identified viruses (for example, morv) and their potential as emerging pathogens in the human population. ethical authorization. ethical authorization for the use of human samples was obtained from the cameroon national ethics committee, yaoundé. all human experiments were performed in accordance with the ministry's national ethics committee guidelines. sample collection and preparation. human fecal samples were collected between february and september , after informed consent was obtained from patients in two different hospitals (lysoka health district and kumba district hospital of the southwest region of cameroon). this region was chosen because here bats are hunted, sold, and eaten. diarrheic patients and/or people who came into contact with bats directly (by eating, hunting, or handling) or indirectly (if a family member was directly exposed to bats) were eligible for sampling. a total of samples were collected from subjects between age and Ͻ years (age group a, samples), and Ͻ (age group b, samples), and Ͻ (age group c, samples), and and older (age group d, samples). all the samples were from people who had symptoms of gastroenteritis, except from age group c who had contact with bats. samples were then placed into labeled tubes containing universal transport medium (utm), placed on dry ice, and stored at Ϫ °c, until being shipped to the laboratory of viral metagenomics, leuven, belgium. the samples were stored at Ϫ °c until used ( ) . fecal samples were first diluted using utm, and equal volumes of the dilutions were pooled based on the location, age, and bat contact status (direct, indirect, or none). each pool contained two to five samples, and for the different age groups (a to d) we had , , , and pools, respectively . the pools were then treated according to the netovir protocol ( ) . briefly, the pools ( % [wt/vol] fecal suspensions) were homogenized for min at , rpm with a minilys homogenizer (bertin technologies) and filtered using an . -m pes filter (sartorius). the filtrate was then treated with a cocktail of benzonase (novagen) and micrococcal nuclease (new england biolabs) at °c for h to digest free-floating nucleic acids. total nucleic acids (both rna and dna) were extracted using the qiaamp viral rna minikit (qiagen) according to the manufacturer's instructions but without addition of carrier rna to the lysis buffer. first-and second-strand synthesis and random pcr amplification for cycles were performed using a slightly modified whole-transcriptome amplification (wta ) kit procedure (sigma-aldrich). wta products were purified with msb spin pcrapace spin columns (stratec), and the libraries were prepared for illumina sequencing using a slightly modified version of the nextera xt library preparation kit (illumina), which is described in detail in reference . samples were pooled in an attempt to obtain an average of approximately million paired-end reads per pool. sequencing was performed on a nextseq high-output platform (illumina) for cycles ( ϫ -bp paired ends). genomic and phylogenetic analysis. ngs reads were analyzed as described in the work of yinda et al. ( , ) . briefly, raw reads were trimmed using trimmomatic (parameters: headcrop: and fastuniq to remove identical reads. the de novo assembly or reads and annotation of reads were performed using spades (with the meta flag) and diamond (with the sensitive option using the genbank nonredundant database), respectively ( , , ) . open reading frames (orfs) of contigs of interest were identified and further analyzed for conserved motifs in the amino acid sequences using ncbi's conserved domain database (cdd) ( ) . nucleotide and amino acid alignments of viral sequences were done with muscle implemented in mega ( ) or mafft ( ) . substitution models were determined using modelgenerator ( ) , and phylogenetic trees were constructed using raxml ( ) , with the automre flag, which enables a posteriori bootstrapping analysis. all trees were visualized in figtree (http://tree.bio.ed.ac.uk/software/figtree/) and midpoint rooted for purposes of clarity. phageome analysis. contig annotation with diamond is dependent on the accuracy of the database used, and in most databases, phages are poorly annotated. however, virsorter uses a manually curated database of virus reference genomes augmented with metagenomic viral sequences sampled from freshwater, seawater, and human gut, lung, and saliva. hence, for further identification of bacteriophages, scaffolds Ͼ kb were classified using virsorter (decontamination mode [ ] ). only scaffolds assigned to categories and were considered bacteriophage contigs and were filtered for redundancy at % nucleotide identity over % of the length using cluster genomes ( ) . then, trimmed reads from each pool were mapped using bowtie ( ) to the bacteriophage contigs, and the generated bam files were filtered to remove reads that aligned at Ͻ % identity using bamm (http://ecogenomics.github .io/bamm/). abundance tables were obtained and normalized for total number of reads of each sample. for the richness comparison, mann-whitney tests were used, and for the clustering, an adonis test was performed. all downstream analyses were done in r ( ) using the vegan package ( ) . furthermore, to identify the potential corresponding bacterial host, a database of these contigs was made to which a nucleotide blastn search ( % identity without gaps) was performed using a fasta file of crispr sequences ( ) as query. these sequences correspond to different bacterial hosts, and their presence in the phage genome highlight the potential host of the phage. to see if the phage community of these humans is related to those of the bats from the same locality, a visualization of the network of both human and bat phageomes was performed using vcontact ( ) . initially, proteins were predicted using prodigal ( ) , and combined with the viral refseq of archaeal and prokaryotic predicted proteins. a database was generated from the contigs of bat pools, human pools, and viral refseq proteins, and blastp was performed against the combined proteins. the output of blast was used to run vcontact, and the output network was visualized in cytoscape ( ). data availability. all sequences were deposited in genbank under the following accession numbers: mh to mh and mh to mh (details in table s ). raw reads were submitted to the ncbi's short read archive (sra) under the project id prjna . supplemental material for this article may be found at https://doi.org/ . / msphere. - . ef _hpev- /bni- st/deu/xx jn /hcosv/a -np /npl gu _mink_astv-sms ay _humanastv -goiania/go/ / /brazil fj _sea_lionastv human-astv_cmrhp d hq _pigeonanv-sh human-astv_cmrhp z _humanastv-newcastle human-astv_cmrhp human-astv_cmrhp fj _humanastv-va ita fj _humanastv-mlb -wd jx _humanastv-va /human/nepal/s hq _bovineastv/b - /hk fj _sea_lionastv z _humanastv-newcastle hq _bovineastv/b /hk hq _bovineastv/b - /hk human-astrov_cmrhp d human-astrov_cmrhp ay . _calicivirus/human/nlv u . _calicivirus/human/nlv fj _pig/f - /can/ /gviii kj _chimp/ijc gu _pig/f kumba_picobirna-like virus_cmrhp a ahx _human_picobirnavirus ans _picobirnavirus_wolf baj _human_picobirnavirus shwc c _shahe_picobirna-like_virus_ global health observatory data repository. gho | world-diarrhoeal diseases. world health organization levels and trends in child mortality . world health organization enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools shift of enterovirus species among children in cameroon; identification of a new enterovirus, ev-a progress and barriers for the control of diarrhoeal disease monitoring of seasonality of norovirus and other enteric viruses in cameroon by real-time pcr: an exploratory study enteric viruses in healthy children in cameroon: viral load and genotyping of norovirus strains a novel picornavirus associated with gastroenteritis new parvovirus in child with unexplained diarrhea new tools for the study and direct surveillance of viral pathogens in water bats, a world of science and mystery the ascension of wildlife rabies: a cause for public health concern or intervention? marburg virus infection detected in a common african bat fruit bats as reservoirs of ebola virus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats middle east respiratory syndrome coronavirus in bats, saudi arabia isolation of nipah virus from malaysian island flying-foxes bats as a continuing source of emerging infections in humans a single bat species in cameroon harbors multiple highly divergent papillomaviruses in stool identified by metagenomics analysis highly diverse population of picornaviridae and other members of the picornavirales, in cameroonian fruit bats novel highly divergent sapoviruses detected by metagenomics analysis in straw-colored fruit bats in cameroon cameroonian fruit bats harbor divergent viruses, including rotavirus h, bastroviruses, and picobirnaviruses using an alternative genetic code high similarity of novel 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infant development early life dynamics of the human gut virome and bacterial microbiome in infants multiple mitochondrial viruses in an isolate of the dutch elm disease fungus ophiostoma novo-ulmi extensive conservation of prokaryotic ribosomal binding sites in known and novel picobirnaviruses ancient invasions: from endosymbionts to organelles evolution of the immune system in humans from infancy to old age immunity to influenza human immune system variation temporal response of the human virome to immunosuppression and antiviral therapy torque teno virus in children who underwent orthotopic liver transplantation: new insights about a common pathogen tt virus in the nasal secretions of children with acute respiratory diseases: relations to viremia and disease severity small anellovirus infections in korean children hepatitis-associated aplastic anemia and transfusion-transmitted virus infection human anelloviruses: an update of molecular, epidemiological and clinical aspects the human gut virome: inter-individual variation and dynamic response to diet viruses in the fecal microbiota of monozygotic twins and their mothers global phage diversity modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis trimmomatic: a flexible trimmer for illumina sequence data spades: a new genome assembly algorithm and its applications to single-cell sequencing cdd: ncbi's conserved domain database mega : molecular evolutionary genetics analysis version . for bigger datasets mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies clustering viral genomes in ivirus fast gapped-read alignment with bowtie r: a language and environment for statistical computing. r foundation for statistical computing community ecology package the crisprdb database and tools to display crisprs and to generate dictionaries of spacers and repeats prodigal: prokaryotic gene recognition and translation initiation site identification integration of biological networks and gene expression data using cytoscape g _rva/bat-wt/cmr/batli / /g p [ ] g _rva/bat-wt/cmr/batli / /g p [ ] g _rva/bat-wt/chn/myas / _g p [ ] g _rva/bat-wt/ken/ke / / /g p [ ] g _rva/cow-wt/jpn/azuk- / /g p [ ] g _rva/horse-tc/gbr/l / /g p [ ] g _rva/human-tc/jpn/au- / /g p [ ] g _rva/cow-tc/ind/hg / /g p [ ] g _rva/bat-wt/cmr/batli / /g p [ ] g _rva/turkey-tc/deu/ v e / /g p [ ] g _rva/human-tc/ind/ m/ /g p [ ] g _rva/pigeon-tc/jpn/po- / /g p [ ] g _rva/human-wt/ecu/ecu / /g p [ ] g _rva/pig-wt/jpn/tj - / /g p [x] g _rva/horse-tc/usa/fi / /g p [ ] g _rva/human-wt/cmr/cmrhp / /g p [ ] g _rva/turkey-tc/irl/ty- / /g p [ ] g _rva/bat-tc/mslh / /g p [ ] g _cow g _rva/bat-wt/cmr/batly / /g p [ ] g _rva/chicken-tc/gbr/ch- / x/g p [ ] g _rva/human-tc/usa/wa/ /g p[ ]a g _rva/human-tc/gbr/a / /g p [ ] g _rva/human-tc/usa/wi / /g p[ ]a g _rva/bat-wt/cmr/batly / /g p [ ] g _rva/sugarglider-tc/jpn/sg / /g p [ ] g _rva/cow-tc/jpn/dai- / /g p [ ] g _rva/pig-tc/mex/ym/ /g p[ ] g _rva/mouse-tc/usa/ew/xxxx/g p [ ] acknowledgments c.k.y. was supported by the interfaculty council for development cooperation (iro) from the ku leuven. n.c.-n. and l.b. were supported by the flanders innovation & entrepreneurship (vlaio). this work was supported by ku leuven grant ejx-c -stg/ / bf. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.c the authors declare no competing financial interests. key: cord- -deq tv d authors: bergström, t.; alestig, k.; svennerholm, b.; horal, p.; sköldenberg, b.; vahlne, a. title: neurovirulence of herpes simplex virus types and isolates in diseases of the central nervous system date: journal: eur j clin microbiol infect dis doi: . /bf sha: doc_id: cord_uid: deq tv d herpes simplex virus (hsv) isolates derived from the central nervous system of ten patients with hsv- -induced encephalitis, one patient with multiple sclerosis, and patients with hsv- -induced meningitis were investigated for neurovirulence by assaying the ld( ) after nose and intracerebral (i.c.) inoculation of mice. hsv- encephalitis strains were significantly more virulent after nose inoculation (i.e. neuroinvasive) when compared with hsv- isolates from patients with oral lesions only, whereas hsv- meningitis strains were significantly more virulent after i.c. inoculation when compared with hsv- isolates from patients with genital lesions only. no correlation between high neurovirulence (defined as low ld( ) for both routes of infection) and replication in cell cultures of neuronal and non-neuronal cell lines was found, but the weakly neurovirulent hsv- strain isolated from a patient with multiple sclerosis gave low replication yields. after nose inoculation, a highly neuroinvasive hsv- laboratory reference strain replicated to high titers in nose tissue, the trigeminal ganglia and brainstem, while a strain with low neuroinvasiveness but high i.c. virulence replicated less well in the brainstem. neuroinvasiveness of the virus strain might be one factor of relevance in the pathogenesis of hsv- encephalitis in man. t. bergstr/ m ., k. alestig , b. svennerholm , p. horal , b. sk/ denberg , a. vahlne t herpes simplex virus (hsv) isolates derived from the central nervous system of ten patients with hsv-l-induced encephalitis, one patient with multiple sclerosis, and patients with hsv- -induced meningitis were investigated for neurovirnlence by assaying the lds after nose and intracerebrai (i.c.) inoculation of mice. hsv- encephalitis strains were significantly more virulent after nose inoculation (i.e. neuroinvasive) when compared with hsv- isolates from patients with oral lesions only, whereas hsv- meningitis strains were significantly more virulent after i.c. inoculation when compared with hsv- isolates from patients with genital lesions only. no correlation between high neurovirulence (defined as low lds for both routes of infection) and replication in cell cultures of neuronal and non-neuronal cell lines was found, but the weakly neuruvirulent hsv- strain isolated from a patient with multiple sclerosis gave low replication yields. after nose inoculation, a highly neuroinvasive hsv- laboratory reference strain replicated to high titers in nose tissue, the trigeminal ganglia and brainslem, while a strain with low neuroinvasiveness but high i.c. virulence replicated less well in the brains|era. neuroinvasiveness of the virus strain might be one factor of relevance in the pathugenesis of hsv- encephalitis in man. when infecting the central nervous system (cns) herpes simplex virus type (hsv- ) can cause focal, necrotizing encephalitis, this virus being considered the most important cause of acute encephalitis in immunocompetent patients ( ) ( ) ( ) . the sporadic occurrence of this disease and the fact that as yet no pathogenetic factor has been singled out as playing a decisive role might indicate that in a particular patient a number of unfortunate circumstances concurr. of relevance to the pathogenesis could be predisposing factors in the patient himself, the virus dose, or the site of replication at primary infection, as well as neurovirulent properties of the virus. data have been presented in support of hsv- strain variability of neurovirulence in mouse models ( - ) and in rabbits ( ). attempts have been made to pinpoint the hsv- genes regulating neurovirulence by the use of recombinant strains ( - ) or deletion mutants ( ) volved. however, whether the pathogenesis in hsv- encephalitis in man is influenced by the neurovirulence of hsv- strains is still not known. herpes simplex virus type (hsv- ) is a rare cause of encephalitis in man ( ) , and in most cases reported the patients have been immunocompromised ( , ) . instead, hsv- may sometimes induce a serous meningitis when hsv- infection spreads from the genital tract to the cns ( , ) . symptoms of meningitis are far more common in primary hsv- infection, where the surface area of involvement is larger and the duration of virus shedding longer, as compared with secondary hsv- infection (i.e. the first episode of hsv- infection preceded by an hsv- infection) ( ) . these findings indicate possible relevance of virus load from the periphery in the development of hsv- meningitis, but the potential virulence properties of hsv- strains causing meningitis have not yet been described. we used a mouse model to assay the ld after infection by different routes with cns-derived hsv- isolates from ten patients with encephalitis and one patient with multiple sclerosis, and hsv- isolates from patients with meningitis, in comparison with clinical reference strains isolated from oral lesions (hsv- ) and genital lesions (hsv- ) in patients without neurological symptoms, and with known laboratory reference strains. the mouse is permissive to cns infections with both hsv- and hsv- , also by peripheral routes ( ) . it has been reported that the neurovirulence of recombinant mouseadapted poliovirus strains in this animal is in concordance with neurovirulence in man ( ) . virus isolation. clinical samples of brain material, cerebrospinal fluid (csf), and secretion from oral and genital lesions were inoculated in cell cultures of green monkey kidney (gmk ah- cells) or veto cells. isolates were typed as hsv- or hsv- by eia using monoclonal antibodies ( , ) . virus strains. ten hsv- strains were isolated from brains of patients with encephalitis (hsv- enc). eight of these strains were derived from biopsies (patients no. - , table ) performed during a trial of antiviral treatment of hsv encephalitis ( ). all biopsies were taken before the commencement of antiviral treatment. in three of these patients (no. , and ), serological data were consistent with a probable primary hsv- infection (low level or absence of hsv- igg antibodies and presence of hsv lgm antibodies in the first serum sample) at the time of the encephalitis, while four had serologic evidence of reactivated hsv- infection. in one patient, serological data were missing. two hsv- strains were isolated from brain material obtained at autopsy of two patients who died of encephalitis before receiving antiviral treatment (patients no. and , table i ) and in whom serologic data (high levels of hsv- igg antibodies at onset) indicated reactivation. as controls, we used ten hsv- strains isolated from oral lesions in patients without neurological symptoms seen at the department of dermatology, university of goteborg, these strains are referred to as hsv- ref strains. also included is an hsv- strain which we isolated from the csf of a patient during the first bout of multiple sclerosis. the isolate was designated hsv- ban and was recently described in more detail ( ) . laboratory reference strains (hsv- labref) used were f (b. roizman, chicago), mclntyre (a. nahmias, atlanta) and our own strain kj ( ) . in fourteen patients with meningitis, hsv- was isolated from csf (hsv- men) at either the department of virology, university of g teborg (six strains) or at the department of virology, stockholm county central microbiological laboratory (eight strains). hsv- clinical reference strains (hsv- ref) were isolated from genital lesions in ten patients with hsv- infection without neurological symptoms seen at the department of dermatology, university of g teborg. hsv- laboratory reference strains (hsv- labref) used were b ur and (s. jeansson, g teborg). in all experiments, low (second or third) passages of virus were used, and virus stocks were prepared by limited dilution of hsv strains in gmk ah- cell-tube cultures followed by low multiplicity infection of monolayers of gmk ah- cell in cm glass flasks with eagle's medium and incubated at °c. flasks were frozen at - °c when an almost confluent cytopathic effect was observed. the hsv-infected cell cultures were thawed, homogenized, centrifuged at low-speed, atiquoted in ml vials, and stored at - °c. in connection with the experiments, plaque titrations of the virus strains were performed on gmk ah- ceils grown in cm plastic dishes with % methyl cellulose as overlaying medium, and the virus strains were quantified by counting plaque forming units (pfus). hsv titers were then expressed as pfus/ml. outbred swiss albino mice were used. we preferred an outbred strain, since clear differences in susceptibility of several inbred mouse strains to hsv have been reported ( ) . groups of five mice of to weeks of age were infected by intracerebral (i.c.) injection of . ml in the left temporal region or, when assessing neuroinvasiveness by nose inoculation, by scraping the region of the virbrissae bilaterally, and inoculation ( x . ml) of log dilutions in hank's medium of the hsv strains. virus quantities per mouse given were for i.e. infection - - pfus for hsv- and - - pfus for hsv- , and for • nose infection i ~-i pfus for both hsv- and hsv- . as negative control, medium alone was used. the groups of mice were coded and observed daily for two weeks after i.e. and for three weeks after nose infection for signs of neurological involvement and the number of deaths were recorded. the ldso was calculated for each strain. pfus/ml). on day to , five mice from each group were sacrificed and, after perfusion through the left heart chamber with eagle's medium, nose tissues, trigeminal ganglia and brainstems were collected and frozen at - °c. the samples were thawed simultaneously, homogenized in . ml of eagle's medium containing % penicillinstreptomycin, and centrifuged for minutes at rpm. supernatants were inoculated on plastic dishes with gmk ah- cells for plaque titration as described above. replication assays. twenty-four hour replication of all hsv strains was assayed by inoculating strains on gmk ah- cells in plastic dishes (inoculum . pfu/celi), and on c cells, a mouse neuroblastoma cell line (inoculum . pfu/cell). after adsorption for h, cetl cultures were rinsed four times with eagle's medium and ml eagle's medium was added to each culture. after incubation at o for h, the cultures were frozen for later quantification in log dilutions by plaque titration on gmk ah- cells as described above. statistical methods. the nonparametric wilcoxon's rank sum test was used throughout for comparisons between the cns-derived hsv strains and the oral or genital hsv strains except for the analysis of time to death after nose inoculation of the hsv- strains, and hsv replication in cell cultures, where student's t-test was used. neurovirulence in mice after cns and peripheral infection. the hsv-infected mice died of encephalitis with seizures and/or paresis after both i.c. and nose inoculation of the hsv strains and the log pfu/lds could be calculated for each strain. by definition, death after nose inoculation was characterized as neuroinvasiveness, since this route constitutes replication and transport along the trigeminal pathway before entrance to the cns. neurovirulence for each strain was defined based on the combination of results of neuroinvasiveness and lds after i.c. inoculation (see below), the results from mice infected with hsv- strains are shown in figure . after i.e. inoculation, mean log pfu/lds for hsv- enc strains did not differ significantly from mean log pfu/lds for hsv- ref strains. the hsv- ban strain isolated from a patient with multiple sclerosis showed a higher pfu/lds ratio than did any of the hsv- enc or ref strains. after nose inoculation, one hsv- enc strain and four hsv- ref strains were non-lethal with the inocula used ( figure ) . seven of the data from i.c. and nose inoculation in mice were combined to classify all hsv- strains into three classes of neurovirulence ( table ). the nature of this difference was further investigated by monitoring virus replication at three levels (skin, trigeminal ganglia and brainstem) after nose inoculation with these two strains and the class iii (a ) hsv- labref strain f. as shown in figure , hsv- replication reached its maximum after to days in the periphery, after to days in ganglia, and after to days in the brainstem with all three strains. however, hsv- ban albl aobl "hsv- strain characteristics expressed as a: neuroinvasiveness after nose inoculation (ao indicates > ; -> al > x ; x -> a - x ; and a < x pfu/ld ) and b: neurovirulence after intracerebral inoculation (b~ indicates > ; >-b >_- ; and b < pfu/lds ). class i (highly neurovirulent) is defined as: a + b = or ; class ii (moderately neurovirulent) as: a + b = or ; and class iii (weakly neurovirulent) as: a + b = or . bserological data indicating a probable primary hsv- infection as the cause of the encephalitis. two different patterns of progression of hsv- infection were found: strain f (class iii) replicated well at the periphery but to low titers at both the ganglionic and brainstem levels, whereas strains mcintyre (class ii)and kj (class i) both replicated to relatively high titers in ganglia and brainstems after high-dose inoculation peripherally. when the infectious dose was lowered, although kj replicated better at all levels, strain mcintyre barely replicated in the brainstem, while the highly neuroinvasive strain kj readily progressed to the brainstem level. in figure , results from the assessment of virulence after i.e. or nose inoculation of hsv- strains are depicted. generally, log pfu/lds values were lower for the hsv- strains than for the hsv- strains. after i.c. inoculation, hsv- meningitis strains were significantly (p < . ) more virulent as compared with the hsv- reference strains, but no difference was found after nose inoculation. replication assays. the replication yields in gmk-ah (mean log pfu/ml + sd) and c neuroblastoma cells (mean log pfu/ml +_ sd) of hsv- enc strains ( . + . the pathogenesis of encephalitis caused by hsv- is largely unknown. the sporadic occurrence and scarcity of underlying immunocompromising conditions found in patients ( ) may suggest pathogenetic relevance of viral alteration by recombination ( ) or mutation. although the existence and genomic locations of hsv neurovirulence have been well documented in experimental studies ( , , , ), the link between neurovirulence in humans in the form of encephalitis and neurovirulence in animal models has hitherto not been established. the hsv- brain isolates used in this study were all proven to be neuropathogenic to man by causing encephalitis with either a fatal outcome or sequelae in spite of antivirat treatment. the peripheral route of nose infection, where virus particles reach the brain via the trigeminal pathway by neuron to neuron transmission ( ) , was chosen to imitate the probable route that hsv- travels when this virus causes encephalitis in humans ( ) . the virulence to mice displayed by encephalitits-inducing hsv- strains by this route of infection suggests that invasiveness is a property of relevance for hsv- neurovirulence. furthermore, we have shown that the same encephalitis-inducing strains also were significantly more invasive as compared with the hsv- ref strains in an in vitro model using cultured rat sensory neurons in a dual-chamber system permitting infection of neuritic extensions only ( ) . in experimentally infected animals, spread along sensory neurons has been found to be the main route by which hsv enters the cns ( , ) . hsv strain variability in capacity to invade the cns has been described ( , ), and non-neuroinvasive strains have been found to be unable to progress from sensory ganglia to the cns ( , ) . in our study, results of viral replication at peripheral, ganglionic and brainstem levels after nose inoculation of mice indicate that the neuroinvasive hsv- labref strain kj differs from the non-neuroinvasive strains mc-intyre and f by its greater ability to reach and to replicate in the brainstem. one of the hsv- isolates in this study was derived from a patient who was part of an epidemiotogicat chain of three subjects who all suffered from meningitis after genital herpes infection. this might suggest the existence of hsv- strains that are more prone to induce meningitis, but studies on virulence characteristics of isolates from patients with meningitis are lacking. the finding in this study that cns-derived meningitis-inducing hsv- strains showed enhanced neurovirulence after i.c. inoculation in mice might indicate the existence of such a strain characteristic. this characteristic was not revealed by the peripheral route of nose inoculation used here. the clinical picture of hsv- induced meningitis described earlier ( , ) with local neurological signs in the lumbosacral region but without spread to the brain or higher regions of the spinal cord may suggest that hsv- is a weakly neuroinvasive virus in adults. however, studies on hsv- invasiveness after genital inoculation in animals with meningitis-inducing strains are needed to further address this question, since a difference exists in tropism between hsv- and hsv- ( ). a connection between hsv- neurovirulence in vivo and replicative capacity has been suggested ( ), but conflicting data have been presented. in later studies, neither an avirulent strain ( ) nor a virulent strain (t ) showed any correlation between virulence and replication in different cell lines. the indications of hsv- and hsv- neurovirulence properties of cns-derived isolates found in vivo in our study were not paratlelled by higher replication yields in either a highly permissive non-neuronal (gmk-ah- ) or a mouse neuroblastoma (c ) cell line giving low replication yields. on the other hand, a tendency towards lower replication yields in c cells was seen for the class iii nonneurovirulent strains in comparison with the class i strains (t = . , student's t-test). likewise, the strain hsv- ban from a patient with multiple sclerosis, which displayed weak neurovirulence in vivo, gave a markedly low replication yield in both cell types. restricted viral replication as a pathogenetic factor in demyelination was found in a study of mice infected with mutants of the coronavirus mouse hepatitis virus ( ) , and has earlier been suggested for hsv in humans in a hypothesis on multiple sclerosis ( ) . however, we have not been able to test this hypothesis properly, due to the lack of additional viral isolates from the cns of patients with multiple sclerosis. in man, hsv- induced cns infections such as encephalitis are rare events although this virus is neurotropic and commonly recovered from sensory ganglia at autopsy ( ) . the suggestion derived from our data that the hsv- strain characteristic of neuroinvasiveness is linked to encephalitis 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molecular analyses of the avirulent wild-type herpes simplex virus type strain kos induction of demyelination by a temperature-sensitive mutant of the coronavirus mhv-a is associated with restriction of viral replication in the brain can a defectwc herpes simplex virus cause multiple sclerosis? recovery of herpes-simplex virus from human trigeminal ganglions this work was financially supported by the gothenburg medical society, the medical faculty, university of goteborg, the swedish medical society and the swedish medical research council. we thank miss ann-sofietyl for skillful technical assistance, and drs marianne forsgren, elisabeth olding-stenquist, monica grandien, jonas blomberg, and gunbritt lowhagen for clinical hsv isolates. key: cord- - di eub authors: biswas, kaushiki; chatterjee, dhriti; addya, sankar; khan, reas s.; kenyon, lawrence c.; choe, alexander; cohrs, randall j.; shindler, kenneth s.; das sarma, jayasri title: demyelinating strain of mouse hepatitis virus infection bridging innate and adaptive immune response in the induction of demyelination date: - - journal: clin immunol doi: . /j.clim. . . sha: doc_id: cord_uid: di eub the presence of immunoglobulin oligoclonal bands in the cerebrospinal fluid of multiple sclerosis (ms) patients supports the hypothesis of an infectious etiology, although the antigenic targets remain elusive. neurotropic mouse hepatitis virus (mhv) infection in mice provides a useful tool for studying mechanisms of demyelination in a virus-induced experimental model of ms. this study uses affymetrix microarray analysis to compare differential spinal cord mrna levels between mice infected with demyelinating and non-demyelinating strains of mhv to identify host immune genes expressed in this demyelinating disease model. the study reveals that during the acute stage of infection, both strains induce inflammatory innate immune response genes, whereas upregulation of several immunoglobulin genes during chronic stage infection is unique to infection with the demyelinating strain. results suggest that the demyelinating strain induced an innate-immune response during acute infection that may promote switching of ig isotype genes during chronic infection, potentially playing a role in antibody-mediated progressive demyelination even after viral clearance. multiple sclerosis (ms) is a demyelinating disease of the central nervous system (cns), pathologically characterized by loss of myelin, oligodendroglial cells, and axonal degeneration [ ] . ms is conventionally believed to be an autoimmune disease, mediated by anti-myelin t cells and autoantibodies. although the etiology is unknown, indirect evidence such as the presence of oligoclonal bands (ocbs) and abnormally increased levels of immunoglobulins (ig's) in cns tissue and cerebrospinal (csf) fluid of ms patients suggest that viral infections play a role in triggering the disease process. various experimental animal models of virus induced demyelination exist, including the demyelinating strain mhv-a , and its recombinant demyelinating strain, rsa induced demyelination in c bl/ mice [ ] [ ] [ ] [ ] . upon intracranial inoculation, rsa causes a biphasic disease that mimics some ms pathology. in the acute phase, rsa causes neuroinflammation including meningitis, encephalitis, myelitis and sparse demyelination. myelin loss starts as early as day post-innoculation (p.i.) but reaches its peak by days - (chronic disease phase), when infectious viral particles are below the limit of detection and viral antigen has resolved, but viral rna persists [ , , ] . demyelination and axonal loss depend on the viral hostattachment spike protein, as rsmhv , a closely related isogenic strain that differs from rsa only in the spike gene, can cause meningitis, encephalitis, myelitis and occasional optic neuritis [ ] but is unable to cause demyelination or axonal loss. thus, spike plays a major role in mhv-induced myelin loss and axonal loss, but it is unknown how differences in spike affect gene expression in order to mediate different host tissue responses. our recent studies using affymetrix microarray analysis revealed that rsa induces ifn-gamma inducible genes and innate immune-activating pathway genes during the acute stage of infection [ ] . recent studies using a high-throughput rna-seq approach also revealed expression of several genes involved in macrophage function, immune infiltration, and changes in cytokine expression levels in response to persistent mhv infection in mice [ ] . however, no high-throughput expression studies are available to identify host immune response genes in the chronic stage of infection, when demyelination and axonal loss are major pathological hallmarks. in the current studies, we examined changes in gene expression in the spinal cord during acute and chronic stage of dm (demyelinating; rsa ) and ndm (non-demyelinating; rsmhv ) mhv infection. four week old c bl/ mice were inoculated intracranially with dm (rsa ) or ndm (rsmhv ) strains using the ld as described in prior studies [ ] [ ] [ ] . to confirm infection, histopathology and immunohistochemistry were performed on liver, brain and spinal cord sections from mice whose spinal cords were processed for microarray analysis. both strains induced hepatitis (score - on a relative scale [ ] ) ( fig. b and c) , and mock-infection induced none (score ) (fig. a) . no encephalitis was observed by h&e staining of control mouse brains (fig. d ), but there was encephalitis, characterized by activated microglia/macrophages (stained for iba- ) [ , ] in olfactory bulb, basal forebrain ( fig. e and f) , hippocampus, basal pons and deep cerebellar white matter at day p.i. with dm and ndm strains. at day p.i., dm strain induced myelin loss, evident by lfb staining (fig. h ), but ndm strain did not (fig. i) . parallel sections stained with iba showed accumulation of microglia/macrophages in demyelinating plaques (fig. k ) of dm strain infected, but not in ndm infected, mouse spinal cords (fig. l) . differentially expressed genes at day p.i. in spinal cords from dm, ndm and mock infected mice. a. venn diagram depicts number of genes whose expression changed by at least . fold during dm strain infection (left circle) compared to mock infected controls, during ndm infection (right circle) compared to mock infected controls, and during dm strain infection compared to ndm infection (bottom circle). overlapping changes between more than one comparison group is shaded. b. increased expression of specific genes found to be upregulated by microarray analysis, including irgm (b), iigp (c), cxcl (d), cxcl (e), and gbp (f) were validated by quantitative real time pcr. the fold change in gene expression with respect to mock infected mice ( −ΔΔct ) was calculated for each of the genes in dm and ndm strain infected mice and plotted. results show significant upregulation of all genes examined ( ⁎ p b . ; ⁎⁎ p b . , ⁎⁎⁎ p b . , ⁎⁎⁎⁎ p b . ) as compared to mock infected mice. only gbp showed significant differences between the dm and ndm strain ( ### p b . ). stars ( ⁎ ) imply the gene expression was statistically significantly upregulated as compared to mock infected mice, whereas # imply that there is a significant difference in the upregulation of certain genes between dm and ndm strain infection. affymetrix microarray analysis was performed on extracts from spinal cords isolated on day p.i. genes that demonstrated significant differences in expression level were identified in comparisons between dm vs. mock, ndm vs. mock and dm vs. ndm infected spinal cords. using a threshold of . fold change in expression level (p b . ), genes were found to be differentially expressed in dm infected spinal cord compared to mock infected controls; whereas, in ndm strain infected spinal cord genes were differentially expressed compared to mock infection ( fig. a) . genes were differentially expressed between mice infected with dm and ndm strains. most differentially expressed genes were upregulated both in dm and ndm strain, with few genes downregulated. analysis revealed that both strains induced innate immune response genes. the majority of upregulated genes are known to be involved in pathways which activate microglia/macrophages and induce interferon-gamma, and thus may be involved in antiviral activities. detailed differential expression data is listed in supplementary table . briefly, genes involved in microglial activation like allograft inflammatory factor (aif ) that encodes iba , macrophage expressed gene , interferon-gamma inducible and activated genes including guanylate binding proteins (gbps), interferon-inducible gtpase, immunityrelated gtpase m proteins (irgms), interferon regulatory factors (irfs), various interleukins, chemokines and chemokine receptors and mhcs were upregulated. cd antigens like cd , cd , cd and cd were also upregulated in both dm and ndm strain infection. toll-like receptor (tlr) family members tlr , , , and and some nlr family genes were upregulated, most prominently nlr family member card domain containing that is implicated in anti-viral immunity and mhc class i expression regulation. overall, most innate immune response genes were upregulated by both ndm and dm, with a trend toward greater upregulation in ndm strain infection, but differences were not significant, except for one gene, gbp . qrt-pcr was performed on the same spinal cord rna used for microarray analysis in order to confirm whether observed changes in expression were significant. expression of selected genes upregulated during acute infection, along with β-actin as endogenous control, was measured. the relative fold change of mrnas in spinal cords of dm and ndm infected mice with respect to mock controls was calculated by the −ΔΔct method. similar to microarray data, qrt-pcr results showed upregulation of irgm ( . fold in dm/ . fold in ndm) and iigp ( fold in dm/ fold in ndm) when compared to mock-infection ( fig. b and c) . cxcl was also upregulated by both dm strain ( . fold) and ndm strain ( . fold) (fig. d ). cxcl was more highly upregulated by ndm strain ( . fold) than dm strain ( . fold) (fig. e ). gbp was also significantly upregulated in both dm and ndm infected mouse spinal cord during the acute stage of infection (fig. f ). among those genes only gbp showed significant differences between the dm and ndm strain ( ### p b . ). affymetrix microarray analysis was performed on extracts from spinal cords isolated on day p.i. genes that demonstrated significant differences in expression level were identified in comparisons between dm vs. mock, ndm vs. mock and dm vs. ndm infected spinal cords. using a threshold of fold change in expression level (p b . ), genes were found to be differentially expressed in dm strain infected spinal cord compared to mock infected controls; genes were differentially expressed in ndm strain infected mice compared to controls; and genes were differentially expressed in dm strain infected mice compared to both mock and ndm infection (fig. a) . differentially expressed genes were common to both dm and ndm strain infection. genes were unique in dm strain infection while unique genes were differentially expressed only in ndm strain infection. a complete list of differentially expressed genes during the chronic phase of infection is shown in supplementary table . interestingly, the genes differentially expressed in dm strain infected mice relative to both mock and ndm strain infection (fig. b ) did not show any differential expression during acute phase infection. ingenuity pathway analysis (ipa) demonstrates that these genes belong to pathways involved in switching of innate to adaptive immunity, including b cell development pathways and communication pathways between innate and adaptive immune systems (fig. a ). evaluation of these genes demonstrates that only genes have functional coding regions, mostly involved in b cell development: igha , ighg , ighm, igj, igk-v , igkv - , igkv - and slpi (fig. b ). further investigation showed that there is also upregulation of cxcl , an important cytokine for recruitment of b cells in the cns, in dm strain infected mice. expression of the igj gene in mock, dm and ndm infected spinal cord was measured by qrt-pcr as a representative gene to validate expression levels, and results confirmed similar expression as identified by microarray (data not shown). the genes that uniquely changed expression in chronic dm strain infection at day p.i. are listed in supplementary table . the canonical pathway analysis program in the ipa software demonstrates these genes are involved in b cell development pathways, t helper cell differentiation, cd signaling, t cell receptor signaling, and ctla signaling, among others ( fig. ) , with the majority implicated in b cell development pathways. ipa network analysis of these genes identifies two important pathways: b lymphocyte activation and ig class switching. the most striking interactions were observed between cd , cd , and mhc-ii, which in turn is able to promote b lymphocyte activation and ig class switching via cd signaling. similarly, ipa path designer network analysis demonstrates ig isotypic molecules overexpressed in dm infection are interconnected in a b cell development network (fig. ). because results show significant upregulation of genes involved in b cell development and ig class switching, antibodies were used to detect proteins produced by specific genes (igj, iga kchain, igha , ighg , ighm, igm and igg) found to be upregulated only in dm strain infection during the chronic phase of infection (day p.i.). protein levels in csf, where oligoclonal banding is seen in ms patients, was measured by immunoblotting, but only - μl of csf could be collected from the infected mice which was insufficient to detect any binding (data not shown). proteins levels were also not high enough in whole tissue lysates to detect meaningful expression above background (data not shown). immunohistochemistry of brain sections demonstrated small areas of increased igg (data not shown) and igm staining in the brain of dm strain infected mice as compared to ndm strain infected and mock infected mice (supplemental fig. ). in spinal cord (supplemental fig. ) , and other parts of the brain, more diffuse increased staining was also observed in dm strain infected mice, but high background staining levels require that these findings be interpreted cautiously. mouse cytokine assay analysis was performed on spinal cord tissue lysates to examine whether changes in protein expression correlate with observed gene expression changes following mhv infection. data show that at day - p.i.(acute phase) markers for innate immune responses, including mkc (kc/gro) and ifn-γ, are upregulated in both dm and ndm infected mice compared to controls ( fig. a and b), with protein levels modestly but significantly higher in ndm strain than dm strain infected mice, similar to microarray results. levels of both proteins significantly diminish by day p.i. cytokine assay for th and th cytokines il- and il- , respectively, also reveal significant upregulation following both dm and ndm strain infection ( fig. c and infection with both dm and ndm strains of mhv-a induce numerous innate immune response genes at the acute stage of infection. results in dm strain infected spinal cord are consistent with prior studies of acute infection [ ] , with current data showing that most of these genes are upregulated to a similar level, and one to an even greater degree, by ndm strain infection. one possible explanation for the observed induction of innate immune system genes by both viral strains may be an anti-viral immune response, as opposed to being involved in mechanisms of demyelination; that is, observed responses in both strains may reflect host attempts to clear the virus from the cns after acute infection. higher levels of innate immune system genes in ndm strain infection, if detected, may provide an explanation for viral persistence of the dm strain, but not the ndm strain, as observed in previous studies [ , , ] and reconfirmed in the current study with viral nucleocapsid specific primer probe set (data not shown). this supposition remains unclear, however, as only one gene (gbp ) was confirmed to be different by rt-pcr between strains, while several proteins (mkc, ifnγ, and il- ) did show significantly higher upregulation between strains. cytokine analysis revealed upregulation of il- that also promotes secretion of ifn-γ, itself known for its anti-viral activity and role in viral clearance, supporting the possibility that these genes are induced specifically to control viral replication and infection. in contrast, the absence of significant differences in many other genes that are upregulated by both strains suggests that other mechanisms are likely fig. b ) were analyzed by ingenuity pathway analysis software, and were found to be involved in six canonical pathways, including the b cell development pathway. the p-value associated with a canonical pathway is a measure of the likelihood that there is significant association between the genes differentially expressed in dm strain infection with the known set of genes involved in that given pathway. the p-value is calculated using the right-tailed fisher exact test. the p-value for a given process annotation is calculated by considering ( ) the number of focus genes that participate in that process and ( ) the total number of genes that are known to be associated with that process in the selected reference set. the more focus genes involved, the more likely the association is not due to random chance and thus the more significant the pvalue. similarly, the larger the total number of genes known to be associated with the process, the greater the likelihood that an association is due to random chance, and the p-value accordingly becomes less significant. the smaller the p-values are, the less likely that the association is random and the association is more significant. p-values b . indicate a statistically significant, non-random association and the threshold line representing the p value is . . canonical pathways are ranked according to their p value and represented as the blue bars. ratio indicates the number of genes differentially upregulated in our data set that overlap with the particular pathway listed. b. ten specific genes that are involved in the b cell development pathway and include various immunoglobulin isotypic genes were found to be upregulated in dm strain infection only. each gene, along with its significant fold-upregulation in dm strain infection is shown. involved in differential clearance; thus, our current mixed results imply that the degree of innate immune responses may only play a minor role in observed differences in viral clearance. during the chronic phase of mhv infection, the level of expression of innate immune response genes is reduced in both strains, more drastically in ndm strain infected mice. unlike acute innate immune response genes, genes involved in adaptive immune responses are upregulated only during the chronic stage of spinal cord infection, and only in mice infected with the dm strain. specifically, significant upregulation of ig genes occurs in the dm strain, and the top canonical pathways whose genes are implicated include the t helper cell signaling pathway, b cell development pathway, and communication between innate and adaptive immune cells. the upregulation of various ig isotypic genes in dm strain infection only in the chronic phase suggests they may play a role in the chronic stage pathology, including a possible role in demyelination or axonal loss, although future studies will be necessary to determine if these genes are indeed involved in mechanisms of demyelination, or whether their expression merely changes as a consequence of the disease. a potential relationship between demyelination and ig's and oligoclonal bands (ocbs), as found in csf and cns tissues of ms patients, has long been suspected [ ] . their presence has been cited as evidence of an infectious etiology [ , ] . though the antigenic targets of the igg's have been difficult to confirm, various studies reported reactivity of intrathecal igg's from ms patients to several myelin epitopes [ , ] as well as to oligodendrocytes themselves [ , ] . the current data is consistent with the possibility that viral infection may trigger cns demyelination and induce ig ocbs without leaving any markers to identify the specific underlying infectious agent. b cells can either be primed in the peripheral immune system with subsequent infiltration in the cns by cytokines like cxcl or alternatively, they can be activated and maintained locally in cns [ ] [ ] [ ] . histopathological analysis of dm and ndm strain infected mouse spinal cords, as seen in prior studies and again here, is consistent with observed patterns of viral rna persistence and changes from innate to adaptive immune responses. in ndm strain infection, when infectious viral particles resolve in the chronic stage, activated microglia return to a quiescent stage, but in dm strain infection, viral rna persists and inflammatory cells also remain in demyelinating plaques [ , ] . differences observed here in expression of adaptive immune response genes lead us to hypothesize that persistence of activated microglia/macrophages identified in prior studies may help initiate the adaptive immune response during chronic dm strain infection. interestingly, current changes in immune responses observed in rsa spinal cord infection are similar to important results reported following infection with the more virulent, yet related, strain mhv-jhm [ ] [ ] [ ] [ ] [ ] [ ] . several groups have shown that the extent of mhv infection plays a critical role in inducing changes in cytokine expression, and rapid viral growth may suppress lymphocytic responses. while a and jhm strains have many similarities, jhm is highly neurovirulent and follows different kinetics of accumulation of virus-specific cd + and cd + t cells. although activated microglia and macrophages make up the vast majority of early infiltrates, up to day following infection in both strains, t cells are most abundant during peak of the inflammation and thereafter in mhv-jhm, whereas in rsa few t cells are present at the peak of inflammation [ ] [ ] [ ] ] . thus, it was not clear whether mhv-a induced changes in immune response genes would be similar to those found in mhv-jhm infection, yet our current results suggests that there indeed may be similar time courses of innate and adaptive immune responses. future direct comparisons may be useful to further examine their similarities and differences. one potential limitation with the current study, as with all animal studies, is how well the findings translate to human disease [ ] [ ] [ ] . the potential translation here is enhanced by the fact that the model used mimics many of the histopathological features of ms, and by some published evidence suggesting ms may be triggered by a viral infection [ , , ] . thus, it might be expected that similar changes in immune regulation as found in our studies could occur in the serum and csf of ms patients in the early phase of the first attack of demyelination versus subsequent attacks in chronic ms. thus, understanding the timing and progressive phases of immune responses may indeed lead to identification of potential points to target with both current and novel immunomodulatory therapies. in addition, the mhv-induced model of cns demyelinating disease used in the current studies shares many histopathologic features with the human disease ms, as well other viral-induced and autoimmune ms animal models [ , [ ] [ ] [ ] [ ] . features include inflammatory cell (fig. a) were analyzed by ingenuity pathway analysis software, and are found to be involved in various canonical pathways, including t helper cell signaling and b cell development pathways. the p-value associated with a canonical pathway is a measure of the likelihood that there is a significant association between the genes differentially expressed in dm strain infection with the known set of genes involved in that given pathway. p-values b . indicate a statistically significant, non-random association and the threshold line represents the p value is . . canonical pathways are ranked according to their p value and represented as the blue bars. infiltration of cns predominantly in white matter, demyelination, and truncation and swelling of axons [ , ] . the best studied virusinduced models of cns demyelination in mice are the picornavirus tmev and certain strains of the coronavirus mhv [ , [ ] [ ] [ ] [ ] . while these models demonstrate similar histopathology, the pathogenesis of tmev-induced demyelination probably differs from that in ms, where persistent viral infection of the cns has not been demonstrated, as is seen with tmev [ ] . in contrast, some neurotropic mhv strains provide models in which viral infection of the cns is cleared and controlled by remarkably well-defined mechanisms [ , , ] . notably, mhv encephalitis carries a sequela of inflammatory demyelination in the absence of detectable pathogen gene expression. the elucidation of this apparent 'hit and run' demyelinating event may hold lessons for understanding ms. . network analysis of genes differentially expressed in chronic dm strain infection. genes that were significantly upregulated in spinal cords of dm strain infected mice were further analyzed using the path designer network analysis function of the ipa software to identify the most significant gene network implicated by the upregulated group of genes. results demonstrating upregulated ig isotypic molecules are interconnected in a b cell development network. nodes in the network represent the genes and the lines connecting the nodes represent the kind (direct is solid line; indirect is dotted line) of relationship/interaction existing between the genes. the intensity of the node color represents the degree of upregulation. genes in uncolored nodes were not identified as differentially expressed in our experiments and are integrated into the network by the ipa software indicating their relevance to the network. in conclusion, the current studies show that innate immune responses induced in the acute phase surrender to development of adaptive immune responses in the chronic phase of rsa dm strain infection, and suggest potential pathways that should be explored as possible precipitating events leading to demyelination. use of animals and all experimental procedures were reviewed and approved by the institutional animal care and use committee at the university of pennsylvania, philadelphia. animal protocols adhered to the guidelines of the united states national institutes of health office of laboratory animal welfare guide for the care and use of laboratory animals, th edition. all experimental methods were carried out in "accordance" with the approved guidelines of universities of pennsylvania, usa. recombinant isogenic dm strain of mhv, rsa , and ndm strain rsmhv were described previously [ , ] . rsa and rsmhv are isogenic except for the spike gene, which encodes an envelope glycoprotein [ ] [ ] [ ] . each strain also expresses enhanced green fluorescence protein (egfp) [ ] . four-week-old, mhv-free, c bl/ mice (jackson laboratory) were inoculated intracranially with % ld dose of rsa ( , pfu) or rsmhv ( pfu) as described previously [ , ] . mock-infected controls were inoculated with an uninfected cell lysate at a comparable dilution. animals were sacrificed (five mice per group) at day or (acute stage) and day or (chronic stage) post-infection (p.i.). we have shown in a series of prior studies that these vastly differing inoculation doses are used because they indeed result in in similar levels of viral replication, infection, liver pathology and viral spread. briefly, our previous studies demonstrated that different doses of the viral strains (rsa , , pfu versus rsmhv , pfu) do not affect neural cell infection and inflammation within the brain [ ] . the two very distinct and different doses of viruses used are based on % of the ld , where previously it has been demonstrated that both strains replicate at a similar rate in the brain and spinal cord and viral titers at day and day p.i. are almost the same [ , , ] . moreover, even at pfus inoculation, rsa can cause demyelination although the number and size of demyelinating plaques formed was less compared to an inoculum of , pfus [ ] . for microarray analysis, total rna was isolated from spinal cords of rsa , rsmhv and mock-infected mice using the qiagen rneasy mini kit and dnase treatment. cdna was synthesized using the ovation pico wta system v (nugen technologies), fragmented, and labeled with biotin followed by hybridization with affymetrix gene chip mouse gene . st array that contains , unique mer oligonucleotide features for , gene level probe sets. arrays were washed and stained using genechip fluidics station and hybridization signals were amplified using antibody amplification with goat igg (sigma-aldrich) and anti-streptavidin biotinylated antibody (vector laboratories). chips were scanned in genechip scanner g. cel files corresponding to scanned spots were created using command console software, and loaded into genespring software (agilent) to identify differentially expressed genes and for statistical analysis. background correction and normalization was performed using robust multichip average in genespring software. volcano plots were used to identify differentially expressed genes using unpaired -sample student t-tests taking p-values ≤ . and a fold change of ≥ . or ≥ as indicated. gene lists were then loaded into the ingenuity pathway analysis software (ipa) for network and functional analysis. total rna was isolated from spinal cord tissue of dm, ndm and mock-infected mice on day and day p.i. using a qiagen rneasy mini kit after transcardial perfusion. cdna was synthesized using high capacity cdna reverse transcription kit (applied biosystems). real time pcr was performed using taqman real time pcr master mix (applied biosystems) and taqman primer probe set (supplementary table ) in an abi prism sequence detection system. average ct values were calculated from triplicate reactions and were normalized with β-actin ct values. the relative fold change in dm and ndm infected mice with respect to mock-infected was obtained by calculating the − ΔΔct values and plotted. one way anova was used to determine the upregulation of genes in dm, ndm infected mice with respect to mock infected mice and between the strains. levels of significance were determined on the basis of multiple comparison testing. mg of flash frozen tissue was lysed in ml of lysis buffer (ripa buffer containing . % sds and . % triton x- with complete mini protease inhibitor cocktail tablets (# ; roche). spinal cord tissues were homogenized by trituration followed by sonication and centrifuged at , rpm for min at °c. protein concentration was measured by bca kit in a microplate reader (wallac victor multicounter; perkin elmer). mouse cytokine assay was performed for th and th cytokines using mesoscale discovery (msd) well multi-spot mouse th /th -plex ultrasensitive kit and il- ultrasensitive kit (msd biomarker assay system). brain and spinal cord homogenates were tested at μg and μg per well. after h incubation, plates were washed three times with pbs + . % tween- . μl of × detection antibody solution was added into each well and incubated for h with shaking at room temperature. plates were washed times with pbs + . % tween- . μl of × read buffer was added to each well. data was acquired in sector® imager and analyzed using msd workbench software. two way anova was used to determine the changes in protein expression in dm and ndm strain infected mice with respect to mock infected mice as well as between the strains. levels of significance were determined on the basis of multiple comparison testing. mice were sacrificed at day and day p.i. and perfused transcardially with pbs followed by % paraformaldehyde. liver, brain and spinal cord were harvested, embedded in paraffin, sectioned, and stained with hematoxylin and eosin [ ] . for evaluating demyelination, spinal cord sections were stained with luxol fast blue. to examine inflammation (microglia/macrophage upregulation), immunohistochemistry of brain and spinal cord sections was performed with iba antibody using the avidin biotin immunoperoxidase technique (abc kit, vector laboratories,) and , ′ diaminobenzidine substrate [ ] . half of liver and spinal cord tissues from each mouse used for rna extraction were also processed for histopathological analysis. data analyses were performed using genespring software version . (agilent technologies, inc., santa clara, ca). probe set signals were calculated with the iterative plier summarization algorithm; with the baseline to median of all samples used as the baseline option. data was filtered by percentile and a lower cut off was set at . the criteria for identifying a gene as being differentially expressed were set at ≥ -fold (for day p.i. data set) and n . -fold (for day p.i. data set) changes as indicated. statistical analysis was performed to compare groups using unpaired t-tests, with significance inferred by p-values less than or equal to . . volcano plots were used to identify differentially expressed genes using unpaired -sample student t-tests taking p-values ≤ . and a fold change of ≥ . or ≥ as indicated. the list of differentially expressed genes was loaded into ingenuity pathway analysis (ipa) . software (http://www.ingenuity.com) to perform biological network and functional analyses per software instructions. supplementary data to this article 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immunological comparison viral infections and demyelinating diseases enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system coronavirus spike proteins in viral entry and pathogenesis endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type spike-mediated entry syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus gliopathy of demyelinating and non-demyelinating strains of mouse hepatitis virus tissue specific optimization of haematoxylin and eosin stain: an experiment accomplished by varying the period of fixation and duration of stain. online journal of biosciences and informatics, online cytomorphological and cytochemical identification of microglia the authors declare no competing financial interests. key: cord- -xq rn j authors: wang, xinyu; li, chunqiu; guo, donghua; wang, xinyu; wei, shan; geng, yufei; wang, enyu; wang, zhihui; zhao, xiwen; su, mingjun; liu, qiujin; zhang, siyao; feng, li; sun, dongbo title: co-circulation of canine coronavirus i and iia/b with high prevalence and genetic diversity in heilongjiang province, northeast china date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: xq rn j to trace the evolution of canine coronavirus (ccov), stool samples from diarrheic dogs in northeast china were subjected to reverse transcription-polymerase chain reactions (rt-pcrs) targeting the partial m and s genes of ccov, followed by an epidemiological analysis. m gene rt-pcrs showed that . % ( / ) of the samples were positive for ccov; of the positive samples, ccov-i and ccov-ii accounted for . % ( / ) and . % ( / ), respectively. a sequence comparison of the partial m gene revealed nucleotide homologies of . %– % among the ccov strains, and . %– . % identity between the ccov strains and the chinese reference strain hf . the ccov-i and ccov-ii strains exhibited genetic diversity when compared with reference strains from china and other countries. the ccov strains exhibited high co-infection rates with canine kobuvirus (cakv) ( . %) and canine parvovirus- (cpv- ) ( . %). the ccov prevalence in diarrheic dogs differed significantly with immunization status, regions, seasons, and ages. moreover, s genes were amplified from the ccov-positive samples, including ccov-iia strains, one ccov-iib strain, and one ccov-i strain. a sequence comparison of the partial s gene revealed . %– % nucleotide identity among the ccov-iia strains, and . %– . % identity between the ccov-iia strains and the chinese reference strain v . the ccov-iia strains showed genetic diversity when compared with reference strains from china and other countries. our data provide evidence that ccov-i, ccov-iia, and ccov-iib strains co-circulate in the diarrhoetic dogs in northeast china, high co-infection rates with cakv and cpv- were observed, and the ccov-ii strains exhibited high prevalence and genetic diversity. canine coronavirus (ccov) was first recognized as an enteric pathogen of dogs in [ ] . ccov is a common infection in young dogs, particularly those housed in large groups [ ] [ ] [ ] [ ] . ccov is an enveloped, single-stranded, positive-sense rna virus, and it belongs to the family coronaviridae, subfamily coronavirinae, genus alphacoronavirus, species alphacoronavirus- [ ] . ccov consists of two distinct genotypes, ccov-i and ccov-ii; the ccov-ii viruses are further divided into two subtypes ccov-iia and ccov-iib [ , ] . the s protein of ccov is a glycoprotein peplomer on the viral surface, and it plays an important role in the induction of neutralizing antibodies, specific receptor binding, and cell membrane fusion. accumulating reports attributed that the increase in the severity of ccov infections in dogs and the emergence of ccov variants to potential recombination events within the s gene, which occur when a host is co-infected with different ccov types [ ] [ ] [ ] . therefore, ccov has received much attention as an emerging cause of infectious disease in dogs [ , [ ] [ ] [ ] [ ] [ ] [ ] . ccov infection is a leading causes of diarrhea in dog population in china. wang et al. ( ) reported that ccov-ii infections were very common in domestic dog, fox, and raccoon-dog populations in china [ ] . ma et al. ( ) reported the molecular characterization of the . -kb region of the ccov - strain [ ] . gao et al. ( ) reported the isolation and identification of a ccov strain from giant pandas in china [ ] . however, information about the epidemiology of ccovs in china is not available in the past five years. in the current study, we conducted a molecular epidemiologic investigation of ccov in heilongjiang province, northeast china. moreover, the genetic evolution and co-infection of the identified ccov strains were analyzed. our aim was to provide insights into the epidemiology and genetic diversity of the ccov strains circulating in northeast china. the animal experiment, sampling, was approved by the animal care and use committee of the harbin veterinary research institute, chinese academy of agricultural sciences, china. the sampling and data publication also were approved by animal's owners. the field study did not involve endangered or protected species. no specific permissions were required for locations of samples because the samples were collected from public areas or non-protection areas. a total of fecal samples were collected in the form of rectal swabs of dogs with diarrhea from animal hospitals in the harbin, daqing, and mudanjiang districts of heilongjiang province in northeast china from may to april , using . -ml commercial virus sampling tubes (yocon biological technology co. ltd., beijing, china). for all samples, animal age, animal breed, animal gender, collection date, and vaccination were recorded, respectively. of the samples, were collected in harbin, were collected in daqing, and were collected in mudanjiang. all rectal swab samples were stored at − °c, and they were also used for etiological investigations in our other studies [ , ] . after ml of fecal samples was centrifuged at , × g for min at °c, the supernatant of each sample was transferred to a . -ml eppendorf tube. viral rna was extracted from each sample using the tianamp virus rna kit (tiangen biotech co., ltd., beijing, china) according to the manufacturer's instructions. the extracted rna were stored at - °c. molecular detection of ccov was conducted using reverse transcription-polymerase chain reaction (rt-pcr) targeting a -bp fragment of the m gene of ccov that was described by pratelli et al. ( ) [ ] . the s gene fragments used for ccov genotyping/subtyping, including a -bp fragment of ccov-i, a -bp fragment of ccov-iia, and a -bp fragment of ccov-iib, were amplified using the primers el f/el r, s f/s r, and cepol- /tgsp- , respectively [ , ] . briefly, first-strand cdna was synthesized using random primers (six nucleotides) using moloney murine leukemia virus (rnase h-) reverse transcriptase (novoprotein scientific inc., shanghai, china) according to the manufacturer's instructions. in this study, the emeraldamp pcr master mix ( × premix) (takara biotechnology co., ltd., dalian, china), and the applied biosystems geneamp pcr system thermal cycler (thermo fisher scientific, waltham, ma, usa) were used for pcr amplifications of all target genes. other pcr amplification conditions were performed according to the protocols described by [ ] and erles and brownlie ( ) [ ] . the purified pcr products were directly subjected to sanger sequencing, and each sample was sequenced three times. sequence analysis was performed using the editseq program in the lasergene dnas-tar™ version . software (dnastar inc., madison, wi, usa). all nucleotide sequences generated in our study were submitted to genbank under accession numbers kt -kt for the m gene of the ccov strains, kt -kt for the s gene of the ccov-iia strains, kt for the s gene of the ccov-i strain, and kt for the s gene of the ccov-iib strain. for the phylogenetic analysis, partial sequences of the m and s genes of ccov strains, including ccov-i, ccov-iia, and ccov-iib strains, were retrieved from genbank. to construct the phylogenetic trees, a multiple alignment of all target sequences was performed using clustalx program version . . furthermore, phylogenetic trees of all target sequences were generated from the clustalx-generated alignments by mega . software using the neighbor-joining method [ ] . neighbor-joining phylogenetic trees were built with the p-distance model, bootstrap replicates, and, otherwise, the default parameters in mega . the ccov-positive samples were screened for cpv- , cakv, canine astrovirus (caastv), canine norovirus (cnov), canine bocavirus (cbov), and group a-rotavirus (crv-a) by either pcr or rt-pcr, followed by sequencing, according to previously described protocols [ ] [ ] [ ] [ ] [ ] . all nucleotide sequences generated in our study were submitted to genbank, and the accession numbers of the target genes of co-infected viruses are shown in s table. results the characteristics of the ccov-positive dogs, the genotyping of ccov strains, and the amino acid substitutions of m protein of ccov strains are shown in s table, and a further analysis of the ccov-positive samples is shown in table . nucleotide sequences of the partial m gene of the ccov strains identified in our study were shown in supporting information (s fig). of the samples, samples ( . %) were positive for ccov following rt-pcr amplification of the partial m gene, and the ccov-positive rates of the harbin, daqing, and mudanjiang districts were . %, %, and . %, respectively (table ). of the ccov-positive samples, ccov-i and ccov-ii accounted for . % ( / ) and . % ( / ), respectively; immunized animals and non-immunized animals accounted for . % and . %, respectively; . % ( / ) were collected from october to december, and . % ( / ) were collected from dogs aged from to months; the total co-infection rate of ccov with any viruses reached . %, and cakv and cpv- accounted for . % ( / ) and . % ( / ), respectively (table ) . of the ccov strains, only s gene sequences were successfully amplified, of which belonged to ccov-iia, one to ccov-iib, and one to ccov-i (s table) . nucleotide sequences of the partial s gene of ccov strains identified in our study were shown in supporting information (s fig). the sequence comparison of the partial m gene revealed nucleotide homologies of . %- % and amino acids homologies of %- % among the ccov strains, while nucleotide and amino acid homologies of . %- . % and . %- . %, respectively, were observed between the ccov strains and the chinese reference strain hf . of the ccov strains, the nine ccov-i strains exhibited . %- % nucleotide identities and . %- % amino acid identities, and the ccov-ii strains showed . %- % nucleotide identities and . %- % amino acid identities (table ) . a total of amino acid substitutions were found in the partial m protein (s table) ; the four substitutions, thr val, met ile, his asn, and gln lys, occurred in all identified ccov-ii strains ( / ), and two substitutions, ile met and asn his, occurred in all identified ccov-i strains ( / ) ( table ). the sequence comparison of the partial s gene revealed nucleotide homologies of . %- % and amino acids homologies of . %- % among the the ccov-iia strains, and nucleotide and amino acid homologies of . %- . % and . %- . %, respectively, between the ccov-iia strains and the chinese reference strain v (table ) . a phylogenetic analysis using the nucleotide sequences of the m gene revealed that the nine ccov-i strains formed two clusters (c and c ), while the ccov-ii strains formed six clusters (c -c ). the ccov-ii strains only exhibited a close relationship to one chinese reference strain, nj , and differed genetically from most of the reference strains from china and other countries (fig ) . a phylogenetic analysis using partial s gene sequences demonstrated that the ccov-iia strains were closely related to three reference strains, (japan), tn- (usa), and -iia (brazil), and differed genetically from reference strains from china and other countries (fig a) . the ccov-i strain hrb-a showed a close relationship to the reference strain from italy (fig b) , while the ccov-iib strain hrb-bb was closely related to reference strains from european countries (fig c) . in our study, of samples ( . %) were positive for ccov, and the ccov-positive rate differed among the three districts of heilongjiang province ( . % for harbin, % for daqing, and . % for mudanjiang). the ccov-positive rate in feces has been reported to be . % [ ] , the republic of korea [ ] , japan [ ] , albania [ ] , brazil [ ] , italy, belgium, the netherlands, germany, the united kingdom, spain, and france, respectively [ ] . these data demonstrated that ccov infections showed clear differences among the geographical regions. the total ccov-positive rate reported here was lower than that in a previous report in in china [ ] . in our study, ccov-ii, accounting for . % of the ccov strains, was the predominant ccov type in northeast china, which is line with most reports [ , , ] . mixed infections of canine enteric viruses frequently occur in diarrheic dogs. in our study, the total co-infection rate of ccov with one or more cakv, cpv- , and cbov strains was . %; single co-infections with cakv, cpv- , and cbov accounted for . %, . %, and . %, respectively. co-infections between ccov and cpv- have been documented in dog populations in western europe, japan, and albania [ ] [ ] [ ] . however, the high co-infection rate of ccov with cakv has not been reported in china and other countries. cakv had been reported to be associated with severe enteritis in a litter of puppies [ ] . it is speculated that the high prevalence of co-infections of ccov with cakv and cpv- may be associated with the occurrence of viral diarrhea in dogs in northeast china. further studies should be conducted to understand the effect of the high co-infection rate with cakv on the severity of clinical symptoms of ccov infections. in our study, ccov-positive rate showed clear differences among seasons and ages. the high prevalence of ccov in the feces of diarrheic dogs aged - months was partially validated in other studies [ , , ] . the high positive rate ( . %) of ccov was found between october and december, which may be associated with seasonal variations in northeast china. at present, most dogs in northeast china are vaccinated for cpv- , canine distemper virus, canine parainfluenza virus, canine adenovirus type , and canine adenovirus type . in our study, of the ccov-positive samples, the immunized and non-immunized animals accounted for . % and . %, respectively. this result demonstrated that vaccination for other canine viruses did not effect ccov infections in the dog population in heilongjiang province, northeast china. given the high prevalence and co-infection rates of ccov, although it is controversial whether ccov vaccines provide adequate immunity [ , ] , the immunization for ccov is recommended in the dog population in northeast china in the future. fragments of the m and s genes have been used to genotype ccov [ , , , ] . in our study, the ccov-i and ccov-ii genotypes of the ccov strains were successfully identified demonstrated that ccov-i, ccov-iia, and ccov-iib co-circulated in northeast china, and that ccov-iia strains predominated in the dog population. the current study is the first to reveal that ccov-i, ccov-iia, and ccov-iib strains co-circulate in northeast china, and that there are high co-infection rate with cakv and cpv- . cco-v-ii strains are predominant, and they exhibit genetic diversity. resulting data increase our understanding of the evolution of ccov strains circulating in northeast china, and they provide valuable epidemiological information for further studies of ccov. however, further studies are necessary to clarify the effect of co-infections and amino acid substitutions on the severity of the clinical symptoms of ccov infections. supporting information recovery and characterization of a coronavirus from military dogs with diarrhea canine coronavirus infections in japan: virological and epidemiological aspects an update on canine coronaviruses: viral evolution and pathobiology canine coronavirus, greece. molecular analysis and genetic diversity characterization cross sectional and longitudinal surveys of canine enteric coronavirus infection in kennelled dogs: a molecular marker for 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circulating in brazil between and identification and characterization of bocaviruses in cats and dogs reveals a novel feline bocavirus and a novel genetic group of canine bocavirus molecular epidemiology of canine norovirus in dogs from portugal phylogenetic analysis of astrovirus and kobuvirus in korean dogs m gene analysis of canine coronavirus strains detected in korea detection and genotyping of canine coronavirus rna in diarrheic dogs in japan detection and genetic characterization of canine parvovirus and canine coronavirus strains circulating in district of tirana in albania western european epidemiological survey for parvovirus and coronavirus infections in dogs efficacy of an inactivated canine coronavirus vaccine in pups safety and efficacy of a modified-live canine coronavirus vaccine in dogs identification of coronaviruses in dogs that segregate separately from the canine coronavirus genotype genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs canine coronavirus highly pathogenic for dogs canine coronavirus: not only an enteric pathogen isolation, tissue distribution and molecular characterization of two recombinant canine coronavirus strains key: cord- -gdra xhj authors: gibbons, henry s.; broomall, stacey m.; mcnew, lauren a.; daligault, hajnalka; chapman, carol; bruce, david; karavis, mark; krepps, michael; mcgregor, paul a.; hong, charles; park, kyong h.; akmal, arya; feldman, andrew; lin, jeffrey s.; chang, wenling e.; higgs, brandon w.; demirev, plamen; lindquist, john; liem, alvin; fochler, ed; read, timothy d.; tapia, roxanne; johnson, shannon; bishop-lilly, kimberly a.; detter, chris; han, cliff; sozhamannan, shanmuga; rosenzweig, c. nicole; skowronski, evan w. title: genomic signatures of strain selection and enhancement in bacillus atrophaeus var. globigii, a historical biowarfare simulant date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: gdra xhj background: despite the decades-long use of bacillus atrophaeus var. globigii (bg) as a simulant for biological warfare (bw) agents, knowledge of its genome composition is limited. furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. we characterized a lineage of bgwith a long history of use as a simulant for bw operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (wgs). results: archival strains and two “present day” type strains were compared to simulant strains on different laboratory media. several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. to trace the microevolutionary history of these isolates, we obtained wgs data for several archival and present-day strains and morphotypes. bacillus-wide phylogenetic analysis identified b. subtilis as the nearest neighbor to b. atrophaeus. the genome of b. atrophaeus is, on average, % identical to b. subtilis on the nucleotide level. wgs of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype. conclusions: our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation. bacillus atrophaeus is a soil-dwelling, non-pathogenic, aerobic spore-forming bacillus related to b. subtilis. for more than six decades, this organism has played an integral role in the biodefense community as a simulant for biological warfare and bioterrorism events (bw) and is commonly referred to by its military two-letter designation ''bg'' [ , ] . b. atrophaeus has served in studies of agent dispersal [ ] , decontamination simulations [ , ] and large-scale process development [ ] . in addition to its historical use as a bw simulant, it is currently in widespread commercial use as a surrogate for spore-forming bacteria [ , ] and is the basis of numerous assays for spore inactivation [ , ] . in addition to its role as a simulant, the organism plays an important role in the biotechnology industry as a source of restriction endonucleases and of the glycosylation inhibitor nojirimycin [ ] . the taxonomic placement of b. atrophaeus has changed dramatically over the years. originally isolated as b. globigii in (migula) as a variant of b. subtilis, it was originally distinguished from b. subtilis by the formation of a black-tinted pigment on nutrient agar and by low rates of heterologous gene transfer from b. subtilis [ ] . it has been alternately known as b. subtilis var. niger, b. niger, and has been confused with b. licheniformis [ ] . other than the formation of the dark pigment, it is virtually indistinguishable from b. subtilis by conventional phenotypic analysis [ ] , and the lack of distinguishing metabolic or phenotypic features has contributed to the confusionin the taxonomic placement of this organism. low interspecies dna transfer frequencies suggested substantial divergence [ ] . based onanalysis of comparative dna hybridization, phenotypicand biochemical tests, nakamura advocated that pigment-producing b. subtilis-like isolates should be classified as a distinct species termed b. atrophaeus [ ] . recently, more sensitive typing methods such as amplified fragment length polymorphism analysis showed that b. atrophaeus strains could be classified into two major biovars: var. globigii encompassing the classical, commonly used bg isolates, and var. atrophaeus encompassing other closely related yet genetically distinct strains [ ] . here we report the definitive molecular typing of several bgstrains using whole-genome sequences, and develop a plausible microevolutionary history of a commonly used lineage based on the accumulation of mutations over time and during transfer between laboratories.the selected strains span more than six decades of development, use, and transfer of bgbetween various institutions and laboratories and offer an unparalleled opportunity to investigate mutation under selection and drift over time. phenotypic analysis revealed substantial heterogeneity both between and within strains, even in type strains, while highthroughput metabolic profiling revealed metabolic ''enhancements'' to a population that had returned to the university of wisconsin (uw) from camp detrick in . whole-genome comparisons of single-nucleotide polymorphisms (snps), small insertion/deletion motifs (indels), and large-scale genomic architecture analysis by optical maps are combined to generate a plausible history of acquisition and use of operationally relevant strains by the american type culture collection (atcc) and by several laboratories within the biodefense community. finally, our analysisof mutation profiles revealed potential signatures of the deliberate selection of strains with properties of enhanced growth and spore yields, properties that were deemed desirable in a simulant [ ] . we also report genetic differences between strains in use in the biodefense community and the commercial sector that argue for adoption of a more uniform standard for b. atrophaeus as a simulant. strains and growth conditions b. atrophaeus strains and their sources are indicated in table . archival strains were maintained as spores in sterile soil at the university of wisconsin (figure ). the lineage, originally founded from the strain, was extensively passaged by serial transfer every - months on agar slants for years. unless otherwise indicated, strains were grown using lb agar plates, lb agar brothor tryptic soy agar containing % sheep's blood (sba, healthlink) at uc. spores were germinated by plating on lb media at uc. plates were examined by stereomicroscopy using indirect lighting and imaged usinga nikon smz with a total magnification of . colonies exhibiting distinct morphologies were repeatedly streaked to confirm stability of the phenotype. genomic dna was prepared from all isolates using the blood and cell culture dna midi kit for bacteria (qiagen) from ml overnight cultures in lb. baci -n was sequenced at the naval medical research center, while all other isolates were sequenced to . -fold coverage at the us army edgewood chemical biological center by massively parallel pyrosequencing on the roche/ gs-flx using the titanium reagent package. draft genome sequences of all isolates were assembled de novo using newbler [ ] (roche) and analyzed using both newbler and lasergene (dnastar, madison, wi). the vogel isolate was designated as the reference strain and was brought to completion using standard finishing techniques. the draft genome of bacillus atrophaeus var.globigii was finished at the department of energyjoint genome institute (jgi) using a combination of illumina [ ] and datasets [ ] . for this genome, we constructed and sequenced an illumina gaii shotgun library which generated reads totaling mb, which was combined with titanium standard library which generated reads totaling mb of data. all general aspects of library construction and sequencing performed at the jgi can be found at http://www.jgi.doe.gov/. the initial draft assembly contained contigs in scaffolds. the titanium standard data were assembled with newbler, version . . the newbler consensus sequences were computationally shredded into kb overlapping fake reads (shreds). illumina sequencing data wereassembled with vel-vet, version . . [ ] , and the consensus sequences were computationally shredded into . kb overlapping fake reads (shreds). we integrated the newbler consensus shreds, the illumina velvet consensus shreds and using parallel phrap, version sps - . (high performance software, llc). the software consed [ , , ] was used in the following finishing process. illumina data was used to correct potential base errors and increase consensus quality using the software polisher developed at jgi (alla lapidus, unpublished). possible mis-assemblies were corrected using gapresolution (cliff han, unpublished), dupfinisher [ ] , or sequencing cloned bridging pcr fragments with subcloning. gaps between contigs were closed by editing in consed, by pcr and by bubble pcr (j-f cheng, unpublished) primer walks. a total of additional reactions and shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. the total size of the genome is bp and the final assembly is based on mb of draft data which provides an average . coverage of the genome and mb of illumina draft data which provides an average coverage of the genome. the complete sequence and wgs were deposited at ddbj/ embl/genbank under accession numbers listed in table . the wgs versions described in this paper are the first versions, e.g. aefm . templated assembly of the remaining strains were mapped to the finished sequence using the gsmapper tool in newbler (roche). high-confidence mutations were selected from newbler ''hcdiffs'' calls (table s ) by applying additional selection criteria that mandated high quality scores in both reference and templated assemblies with . % of the sequencing reads differing from the reference, elimination of mutation calls associated with homopolymer tracts (with the exception of tracts that were formed by a deletion -see below), and a minimum coverage depth of with bidirectional sequence reads. finally, the raw reads from the isolate were mapped to the finished sequence to assess error bias in the process and to correct for residual sequencing errors in the finished sequence. accession numbers of the relevant wholegenome shotgun sequences are found in table . phylogeny was calculated using paup . b . fifty-eightnucleotide positions were used with gaps being treated as a '' th base'' and all characters assuming equal weight. one thousandbootstrap replicates were computed using a heuristic search with the optimal criterion set to ''parsimony''. the tree was created using stepwise addition. nineteen loci in which putative mutations were identified from the dataset were re-sequenced from pcr products by standard sanger dye-terminator methods. no false-negatives or false-positives were identified among the re-sequenced loci; however resequencing of the apparent mutation at position revealed mixed genotypesin several isolates that are artifacts of a large duplication in the chromosome. therefore, this signalcannot be considered a true snp. annotation, comparative genomic analysis, and multiple alignments preliminary annotations were generated using a combination of the rast [ ] algorithm (rast.nmpdr.org). loci containing mutations were used to query the non-redundant (nr) databases and refseq protein databases at ncbi using directed blastx and blastp. the comparative blast tool from rast was utilized for genome-wide protein sequence comparisons to b. subtilis. results were filtered for bidirectional hits. multiple alignments were generated by megalign from the lasergene software package using the custalw algorithm. genomic dna was prepared from live bacteria on agar slants to maximize the yield of extremely high-molecular weight dna. optical maps were generated by digestion with ncoi of dna arrayed linearly on glass slides and the resulting maps were aligned and compared with the mapsolver software package (opgen, inc., gaithersburg md). using an information-based method for genomic classification [ ] , the sequence contigs from bg isolates , - and were analyzed in order to map the phylogenetic relationships of these isolates to other bacillus species. in this method, genomic content is characterized by the frequencies of occurrence of short n-mers contained within each sequence (n typically from to ). these n-mers are then rank ordered by genome. the pair-wise comparison of the rank of n-mers within two different genomes is then used to compute an informationbased genetic distance (ibgd), where the sum of the differences in rank for all possible n-mers is weighted by an entropy factor that depends on the frequencies of occurrence of the respective n-mers in the two genomes. the pair-wise ibgd values are then used to construct a phylogenetic network [ ] . bacilli genomes were obtained from genbank. this method for phylogenetic characterization enables computation even with the unassembled reads, and it can be applied to draft or partial genome sequence data, which was the case for the three b. atrophaeus genomes studied here. the first seven bgstrains listed in table were streaked for single colonies on bhi plates and incubated at uc overnight, followed by subculturing a second time under the same conditions. subsequently, cell suspensions were prepared according to biolog specifications, with od readings ranging between . - . at nm. biolog phenotypic microarray plates pm through pm , were inoculated according to the manufacturer's specifications, and incubated at uc for hours. readings were taken every minutes, and data processed by omnilog phenotype microarray file management/kinetic plot and parametric modules. two biological replicates of the experiment were conducted for each strain. pm - contain single wells for each growth condition whereas pm - contain quadruplicate wells for each condition. the area under the curve (auc) values were computed by adding all omnilog values at all time points for each of the distinct phenotypes produced from the omnilog software. the auc values from the two different biological replicates for each unique phenotype were averaged. the ratio for each auc was calculated between the query strains (detrick- , detrick- , detrick- , - , - , and dugway) and reference parent strain ( ) . for the purpose of visualization, phenotypes were included in the heatmap (i.e. this better represents the locations of the phenotypes which correspond to different modes of action categories). the same ratios were used for the phenotypes that have replicates. the ratio values were formatted as pm to pm for each strain across the columns and wells a i to h i , where i = to for the rows. the results were plotted in a heatmap using r [ ] . positive growth wells are represented by greenblocks while negative growth wells are represented by red blocks. catalase activitywas assayed by spotting drops of hydrogen peroxide ( %) onto isolated colonies on lb agar plates. colonies were monitored for bubble formation, signifying the release of water and oxygen. a colony was considered to be catalase positive by observation of bubbles. streaks of detrick , detrick , and strains were grown for two days on tsa plates containing sba.bacterial cell mass was scraped using an inoculating loop ( ml) from the streak and resuspended in pbs. sporulation was evaluated by bright field phase-contrast microscopy. phase-bright free sporesand phasedark vegetative cellswere counted. five representative viewing fields were counted from each strain for each experiment. this experiment was completed in triplicate by repeating once per day over the course of three consecutive days. in order to compare the percent sporulation between detrick and detrick , and detrick and , a mixed analysis of variance (anova) was used to complete the analysis. strain and viewing field were evaluated as fixed factors, and replicate was included as a random factor. the natural log of the percent sporulation was taken to obtain a normal distribution of the residual error. tukey's method was applied to compare the difference between the mean log percent sporulation. we traceda potential provenance of the commonly used bgstrains through an exhaustive search of the open literature and the archives of the university of wisconsin,which suggested a possible lineage from which the ''military'' bgstrains were derived. the original source of the strains were the collections at the university of wisconsin during the s and s, from which the strains were transferred to camp detrick at the initiation of the us army's bw program at the beginning of the second world war [ , ] . at camp detrick, bg was used as a non-pathogenic surrogate in process development for sporeforming bacteria it is tempting to speculate that the university of wisconsin supplied bg to porton down: a note found in the archive of dr. baldwin's papers, dated february , , contained an order from dr. fildes (presumably sir paul fildes, a noted bacteriologist active in the british bw program at the time), for a batch of b. subtilis spores. it is not clear whether bg or b. subtilis subsp. subtilis was supplied, or whether this material was actually delivered. unfortunately, original records describing in detail the maintenance of the strains during the period - were destroyed as per us army policy at the time (dr. mark wolcott, usamriid; personal communication), and the personnel who had first-hand knowledge of the strain passage histories and methods are deceased. therefore, the actual source of the camp detrick isolates must be inferred from published work [ ] , limited available documentation (e.g. atcc ) and the genome sequences presented herefrom camp detrick the isolates were eventually transferred to atcc as b. subtilis var. niger ''red strain.'' the desire to maintain a phenotypically and genotypically uniform simulant throughout the biodefense communityprompted us to elucidate whether significant phenotypic and/or genomic differences had accumulated in any of the commonly used isolates during the growth and transfer of strains to different institutions and to compare the isolates in broad use today to the so-called ''mil-spec'' strain (atcc ).in contrast, the origin of atcc prior to acquisition f. young's laboratory (the depositor) is unclear. we obtained isolates from archival spore suspensions in sterile soilfrom the university of wisconsin with legible labels dating back as far as ( figure ; table ). these isolates included an archival stock dated that likely predated the transfer to camp detrick, as well as material that had been returned to the university of wisconsin from camp detrick in . a derivative of the strain that had been repeatedly passaged in vitro on agar slants over a period ofapproximately years allowed us to compare the genomic signatures of deliberate selection with the effects of long-term in vitro passage. in addition, a sample of strain nrs- [ ] , which is mentioned as a possible parent strain in correspondence between various academic laboratories and camp detrick, was also obtained from the same source as the ''vogel'' strain. these isolates were subsampled, germinated on lb plates, screened for colony morphology variation (see below). genomic dna was prepared from these isolates for sequencing. upon initial plating of the archival and modern-daybg stocks, we noted distinct colony morphotypes for many of the strains, with some strains containing multiple variants ( figure , table ). some of these morphotypes were consistent with those observed by hayward et al. [ ] whooriginally described the emergence of colony variants in ''b. globigii.'' as in the earlier report, individual morphotypes were stable and did not interconvert with high frequency (data not shown), suggesting that these morphotypes were the result of relatively rare chromosomal mutations, although - occasionally threw off papillae in heavier streaks (not shown). multiple morphotypes were noted for atcc , atcc , detrick, and , while the archival isolate, the isolate obtained from dugway proving ground (dugway) and baci appeared to be pure populations on lb. all strains tested positive for bgusing real time-pcr primers specific to the recf gene (methods s ) [ ] . the appearance of multiple colony morphotypes even within single ''strains'' strongly suggested an asyet undescribed level of genetic diversity within these samples that likely affected the expression of cell-surface components and/or sporulation. the intra-strain colony morphology variation was particularly dramatic in the in vitro passaged and atcc isolates, in which one variant of each lineage had lost the production of color on lb orsbaplates (figure ), suggesting more dramatic alterations to the genome. draft genome sequences were generated from several bgstrains in our collection. a summary of the results from the sequenced isolates is indicated in table . all of the ''military'' isolates (detrick clones through , baci , dugway) were extremely closely related to each other and to both atcc variants. the atcc isolates possessed additional mutations that were absent in the ''military'' isolates. the size of the finished and closed genome of b. atrophaeus var. globigii was , , bp, and annotation using rast [ ] revealed features, including proteincoding sequencesand rna molecules [ ] . the preliminary annotations derived from rast are available as genbank .gbk files in the supplementary material. on average, the genome of b. atrophaeus is approximately % identical to b. subtilis on the nucleotide level,supporting its delineation as a distinct species and agreeing well with previous estimates [ ] . analysis of the ibgd using whole-genome sequences (n-mer length . ) supported the identification of b. subtilis as the closest relative among sequenced bacterial genomes ( figure ). for this particular case, n = (i.e., there were = total -mers used to compute the ibgd). the ibgd values were relatively insensitive to the choice of n over the range of - . thethree bggenomes analyzed grouped closely together, and our analysis of the bacillus-wide phylogeny using ibgd revealed the phylogenetic distance of that b. subtilis/b. atrophaeus species from b. anthracis, supporting the inferences published elsewhere from rrna sequence analysis ( figure ) [ ] . primary amino acid sequences of rast-annotated proteins are on average % (median %) identical between b. atrophaeus and b. subtilis. when only the proteins that yielded bidirectional blast hits in rast are examined, the predicted proteome of b. atrophaeus is, on average, % identical ( % median) to b. subtilis. we utilized the finished sequence of the isolate as a reference strain for templated assembly of the remaining bg draft sequences. two additional atcc isolates of b. atrophaeus ( and ) were distinguishable from var. globigii on the basis of very high snp/indel counts, lower coverage ofand percentage of reads mapping to the reference, and unique genomic features which supported their proposed classification as var.atrophaeus [ ] . the distinguishing genomic features of var.atrophaeus strains and the delineation of the b. atrophaeus clade from b. subtilis will be published elsewhere. optical restriction mapping [ , , ] was used to compare the overall genomic structure of selected isolates. no differencesin overall genome architecture between the ''military'' bg isolates, the archival isolate, or - were observed (figure , data not shown), suggesting that the global architecture of these isolates is relatively stable, even over years of serial in vitro passage.however, the optical maps and sequence coverage analysis of - and - revealed substantial deletions of approximately , and , bases, respectively, of genomic materialspanning from positions , , to , , ( - ) or , , - , , (atcc - ) (figure ; table s ). the genes within this deleted region are listed in table s but notably contain genes encoding for nitrite reduction, germination (gerkabc), and biosynthesis of the lipopeptide surfactin (srfcab) [ , ] . a defect in surfactin production is a particularly intriguing candidate for the morphology and pigmentation variations in - and atcc - , since disruption of srfa has been shown to have dramatic effects on spreading motility on semisolid media, on biofilm formation [ , ] , and low-grade hemolytic activity. using the de novo assembled draft sequence from the isolate as a template for subsequent analysis of snps and small indels in the other ''military'' isolates, we generated a list of highconfidence, discriminatorymutations that differentiate the strains ( figure a ). the nature and annotation of the mutations are found in table and can be assigned an approximate temporal order in which they occurred ( figure b ). based on this analysis, is the most likely parental strain for all of the isolates in this study, with the lineage diverging earliest, followed by , then the ''military'' lineage prior to the transfer to camp detrick. this conclusion is based on the observation that shares three snps with detrick- . the latter is the most likely progenitor of the other ''military'' isolates, since it has the fewest mutations relative to strain . detrick- can be differentiated from other ''military'' isolates by possessing the parental allele of spo f rather than the h r allele (position ) that is characteristic of all of the other ''military'' bgisolatesand the atcc strains. the two colony morphology variants of atcc each exhibited distinct mutation profiles indicating that the reference strain is in fact a mixed population of at least two genetically distinct substrains. the kb deletion in - included the structural genes for biosynthesis of surfactin, a cyclic lipopeptide with a mild hemolytic activity [ ] . to test whether the ''military'' and in vitro passaged strains possessed low-grade hemolytic activity, we streaked these variants on rich agar media containing % sheep's blood and looked for hemolysis. to our surprise, all strains exhibited striking variation in their coloration (figure a) , with the , - and detrick- isolates considerably darker on blood agar than the other ''military'' and in vitro passaged isolates. in addition, on lb the - and - isolates appeared white and off-white, respectively. pigmentation of b. subtilis colonies is associated with production of a melanin-like pigment by the cota protein, a major component of the spore coat [ ] . in addition to the variations in pigmentation, streaks of the and detrick- isolates were consistently translucent under transillumination ( figure b ). these zones of translucency are suggestive of weak b-hemolysis, which has previously been observed in b. subtilis strains that produce high levels of surfactin [ , , ] . the other strains exhibited either weak a-hemolysis or none at all, with the exception of the strongly hemolytic - variant. at least in the ''military'' lineage, the quasi-hemolytic phenotype and darkbrown colony pigmentation correlated with the presence of a wildtype spo f allele, suggesting that the ability of b. atrophaeus to lyse red blood cells may be regulated in part by spo f. however this was not universally the case; the baci strain had two discernible variants on sba (not shown), one of which appeared to have recovered partial hemolytic activity ( figure b ). to gain insight into the effects of genetic divergence of the adapted isolates on their metabolic capacity, the detrick isolates and the separate isolates were compared by multiphenotype analysis using the omnilog system, which allows the highthroughput comparison of discrete growth conditions, including carbon, nitrogen, phosphorus, sulfate, nutrient supplements, ph, osmolytes as well as a broad class of growth inhibitors. the growth of the strain was used as a reference for determining relative growth rates of the other strains. the results of mutations exhibiting high quality scores in both reference and query sequences and with differences from the template exhibited in . % of the individual sequencing reads are indicated as a blackened box. in one case (position in atcc - ) an initial false-negative due to the formation of a homopolymeric tract was found by direct inspection of the assemblies. the genes whose functions are altered by the given mutation are indicated in table these experiments are summarized in figure and table s . in general, growth of the isolates was significantly diminished relative to the in many different growth conditions, most notably in the ability to use amino acids and peptides as carbon and nitrogen sources, to withstand osmotic stress, and to grow under reduced ph. in addition, the strains had developed sensitivity to beta-lactams, quinolones, and membrane-disrupting activities. these results suggested broad combined effects of several mutations on the phenotype of the strains. in addition to the spo f(a p) allele, which is a likely candidate for highly pleiotropic effects on the decision to sporulate under many different conditions, both strains contain substitutions in the yetf and yqge genes that may be contributing to the phenotypes observed. the more pronounced defect in - may be attributable to defects in the gerab and gerac genes and the large kb deletion which contains several genes involved in germination. by contrast, the detrick isolates in general grew more robustly than the strain under multiple growth conditions. increased relative growth rates were particularly pronounced for acidic conditions and media containing osmolytes, but particularly for wells containing sodium lactate [ ] . another isolate in the ''military'' lineage, dugway, is clearly derived from the detrick lineage by snp/indel profiling yet has a metabolic profile that is much closer to the parental strain. like the detrick isolates, the dugway strain grows better at low ph, but many of the other conditions do not promote elevated growth relative to . only one mutation differentiates that isolate from the detrick- isolate -a -bp insertion in the yojo gene encoding a putative activator of nitric oxide (no) synthesis. again, the physiological role of this mutation is unclear, although nitric oxide synthesis plays a critical role in modulating antibiotic resistance in bacillus spp. [ ] . in addition to its role in promoting resistance to antibacterial drugs, no is known to modulate b. subtilis genes involved in nitrate respiration when oxygen is limited [ ] ; thus the lowered growth in this strain may reflect the inability to grow to higher densities and overcome the resulting lower oxygen tension. an additional isolate, baci is clearly derived from dugway, yet two variants have accumulated additional mutations in sigh (spo h), hpr/scoc, and ebrb. notably, the phenotype of baci -e on plates more closely resembles the strain ( figure b ). sequencing of the ''military'' isolates revealed a frameshift mutation in the kata gene encoding the major vegetative catalase [ ] . the absence of catalase activity in ''military'' isolates was confirmed by adding a solution of % h o to smears of various strains. in contrast to the strain, which exhibited immediate and robust catalase activity, the strains containing the frameshift lacked this activity. a small amount of bubbling could be seen, probably due to the presence of a second catalase normally packaged in spores [ ] . to test whether the phenotype observed on blood agar was associated with differences in sporulation, selected strains were grown for two days as patches on blood agar, resuspended in pbs annotations are a combination of rast and directed tblastn and blastp searches vs bacillus databases. forms part of a large polypeptide synthase containing highly homologous regions. also shared with strain atcc . the conserved start codon of the rada gene (bg ) of b. atrophaeus and b. subtilis falls within the bg orf. therefore bg as called by rast is not deemed likely to be a protein-coding gene. in putative transmembrane region. doi: . /journal.pone. .t table . cont. and counted directly. strain detrick- exhibited significantly higher percentages of phase-bright spores than the detrick- strain (figure , mean +/ standard error of the mean). similar results were observed for the and dugway strains (not shown). the - strain exhibited an even higher degree of sporulation than the detrick- strain under identical conditions ( figure ). bacillus atrophaeus has historically been grouped with b. subtilis, and is usually described as a black-pigmented variant (var. niger) because of its many phenotypic similarities to the bettercharacterized b. subtilis. both organisms are soil-dwelling, nonpathogenic saprophytes, but have been differentiated by the ability to produce pigment on nutrient media containing an organic nitrogen source [ ] . the orange pigmentation of b. atrophaeus var.globigii spores made it an attractive simulant for b. anthracis, facilitating the detection of dispersed spores in complex environmental samples. recently, more sensitive phylogenetic approaches using aflp have delineated b. atrophaeus as a separate species [ , ] . the taxonomic confusion has arisen due to inadequately sensitive typing methods, and has led to misattribution of pathogenic qualities associated with some b. licheniformis strains to the b. atrophaeus strains currently in use as simulants [ ] , for which no direct evidence of pathogenicity exists. this report defines the genomic composition of b. atrophaeus var.globigii and clearly separates the species by wholegenome phylogenetic analysis. in this study, we generated a high-quality, closed reference genome for the isolate using a combination of , illumina, and directed sanger sequencing. we expect the final genome to have an error rate of , in , basepairs. when we mapped the datasets for all of the isolates back to the finished sequence that was generated using the same dna, we noted several putative snps that were common to all datasets (table ) . we believe these represent errors introduced during generation of the final consensus sequence, as they did not appear when the isolates were mapped against draft sequence generated exclusively using the platform; these are currently being verified and the final sequence will be updated. our sequences of multiple, closely related strains of this organism allow us to trace the derivation of the ''military'' bg isolates currently in use to a culture present at camp detrick during the s and s. the origin of atcc is not as clear, but a publication from that era suggests a possible common origin at the university of wisconsin [ ] . while that strain is unlikely to be nrs- itself, given the presence of several strain-specific snps in our sequence, the snps common to both and the ''military'' lineage suggest a common ancestor that is not represented among the strains sequenced for this study. given the lack of original records, it is unclear whether the nrs- variant in this study might have passed through camp detrick and been returned to the university of wisconsin. however, given the date on the label and the general secrecy of operations at camp detrick during the second world war [ ] we consider this possibility unlikely. during development of bgas a simulant for b. anthracis, strains were selected that exhibited the most desirable characteristics, those being rapid growth, high spore yield, and experimental reproducibility. without being aware of the nature of the genetic alterations in their ''optimized'' strains, bw workers at camp detrick selected a mutant that provided dramatically higher total and relative spore yields, and generated consistent experimental results [ ] . these strains were adopted into the inventories of numerous biodefense laboratories and have been used for many figure . the spo f(h r) and spo f(a p) alleles are associated with hypersporulation. phase-contrast microscopy of bg strains after two days of growth on sba. vegetative cells appear as phase-dark rods, while spores appear as round, phase-bright globules. the mean percentage sporulation of each strain in a representative experiment is given sem. the experiment was repeated on three consecutive days; representative results of a single experiment are shown. statistical significance was determined by mixed anova (tukey's method, p, . ). doi: . /journal.pone. .g figure . omnilog phenotypic arrays of b. atrophaeus subsp. globigii strains. six strains were each inoculated into twenty -well omnilog plates and grown at uc. reduction of tetrazolium dye by respiring cells was measured every minutes by optical density. dye reduction relative to the strain is shown; the red ratio values indicate less respiration while the green ratio values indicate more respiration as compared to the strain. individual arrays or strains are displayed in each of the six major columns labeled detrick , detrick , detrick , - , - , and dugway. a) heat map of all conditions for each strain. each of the twenty plates for each strain is represented by the notation pm -pm (left-toright for each strain) along the x-axis. the rows represent the well position, and are denoted as a i to h i (i = to ) from the bottom to the top of the plot in each array along the y-axis. each cell ratio value represents the average of two biological replicates for each strain. plates pm -pm contains single wells for each growth condition, while plates pm -pm contain quadruplicate wells for each growth condition. solid circle indicates wells containing sodium lactate; dotted circle indicates well containing l-serine at ph . . the details of the growth conditions can be found in the first worksheet labeled ''all strain auc data'' in table s . b) most significant phenotypes for each of the six test strains as compared to the strain. the phenotypes with statistically significant increases and/or the decreases in ratio values for each of the six strains are presented. for the isolates only the conditions giving the five largest changes are presented. the number in each color block indicates the ratio for the test strain relative to the parent strain for the phenotype specified. the details of all significant phenotypes for each test strain can be obtained in table s . bold italic font indicates p, . . doi: . /journal.pone. .g decades in simulations of decontamination and dispersal [ ] . by applying a combination of genomic and biochemical profiling techniques, our data demonstrate that the bg isolates were ''enhanced'' by researchers at camp detrick during the development of the organism as a simulant. the selection of a strain with the desired properties appears to have occurred in at least two discrete steps, as shown by the genome sequences and metabolic profiles. the initial step appears to have been the adaptation of a strain to growth in corn steep liquor, an acidic medium rich in protein and lactate [ ] . the robust growth of the detrick strains relative to in low-ph medium containing high lactate levels is likely due to mutations in mmgd ( -methylcitrate synthase, position ), or a short-chain -oxoacyl-[acyl-carrierprotein] reductase (position ), or both. the most likely candidate for a mutation in the detrick isolates that increases growth is the frameshift in mmgdthat occurred following the divergence from the lineage and results in an altered c-terminus ( figure s ). the mmgd geneencodes a -methylcitrate synthase that is expressed in the mother cell at the intermediate stages of sporulation [ ] . a null mutation in mmgd had no perceptible effect on sporulation, although other tca-cycle enzymes when mutated led to a loss of sporulation [ ] . the effects of the frameshift mutation on sporulation and cellular physiology on the function of the enzyme are not clear at this time. we speculate that the frameshift mutation alters the substrate specificity of mmgd in favor of citrate, thus increasing the flux of lactate-derived intermediates through the tricarboxylic acid cycle. evidence for this possibility includes the observations that -methylcitrate synthases can have partial citrate synthase activity [ ] and that the b. subtilis mmgd gene can complement a glta (citrate synthase) mutant of e. coli [ ] . alternatively, alteration of function of mmgd may have predisposed the lactate-adapted strain to acquisition of a hypersporulating phenotype, which is not readily isolated or stable in b. subtilis (see below); however the presence of a hypersporulating phenotype in an independently evolved lineage ( ) of bg indicates that the species may have an intrinsic predisposition to evolving such a phenotype in vitro. the ''military'' strains also grow more readily on media containing d,l-diaminopimelic acid (meso-dap), a major component of bacterial peptidoglycan. corn steep liquor is derived from the incubation of corn in water at - uc, during which a lactic fermentation by a community of wild organisms including numerous uncharacterized bacillus spp. occurs. total bacterial counts at the conclusion of csl production can be quite high [ ] , thus the availability of such compounds for growth is not surprising. another potential source of meso-dap could be bacterial autolysis during sporulation. the relative roles of each of the alleles in growth on lactate and/or meso-dap is the subject of current investigation in our laboratory. the second step in the development of bg as a simulant appears to have been the deliberate selection of a hypersporulating variant [ , ] . importantly, the selection of a strain optimized for spore yield resulted in the fixation of a new spo f allele that has no counterpart among the available spo f sequences (figure ). the sole spo fsequence that differs at position is that of b. clausii, in which tyrosine replaces histidine. notably, the spo f(h r) mutation is distinct from a separate spo f(a p) mutation present in the in vitro passaged isolates. given that the amino acid sequence of b. atrophaeus spo f is identical to that of b. subtilis but for two conservative substitutions, it is likely to have very similar if not identical biochemical properties. detrick- and likely represent one of the two r colony morphotypes described by hayward et al. [ ] , whereas the hypersporulating f morphotypes likely arose due to the emergence of the spo f(h r) mutation. however, the possibility that detrick- represents a reversion mutant at this locus from detrick- cannot formally be excluded, but since it represented the dominant morphotype in the detrick vial we believe this is unlikely. the presence of the spo f(h r) allele in the atcc strains suggests that these strains were acquired by atcc after this mutation appeared within the detrick lineage. experiments to verify the roles of each allele in modulating sporulation are currently in progress. preliminary results indicate that transformation of b. subtilis dspo f with b. atrophaeus dna and selection of spo+ cells dramatically alters colony morphology independently of the spo f allele introduced; additional studies to verify the effects of each allele are currently in progress (james hoch, personal communication). the h r and a p allelesare likely to alter the response to signals promoting sporulation. aspo f(h a) allele results in a sporulation-proficient strain that throws off sporulation-deficient papillae [ ] , and the same mutation has been shown to suppress the spo phenotype of a strain containing a defective kina allele. h has been proposed as a potential metal-binding site with particular affinity for cu + [ ] . binding of cu + (or another divalent metal) at this site may modulate interaction with one or more sensor kinases that promote sporulation. substitution of positively charged arginine at this position could potentially mimic the binding of a metal cation in the loop containing h , resulting in altered sporulation of the strains due to a change in the interaction with the kinases governing sporulation. it is unclear why, given the proposed role of divalent cu + in suppressing sporulation, h r would result in a hypersporulation phenotype. the mechanistic relationship between spo f(h r) and the hypersporulation phenotype will be tested in future experiments. both variants in the lineage possess an a p allele in spo f. although the presence of several other mutations within this lineage confounds the attribution of the hypersporulating phenotype to this allele at this time, the presence of a mutation in the same gene as another hypersporulating mutant is highly suggestive. the effect of proline substitution at position on spo f functionis not immediately obvious, but the relatively inflexible proline residue can disrupt alpha-helices in protein structures. the - lineage exhibits a hypersporulating phenotype even more pronounced than spo f(h r) strains in the ''military'' lineage. the observation that hypersporulating phenotypes have emerged during cultivationof two independent b. atrophaeus lineages point to the possibility that certain in vitro selection pressures may actually favor hypersporulating variants. the selection pressures acting on the sporulation pathwayare highlighted by the sheer number of mutations discovered within the entire data set that occur in proteins known to play roles in sporulation. nine of the mutations ( %) found in all lineages were in genes that directly or indirectly regulate either entry into stationary phase or sporulation; this number exceeds the number that would be expected if mutations were to occur by chance, since less than % of b. subtilis genes are dedicated to regulatory processes of any kind [ , ] . in addition to the mutations found within the ''military'' lineage, the two variants of atcc shown in figure differ by mutations in rpob (table s ) which also plays a role in entry into sporulation [ ] . null mutations in spo f resulting in asporogenous phenotypes contribute to colony morphology variation in b. anthracis, b. thuringiensis and b. subtilis [ , , ] . enhanced in vitro ''fitness'' is also a likely driver behind the recovery of asporogenic b. anthracis mutants that were discovered during the investigation into the b. anthracis attacks of [ ] . because the process of sporulation is highly energy-intensive and irreversible once commenced, mutants that delay sporulation (or fail to sporulate altogether) to take advantage of remaining nutrients would out-compete wild-type cells during repeated passage in vitro in the absence of other selection pressures, as has been demonstrated in extended in vitro evolution studies with b. subtilis under relaxed sporulation conditions [ ] . this may not be universally the case, since gain-of-function mutations in sporulation such as those observed in this studymay compete favorably with wild-type cells if cannibalism of vegetative cells by sporulating bacteria is the dominant selective pressure [ ] . finally, horizontally transferred genetic elements can have dramatic effects on sporulation: for example, recent studies of phage lysogeny in b. anthracis have revealed the ability of several integrated phages to positively affect the kinetics of sporulation upon lysogeny of commonly used b. anthracis strains [ ] . this study identifies the spo f(h r) allele as the signature of a deliberate selection during the development of b. atrophaeus as a simulant. however, without the knowledge of the history and the analysis of the phenotypes of the strains originating from ''camp detrick'' as published in the open literature, attribution of this genotype to a deliberate selection event would not have been definitive, since a similar phenotype is observed in the lineage which to our knowledge was not deliberately selected for any specific trait. any study designed to determine genomic ''signatures'' of deliberate enhancement or selection is likely to require an analysis of the baseline likelihood that mutations conferring a similar phenotype would emerge and become fixed by natural processes within an evolutionary timeframe consistent with a known time interval or number of passages. available evidence suggests that hypersporulation is not easily evolved in vitro. maughan and coworkers attempted to evolve populations of a laboratory strain of b. subtilis with a hypersporulating phenotype by repeatedly heat-shocking cultures. while their efforts to enrich for hypersporulators failed, other studies revealed that asporogenous mutants evolved readily [ , ] , confirming many early studies ( [ ] and references therein). with the exception of the studies by maughan et al., most ofthese investigators applied selections intended to inhibit sporulation rather than to enrich for strains with elevated sporulation rates. the lineage was never heat-shocked during its many transfers; thus the adaptations seen in this work are the result of balancing sporulation versus vegetative growth for prolonged periods on agar slants. however, because undomesticated isolates were observed to sporulate to - % [ ] , we cannot formally exclude the possibility that in vitro culture of the strain following its isolation for an unknown period by the university of wisconsin might have selected for a hyposporulating variant. in this scenario, the h rand a p mutations would represent suppressor mutations. we consider this possibility unlikely, given the phenotypic similarity of two environmental isolates in the uw collection ( and nrs- ). furthermore, a progression toward darker pigmentation and greater hemolysisis evident in the ''military'' lineage ( figure b ). these phenotypic changes are associated with the accumulation of additional mutations including a p l substitution mutation in sigh, a positive regulator of sporulation [ , ] and an a p mutation in scoc, a negative regulator of sporulation [ ] . together, the strains analyzed in this study suggest strong selective pressures on the genes in the sporulation pathway, and more carefully controlled studies should be carried out to determine the dynamics of in vitro evolution and adaptation of spore-forming organisms, as has been done extensively in e. coli [ , , , , ] . unexpectedly, the ''military'' lineages were also marked by the loss of catalase activity, whose presence is an identifying feature of both b. subtilis and b. atrophaeus [ ] . this activity was present in a separate lineage of in vitro passaged organisms, so it is not immediately clear why ''military'' isolates, i.e. those subjected to selection within the early days of the development of bg as a simulant organism, would have lost the catalase activity characteristic of the parental isolate. because the kata gene product is not found in spores [ , ] , we consider it unlikely that the absence of this activity would impact the resistance of spores to decontamination reagents, and thus any antioxidant resistance phenotype exhibited by spores of ''military'' isolates would likely have gone unnoticed. however, direct comparisons of the ''military'' b. atrophaeus lineages to the progenitor strains have not been done, and pleiotropic effects of a spo f mutation on spore physiology cannot currently be excluded. whole-genome approaches are becoming critical components of microbial forensics. the snps and indels identified in the analysis of evidentiary materials currently become the basis for higherthroughput assays to screen large numbers of samples [ , ] . decreasing costs of whole-genome sequencing, and the comprehensive nature of the analysis, may make this the preferred method of forensic analysis of microbial samples in the future. with recently developed techniques of allele quantitation within populations by mass spectrometry [ ] , real-time pcr [ ] , and census-bysequencing [ , ] , it may be possible to quantitate accurately rare alleles within any given microbial population. we are particularly intrigued by the possibility that, given a mixture of different variants and sufficient sequencing power, ultra-high coverage sequencing may prove to be a more quantitative means of enumerating the relative populations in a sample even before the presence of variants has been established. the results from sequencing two strains of baci in this study provide evidence of such hidden diversity. the genomic basis of interlaboratory strain variation is only beginning to become evident, with recent studies tracing the histories of commonly used lab strains of b. subtilis , e. coli, salmonella enterica serovar typhimurium s, pseudomonas aerugi-nosapa and mycobacterium tuberculosis h rv [ , , , , , ] . these have revealed significant divergence of putatively identical strains from one laboratory to another, largely arising from mutations that accumulate during serial passage. like the earlier work, our study highlights the utility of approaches based on wholegenome sequencing for the discrimination of closely related strains, especially when investigating the provenance for a given isolate. tragically, at least institutions are known to have destroyed archival collections of select agents [ ] following the implementation of mandatory monitoring and reporting requirements, representing an incalculable loss of phenotypic and genomic diversity. this report underscores the importance of maintaining the genetic heritage preserved in the culture collections of individual investigators and institutions. discovery of phage display peptide ligands for speciesspecific detection of bacillus spores a miniature biochip system for detection of aerosolized bacillus globigii spores anthrax letters: personal exposure, building contamination, and effectiveness of immediate mitigation measures the sterilizing action of gaseous ethylene oxide; sterilization of contaminated objects with ethylene oxide and related compounds; time, concentration and temperature relationships virulent spores of bacillus anthracis and other bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants strain variation as a factor in the sporulating properties of the so-called bacillus globigii difference between the spore sizes of bacillus anthracis and other bacillus species comparative sterilization effectiveness of plasma in o -h o mixtures and ethylene oxide treatment biological indicator for ethylene oxide sterilization, paper strip. the united states pharmacopeia/the national formulary. rockvillemd: us pharmacopeia characterization of bacillus subtilis dsm and its production of -deoxynojirimycin interspecies transformation in bacillus: sequence heterology as the major barrier advisory panel for the study of long-term health effects of participation in project shad ( ) long-term health effects of participation in project shad (shipboard hazard and defense) taxonomic relationship of black-pigmented bacillus subtilis strains and a proposal for bacillus atrophaeus sp. nov detection of molecular diversity in bacillus atrophaeus by amplified fragment length polymorphism analysis genome sequencing in microfabricated high-density picolitre reactors velvet: algorithms for de novo short read assembly using de bruijn graphs base-calling of automated sequencer traces using phred. ii. error probabilities base-calling of automated sequencer traces using phred. i. accuracy assessment consed: a graphical tool for sequence finishing finishing repeat regions automatically with dupfinisher the rast server: rapid annotations using subsystems technology genomic classification using an information-based similarity index: application to the sars coronavirus application of phylogenetic networks in evolutionary studies r: a language and environment for statistical computing the biology of doom: the history of america's secret germ warfare project evaluation of the biological sampling kit (biskit) for large-area surface sampling the national microbial pathogen database resource (nmpdr): a genomics platform based on subsystem annotation reclassification of bioindicator strains bacillus subtilis dsm and bacillus subtilis dsm as bacillus atrophaeus identifying experimental surrogates for bacillus anthracis spores: a review optical mapping as a routine tool for bacterial genome sequence finishing optical mapping and sequencing of escherichia coli o : h isolates linked to the us spinach-associated outbreak optical maps distinguish individual strains of escherichia coli o : h identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in bacillus subtilis srfa is an operon required for surfactin production, competence development, and efficient sporulation in bacillus subtilis rapid surface motility in bacillus subtilis is dependent on extracellular surfactin and potassium ion cota of bacillus subtilis is a copper-dependent laccase cloning and characterization of srfb, a regulatory gene involved in surfactin production and competence in bacillus subtilis endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics induction of resde-dependent gene expression in bacillus subtilis in response to nitric oxide and nitrosative stress alkyl hydroperoxide reductase, catalase, mrga, and superoxide dismutase are not involved in resistance of bacillus subtilis spores to heat or oxidizing agents the katx gene, which codes for the catalase in spores of bacillus subtilis, is a forespore-specific gene controlled by sigmaf, and katx is essential for hydrogen peroxide resistance of the germinating spore autolysis of cell walls of bacillus subtilis corn steep liquor in microbiology a sigma e dependent operon subject to catabolite repression during sporulation in bacillus subtilis citrate synthase and -methylcitrate synthase: structural, functional and evolutionary relationships development of processes for the production and use of bacillus globigii spores as a simulant molecular recognition in signal transduction: the interaction surfaces of the spo f response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis structural analysis of divalent metals binding to the bacillus subtilis response regulator spo f: the possibility for in vitro metalloregulation in the initiation of sporulation the complete genome sequence of the gram-positive bacterium bacillus subtilis sporulation of bacillus subtilis isolation and characterization of sporulation-initiation mutation in the bacillus subtilisprfb gene transduction of sporogenesis in bacillus subtilis analysis of the morphological variants arising during s isolation of an asporogenic (spooa) protective antigen-producing strain of bacillus anthracis whole-genome typing of bacillus anthracis isolates by nextgeneration sequencing accurately and rapidly identifies strain-specific diagnostic polymorphisms the roles of mutation accumulation and selection in loss of sporulation in experimental populations of bacillus subtilis cannibalism by sporulating bacteria the secret life of the anthrax agent bacillus anthracis: bacteriophage-mediated ecological adaptations stochastic processes influence stationaryphase decisions in bacillus subtilis the population genetics of phenotypic 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environment formation and composition of the bacillus anthracis endospore strain-specific single-nucleotide polymorphism assays for the bacillus anthracis ames strain high-throughput oncogene mutation profiling in human cancer rapid quantification of single-nucleotide mutations in mixed influenza a viral populations using allele-specific mixture analysis detecting snps and estimating allele frequencies in clonal bacterial populations by sequencing pooled dna high-precision, whole-genome sequencing of laboratory strains facilitates genetic studies tracing ancestors and relatives of escherichia coli b, and the derivation of b strains rel and bl (de ) genome diversity of pseudomonas aeruginosa pao laboratory strains genomic sequencing reveals regulatory mutations and recombinational events in the widely used mc lineage of escherichia coli k- short-term signatures of evolutionary change in the salmonella enterica serovar typhimurium variation among genome sequences of h rv strains of m. tuberculosis from multiple laboratories destruction of microbial collections in response to select agent and toxin list regulations we thank dr. kevin p. o'connell for helpful discussions and insights into the manuscript and for facilitating the collaboration with the university of wisconsin. we also thank kristin willner and amy butanifor assistance with sequencing. we thank gary ouellette for help with phylogenetic analysis and drs. mark wolcott (usamriid) and james hoch (scripps) for helpful discussions. the opinions presented here are those of the authors and are not necessarily those of the u.s. government or any of its agencies. information in this report is unclassified and cleared for public release. key: cord- -qzd hf y authors: alhatami, abdullah o.; alaraji, furkan; abdulwahab, husam muhsen; khudhair, yahia ismail title: sequencing and phylogenetic analysis of infectious bronchitis virus variant strain from an outbreak in egg-layer flocks in baghdad, iraq date: - - journal: vet world doi: . /vetworld. . - sha: doc_id: cord_uid: qzd hf y background and aim: infectious bronchitis (ib) has an influential economic impact on the poultry industry, causing huge losses each year due to the condemnation of infected chickens. despite the use of many kinds of vaccines in iraq, it is common to find ib problems in vaccinated chickens. information about the strains that affect iraqi chickens is very limited. therefore, we aimed to detect the currently circulating strains of ib virus that cause frequent outbreaks in egg layers despite the use of vaccination against the virus. materials and methods: isolate detection, sequencing, and phylogenetic analysis were performed using a rapid ib virus antigen kit ( tracheal swabs), flinders technology associates (fta) card ( tracheal swabs), and partial gene sequencing ( positive fta samples). results: the isolated strain was different from other strains, especially the strain isolated in the north of iraq (sulemania strain) and shares % homology with an israeli strain (israel variant , is ). conclusion: although more studies are needed to detect ib virus strains circulating in iraq, this work lays the foundation for making a good strategy to control the disease and selecting vaccines that should be used in farms. an important viral disease affecting the poultry industry globally and locally in iraq is the infectious bronchitis virus (ibv), which causes an acute, highly contagious respiratory disease. the identification of endemic ibv strains promotes better control of the disease and prevents production losses [ ] . ibv belongs to the family coronaviridae and the genus gamma-coronavirus. the single-stranded rna (positive-sense, ~ kb) of this enveloped virus has several open reading frames [ ] . although the nucleocapsid (n) gene is highly conserved among coronaviruses, the spike (s) glycoprotein (particularly s ) gene contains highly variable components in these viruses [ ] . ibv usually is subjected to changes due to mutations and genetic recombination. therefore, a mutation in the s gene may affect virus tissue tropism and virulence [ ] . consequently, ibv can spread widely to affect different regions of the world, creating huge difficulties in controlling the disease due to these newly modified strains [ , ] . even with high numbers of the globally identified genotypes or serotypes of ibv, cross-protection generated by those strains is barely present [ ] . the highly variable s gene represents the ideal genetic target for monitoring viral evolution occurring in ibv, especially in strains with high rates of serotype correlations [ ] . therefore, sequencing the s gene of ibv strains detected in the field is critical for control programs and epidemiological purposes. the spike protein is a major structural protein of amino acids that are cleaved into -aa-sp (s , n-terminus) and -aa-sp (s , c-terminus). the s gene induces serotype-specific based antibody neutralization through its two hypervariable regions [ ] . many polymerase chain reaction (pcr)based techniques have been developed to monitor the prevalence of ibv by targeting and analyzing the s gene. reverse-transcriptase pcr (rt-pcr) and nucleotide sequencing of the s gene are routinely used to detect ibv genotype; in addition to real-time quantitative pcr (rt-qpcr), which also is used to identify ibv from field samples. these molecular methods are highly sensitive and specific compared with other diagnostic methods [ ] [ ] [ ] . since its discovery in , and despite massive routine vaccination, ibv continues to be one of the major concerns in the poultry industry worldwide. this may be due to many reasons, one of which is the continuous emergence of new ibv strains [ ] with unique genetic determinants identified in each geographical area. to the best of our knowledge, only one study to date reports the molecular characterization of ibv that resulted in a broiler strain (sul/ / ), identified in the kurdistan region of iraq. therefore, the current report describes the role of ibv during an outbreak of the respiratory disease in an egg-layer farm in the baghdad region of iraq, investigates the genetic characteristics of this field strain by analyzing the s gene and compares it with other isolates registered globally for developing significant vaccines to control this disease. this study does not require ethical approval. however, all birds in the current work were treated humanely following international and national criteria of animal care and use. an outbreak occurred during january in al-janoob company, located in the al-wahda district of baghdad, iraq. this was composed of eight caged houses of egg layers with a total number of , hens and , rearing pullets. the outbreak was not reported to the national authority. all flocks were vaccinated with commercial live-attenuated nobilis ib - . respiratory symptoms began in -day-old birds of an isa brown flock composed of , birds per house. clinically, the symptoms were suspected to be ibv infections. necropsies were performed, and gross lesions were evaluated. sixty-four tracheal swabs (two swabs per bird) were collected during january , from birds that showed such respiratory manifestations. thirty-two tracheal swabs were used for serological diagnosis of ibv antigen using a rapid ibv ag test kit (rg dd, bionote, korea), which is a chromatographic immunoassay for qualitative ibv antigenic detection in avian swab samples. tests were performed according to the manufacturer's instructions. samples were pooled on flinders technology associates (fta) cards (whatman ® fta ® card technology) with four sample areas per card (total homogenate volume up to μl) containing chemicals for lysing cells, denaturing proteins, and protecting nucleic acids against damage from nucleases, ultraviolet radiation, and oxidation [ , ] . fta cards containing viral rna were outsourced to anicon labor gmbh (muehlenstraße a hoeltinghausen, germany), where the extraction of the ibv rna was performed using kylt ® rna/dna purification kit through the manufacturer's protocol. rt-pcr runs were generated by anicon labor gmbh. briefly, species-specific and variant-specific rt-pcr methods were performed to detect avian coronavirus (acov, including ibv) and ibv variants. hybridization probe-based chemistry was used with the following primers: and kylt ® ibv-ib (anicon labor gmbh). rt-pcr, cfx , and cfx (bio-rad, hercules, ca, usa) systems were used according to the following conditions: °c for min and °c for min (initial denaturation), then cycles of °c for s, °c for min, and read. the amplified rt-pcr products were sequenced by anicon labor gmbh. sequences of the iraqi-ibv-strain s gene were deposited in the genbank database available from the national center for biotechnology information (ncbi) website to collect accession numbers. ibv s gene sequences obtained herein were compared with sequences of ibv deposited globally in the genbank database using the ncbi-based blast search. the identities of the sequences were analyzed by dnastar software (dnastar, madison, wi, usa; https://www.dnastar.com/), and molecular evolutionary genetics analysis (mega) x software (https://www.megasoftware.net/) were used to construct the phylogenetic tree. the flock suffered from signs of infectious bronchitis (ib) disease, and the mortality rates reached % for approximately days. birds suffered the following symptoms: coughing, sneezing, and rales with depression. postmortem findings included tracheal congestion, and caseous materials that partially obstructed the trachea and the tracheal bifurcations, and pneumonia and fibrinous airsacculitis ( figure- ). kidneys were enlarged and congested and contained an accumulation of urate in the nephrons and ureters. thirty-two ( %) of the examined samples were positive for the presence of the ibv antigen. viral rna from the fta cards (four spots pooled) with the sample number (a . ) showed positive results with species-specific and ibv-variant-specific rt-qpcr (table- ) . available at www.veterinaryworld.org/vol. /july- / .pdf a -bp and -bp fragments of the s protein-coding gene were sequenced by anicon labor gmbh. the sequences were deposited in the ncbi genbank under accession numbers mh and mh with the name acov strain iq and iq spike glycoprotein (s ) gene, respectively. the genetic relationship between the s gene sequence of the mh . ibv-iq strain and sequences of vaccine and other virulent strains are presented in figure- and table- . the mh . strain was closely related ( % similarity) to eu . _ibv_isolate_is/ / (israeli variant ), whereas the percentage of sequence identity was % compared with gq . ibv_isolate_ sul/ / _s (sulaimania isolate). moreover, the nucleotide similarity was % and % compared with fj . ibv strain h and af . ibv strain / , respectively. ib has an influential economic impact on the poultry industry, causing huge losses each year due to the condemnation of infected chickens [ ] . since ibv was identified during the early s, ibv ( / type) has frequently been reported in europe and many countries around the world [ ] . ibv vaccine strains can perform recombination with field strains, reversing virulence [ ] . such characteristics have encouraged verification of the relationship between vaccine and field strains [ ] . although a /b-serotypebased attenuated vaccine is available, some /b-serotype viruses remain active in europe and several countries [ ] , and the live-attenuated or killed massachusetts (mass) strain-dependent vaccines are most widely used for vaccination programs throughout the world. nevertheless, there is an increased failure of ibv vaccination programs, particularly against the / ibv strain, in addition to the circulation of many ibv-vaccine-related strains [ , ] . most of the ibvvariant strains have distinctive characteristics due to their global distribution; however, some strains are unique to certain regions, and these properties occur for unknown reasons. the current work represents the first identifying and genotyping report of ibv that resembles the israeli isolate in iraq. the results of the sequencing and phylogenetic tree analysis indicate that our isolate shares % homology with the israel variant (is ). in , zana et al. [ ] reported a newly isolated strain along with other regionally identified isolates from other israel strains (is/ / , is/ ), and waleed et al. [ ] also registered strains /b and mass from infected broilers in the south of iraq. thus, isolates from different parts of iraq demonstrate large differences in homology. in addition, in the sulaimaniah isolate (sul/ / ), the birds displayed nephron pathological lesions, and the virus was detected from kidney samples but not from tracheae. therefore, the sulmania (iraqi strain) that affected broilers differs genetically from our strain that infected layers. furthermore, our isolate shared < % and % in nucleotide sequence with vaccine strains fj . and kx . , respectively, which may explain the occurrence of infection despite vaccination. therefore, our work lays the main foundation including rna-based s protein transcript sequencing and the related phylogenetic analysis, to initiate launching strategies for control of ibv in the field. despite the intensive vaccination program in iraq, ib outbreaks have occurred for many decades. in this study, we isolated a strain of ibv very different from that of local north iraq (sulemania strain) but similar to an israeli strain (israel variant , is ). genetic characterization of the iraqi circulating ibv strains is critical because of inadequate data from this location. more studies revealing such strains will pave the way for a good strategy of controlling the infections and understanding or developing types of vaccines that should be used in the future. aoa and hma visited the infected farm, collected the samples, and performed the rapid ibv antigen detection. fa and yik visited the infected farm, collected the samples, run the rapid ibv antigen detection, confirmed the results using fta card that was sent to germany, deposited the genetic information into the ncbi database, analyzed the differences between the strains of the virus, and drafted the manuscript. all authors revised the manuscript. all authors have read and approved the final manuscript. pathogenesis and diagnostic approaches of avian infectious bronchitis changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification delmarva (dmv/ ) infectious bronchitis virus (ibv) variants isolated in eastern canada show evidence of recombination using phylogenetic analysis to examine the changing strains of infectious bronchitis virus infections in ontario over time a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain the avian coronavirus spike protein comparison of sybr green i realtime rt-pcr with conventional agarose gel-based rt-pcr for the diagnosis of infectious bronchitis virus infection in chickens in morocco the first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in algeria genotyping of avian infectious bronchitis virus in afghanistan identification of a novel recombinant virulent avian infectious bronchitis virus evaluation of full s gene sequencing of classical and variant infectious bronchitis viruses extracted from allantoic fluid and fta cards genotypes of infectious bronchitis viruses circulating in the middle east between prevalence of avian infectious bronchitis virus in broiler chicken farms in south of iraq infectious bronchitis virus variants: molecular analysis and pathogenicity investigation progress and challenges toward the development of vaccines against avian infectious bronchitis molecular and antigenic characterization of gi- and gi- avian infectious bronchitis virus isolated in chile from to regarding / vaccine introduction molecular characterization of pathogenic / -like and qx-like infectious bronchitis virus infecting commercial poultry farms in indonesia vaccine or field strains: the jigsaw pattern of infectious bronchitis virus molecular epidemiology in poland isolation and molecular characterization of sul/ / avian infectious bronchitis virus, indicates the emergence of a new genotype in the middle east prevalence of avian infectious bronchitis virus in broiler chicken farms in south of iraq the authors thank anicon laboratory in germany for their assistance in the diagnosis of the virus. the work was performed in the college of veterinary medicine, university of al-qadisiyah, available at www.veterinaryworld.org/vol. /july- / .pdf diwaniyah city, iraq, and the anicon labor gmbh (muehlenstraße a hoeltinghausen, germany). the authors did not receive any funds for this study. the authors declare that they have no competing interests. veterinary world remains neutral with regard to jurisdictional claims in published institutional affiliation. key: cord- -npefpo t authors: yinda, claude kwe; zeller, mark; conceição-neto, nádia; maes, piet; deboutte, ward; beller, leen; heylen, elisabeth; ghogomu, stephen mbigha; van ranst, marc; matthijnssens, jelle title: novel highly divergent reassortant bat rotaviruses in cameroon, without evidence of zoonosis date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: npefpo t bats are an important reservoir for zoonotic viruses. to date, only three rva strains have been reported in bats in kenya and china. in the current study we investigated the genetic diversity of rvas in fecal samples from straw-colored fruit bats living in close contact with humans in cameroon using viral metagenomics. five (near) complete rva genomes were obtained. a single rva strain showed a partial relationship with the kenyan bat rva strain, whereas the other strains were completely novel. only the vp and vp genes showed significant variability, indicating the occurrence of frequent reassortment events. comparing these bat rva strains with currently used human rva screening primers indicated that most of the novel vp and vp segments would not be detected in routine epidemiological screening studies. therefore, novel consensus screening primers were developed and used to screen samples from infants with gastroenteritis living in close proximity with the studied bat population. although rva infections were identified in % of the infants, there was no evidence of zoonosis. this study identified multiple novel bat rva strains, but further epidemiological studies in humans will have to assess if these viruses have the potential to cause gastroenteritis in humans. rva/bat-wt/ken/ke / /g p [ ] , and possesses the following genotype constellation: g -p[ ]-i -rx-c -mx-ax-n -t -e -h . two other bat rvas were found in china in a lesser horseshoe bat (rhinolophus hipposideros) and a stoliczka's trident bat (aselliscus stoliczkanus) named rva/bat-tc/chn/mslh / / g p [ ] and rva/bat-tc/chn/myas / /g p [ ] , respectively , . phylogenetic analysis showed that strains mslh and myas , although sampling sites were more than km apart, shared the same genotype constellation (g -p[x]-i -r -c -m -a -n -t -e -h ) except for the p genotype which was p [ ] for mslh and p [ ] for myas . to further study the genomics of rva in bats and their zoonotic potential in humans, we screened stool samples of straw-colored fruit bats (eidolon helvum) living in close proximity with humans in the south west region of cameroon (fig. ) , as well as samples from infants with gastroenteritis. our choice of this region is due to the fact that bats are considered a delicacy and the species sampled are the most commonly eaten bat species in these localities. sample characterization. a total of pools of - bat fecal samples were constituted, enriched for viral particles and sequenced. illumina sequencing yielded between . and . million reads per pool, and diamond classification of the obtained contigs indicated that five pools contained a significant amount of rva sequence reads. the percentage reads mapping to rva in each pool ranged from . - . % (table ) . partial segments were completed by regular pcr and sanger sequencing, to obtain at least the entire orf for each of the obtained variants. obtained sequences were used for phylogenetic comparison with a selection of representative members of each genotype. the rva strains discovered in this study were named rva/bat-wt/cmr/batli / / g p [ ] , rva/bat-wt/cmr/batli / /g p [ ] , rva/bat-wt/cmr/batli / /g p [ ] , rva/bat-wt/ cmr/batly / /g p [ ] and rva/bat-wt/cmr/batly / /g p[ ] hereafter referred to as batli , batli , batli , batly and batly , respectively. all the obtained sequences were highly divergent from established genotypes and were therefore submitted to the rotavirus classification working group (rcwg) for novel genotype assignments (see below) and to genbank (accession numbers: kx -kx ). phylogenetic analysis. the vp gene of batly was % identical (on the nucleotide (nt) level, supplementary data s ) to the kenyan bat rva strain ke counterpart, which had been previously classified as a g genotype (fig. ). batli and batli were % identical and also clustered closely with strain batly ( % similar). this cluster was only distantly related to all other known vp rva sequences as well as to strain batli , which also formed a unique long branch in the phylogenetic tree. both clusters only show similarities below % with established genotypes (fig. ) . the vp of these strains (batli , batli , batly and batli ) did not belong to any of the established rva g-genotypes, according to the established criteria , and were assigned genotypes g (batli , batli and batly ) and g (batli ) by the rcwg. for vp , vp and vp , all five cameroonian bat rvas strains were distantly related to other known rva strains, including the kenyan and chinese rva strains and were therefore assigned to novel genotypes according to the rcwg classification criteria (fig. ) . the vp gene of strains batli , batli and batli (representatives of the novel genotype p[ ]) were almost - % identical to each other and only - % identical to any other p-genotype. that of strains batly and batly had % nt dissimilarity to each other and their nt identity ranged from - % with other p-genotypes and therefore were assigned the genotypes p[ ] and p[ ], respectively. the vp and vp genes of batli , batli , batli and batly were nearly identical (nt identity range - %) and clustered together but distinct from other established r and m-genotypes, thereby representing the new genotypes r and m , respectively (fig. ) . the vp and vp genes of batly were only distantly related to the other four cameroonian bat rvas ( - % nt identity) and are the sole member of the newly assigned genotypes r and m , respectively (fig. ) . the vp , vp , nsp , nsp and nsp gene segments of of our strains (batli , batli , batli and batly ) were distantly related to their counterparts of other mammalian and avian rvas fig. ) . for all the strains, these gene segments clustered together and were - % identical to each other and consequently they constitute new genotypes for the different gene segments (i , c n , t and h , respectively). the vp , vp , nsp , nsp and nsp gene segments of batly phylogenetically clustered together with the kenyan bat rva strain ke in the previously established i , c , n , t and h genotypes, respectively (fig. ) . for nsp , the cameroonian bat strains batli , batli , batli and batly clustered closely together ( - % nucleotide sequence identity) in the novel genotype a , and showed only % nucleotide similarity to strain batly (a ). these new nsp gene segments were only - % identical to that of the most closely related established nsp genotype a and a (from cow) (fig. ) . the nsp gene segments of all the rvas discovered in this study were quite divergent to those of other known bat rotaviruses (at most % nucleotide sequence identity) and other rvas (approximately - % nucleotide similarity) forming two distinct clusters. the nsp gene segments of strains batli , batli , batli and batly (genotype e ) were % identical but all were - % divergent from that of batly (e , fig. ). batli (p ) batlyp (p ) batli (p ) batli (p ) batlyp (p ) bat rotaviruses in humans? several different primer pairs are currently being used to detect human rva vp and vp gene segments, to determine the g-and p-genotypes using sequencing or multiplex pcr assays [ ] [ ] [ ] [ ] . in order to find out if the currently used human rva screening primers would detect the bat rva strain from this study in case of zoonosis, we compared these primers with their corresponding sequences in the respective gene segments (table and supplementary data s ). overall, the similarity percentages for the vp forward and reverse primer between the bat rva sequences and the human primers were . - % and . - %, respectively. for vp , the percentage similarity ranged from . - . % and . - . % for the forward and reverse primers, respectively. vp forward primers beg , sbeg and con -l showed a (near) perfect match with batly -g , whereas strain batli -g and batly -g (first nt are missing for this strain), showed up to and nucleotide mismatches at the ′ end of the primers, respectively. vp forward primer con -l showed a perfect match with all the genotypes (g , g and g ). considering the vp reverse primers enda, vp -rdeg, end and rvg , batly -g did not show a perfect match as there were , , and mutations, respectively. the mismatches with enda, vp -rdeg and rvg were near the middle or at the ′ end of the primer, whereas of those of end were close to the ′ end. comparing the same vp reverse primers with strain batli -g and batli -g also showed mismatches. for enda and vp -rdeg maximum mismatches are located in the middle or near the ′ -end, whereas for end and rvg, there were multiple mismatches of which and mismatches, respectively were right at the ′ -end. for vp forward primer vp - additionally, to determine if any of these bat rvas could cross species and infect humans, we designed primers (rva-vp _ f and rva-vp _ r) from an alignment of both human and bat rva vp segments to screen diarrheic infant samples (infants living around the same region where the bat samples were collected). thirty-six percent of human samples were positive for rva, however, none of them was of bat rva origin. they all possessed the typical human genotype i and were % identical to the gambian, senegalese, belgian and brazilian wa-like g p [ ] (table ) . screening human samples for these bat rvas indicated no interspecies transmissions and primer comparison showed that not all the strains can be picked up with the currently used screening primers. bats have been proven to harbor several human pathogenic viruses including sars, mers-related coronaviruses, as well as filoviruses, such as marburgvirus, or henipaviruses, such as nipah and hendra virus [ ] [ ] [ ] , but bat rvas [ ] , were isolated from a lesser horseshoe bat, and a stoliczka's trident bat in china, respectively , . to better understand the spread and diversity of rva in bats, we performed an rva screening in cameroonian bats, after trapping both male and female, young and adult bats close to human dwellings in muyuka, limbe and lysoka localities of the south west region of cameroon (fig. ) . using an unbiased viral metagenomics approach, we identified divergent novel bat rva strains, of which were genetically similar to each other. the fifth strain was related to the kenyan bat strain. interestingly, all these rvas were identified in adult (both female and male) straw-colored fruit bats (eidolon helvum) which is in contrast to human and other animals whereby rva (symptomatic) infections occur mostly in juveniles . also, diarrhea or other obvious signs of sickness were not noticed in these bats. this may suggest that bats may undergo active virus replication and shedding without obvious clinical signs , which potentially could increase human exposure. even though there exists a considerable genetic divergence between bat rva and human rva, suggestions have been made about potential interspecies transmission of chinese and kenyan bat rva strains. the two table . nucleotide comparison between the sequence of human rva screening primers for vp (beg , sbeg , con -l, enda, vp -rdeg, end and rvg ) and vp (vp - - f, con and con ) with their corresponding region of the new bat rva genotype. black shaded nucleotides indicate dissimilar nucleotides between a strain segment sequence and primer and bold numbers at the beginning and end of sequence indicate nucleotide positions for different strains. for clarity the reverse complement sequence of the reverse primers is used for comparison with bat rva sequences. chinese rva strains are genetically quite conserved (all segments of both strains have the same genotype except for their vp gene). based on genome comparisons of chinese bat and partial human rva strains from thailand (cmh and cmh ) and india ( m, m and mcs ), xia and colleagues speculated that asian bat rvas may have crossed the host species barrier to humans on a number of occasions . in addition, the unusual equine strain e shares the same genotype constellation with either myas and/or mslh in all segments except vp . this data therefore suggests that this equine rva strain most likely share a common ancestor with asian bat rvas. furthermore, the genotype constellations of these asian bat rva (table ) are reminiscent to the au- -like genotype backbone of feline/canine-like rva strains, as well as to the genotype constellation of two unusual simian rvas (rrv and tuch) , suggesting that interspecies transmissions might have also occurred in the distant past. moreover, an unusual ecuadorean human rva, ecu is closely related to bat sequences from brazil recently submitted to genbank. similarly, possible interspecies transmission trends were also suggested by he and colleagues between bovine strain rva/cow-wt/ind/rubv / /g p [ ] and the bat strain mslh . given the novelty of the bat rva strains described here, it is questionable if the currently used human rva screening primers (for vp and vp ) will pick up these divergent strains in case an interspecies transmission from bats to humans would occur. comparisons (table and supplementary data s ) of these primers with the corresponding sequences showed that the primer combination con -l and vp -rdeg , , would most likely detect both g and g rva strains. also, the combination of either beg or sbeg with end or rvg might be successful in amplifying g , but the same combinations might not be able to pick up the novel g and g genotypes in pcr screening assays. furthermore, the primer combination beg /sbeg and enda are likely to detect g , but will be unsuccessful in case of g and g especially if the forward primer beg is used. considering vp , both forward primers, vp - - f and con in combination with the reverse primer con will be sub-optimal in detecting any of the p genotypes. generally, with the exception of strain g , detection of most of the bat strains will be sub-optimal or not successful at all for the different available primer combinations. therefore, zoonotic events of bat rva strains could easily be missed with the current screening primers depending on the primer combinations, pcr conditions and/or circulating zoonotic strains. in order to investigate the possibility of bat rva infecting humans who are living in close contact with bats, we used novel primers (rva-vp _ f and rva-vp _ r) designed from an alignment of both human and bat vp rva segment to screen infant samples from patients with gastroenteritis, living around the same region where the bat samples were collected. interestingly, % of human samples were positive, however, none of these were positive for bat rvas. all were of the typical human rva genotype i and therefore there is no evidence for interspecies transmissions of bat rva to humans. however, this result is not conclusive as only a small sample size was considered here. sampling a larger number of subjects and from different localities around the region might result in more conclusive answers with respect to the zoonotic potential of these bat rva strains. the genotype constellations of the two chinese bat rvas showed clear indication of recent reassortment event(s) because they possessed different p genotypes (p [ ] for myas and p [ ] for mslh ), and some gene segments were nearly identical whereas others were not , . their genotype constellation differs markedly from the kenyan straw-colored fruit bat strain (ke ). although this strain showed a unique genotype backbone, some of its segments were similar to some human and other animal rvas . moreover, ke share the same genotypes in several gene segments (vp , vp , vp , nsp , nsp and nsp ) with our bat rva strain batly indicating possible reassortment events between different bat rva strains, as well as a large geographical spread of this virus. furthermore, batli , batli , batli and batly had conserved genotype constellations (in vp , vp -vp , nsp -nsp ) with - % nucleotide sequence similarity except for the vp of batli and vp of batly , again confirming reassortment events within bat rvas. the high genetic divergence and partial relatedness of most of the segments of the different bat rva strains and the ones identified in this study indicate the frequent occurrence of reassortment events in the general bat population and those of cameroon in particular. also, with the current knowledge of the genetic diversity, there seems to exist several true bat rva genotype constellations, as has been previously described for humans, and cats/dogs , . however, this needs to be further confirmed by identification of a larger number of rvas from bats from different age groups and different geographical locations. ethical authorization. ethical authorization for the use of human samples was obtained from the cameroon national ethics committee, yaoundé. all human experiments were performed in accordance with the ministry's national ethics committee guidelines. ethical authorization for the protocol and the use of animal samples was also obtained from the cameroon national ethics committee, yaoundé. all animal experiments were performed in accordance with the ministry's national ethics committee guidelines. all experimental protocols used in this study were approved by cameroon national ethics committee. administrative authorization was obtained from the delegation of public health for south west region, cameroon. informed consent was obtained from human subjects or their parents or guardians. bat sample collection. bat samples were collected between december and may using a previously described method . briefly, bats were captured in different regions (lysoka, muyuka and limbe) of the south west region of cameroon (fig. ) using mist nets around fruit trees and around human dwellings. captured bats were retrieved from the traps and held in paper sacks for - min, allowing enough time for the excretion of fresh fecal boluses. sterile disposable spatulas were used to retrieve feces from the paper sacks, and placed into tubes containing ml of universal transport medium (utm, copan diagnostics, brescia, italy). labeled samples were put on ice and then transferred to the molecular and cell biology laboratory, biotechnology unit, university of buea, cameroon and stored at − °c, until they were shipped to the laboratory of viral metagenomics, leuven, belgium where they were stored at − °c. each captured bat was assessed to determine species, weight (g), forearm length (mm), sex, reproductive state, and age. all captured bats were then marked by hair clipping to facilitate identification of recaptures, and released afterwards. trained zoologists used morphological characteristics to determine the species of the bats before they were released. no clinical signs of disease were noticed in any of these bats. sample preparation for ngs. eighty-seven fecal samples were grouped into pools each containing three to five samples and treated to enrich viral particles as follows: fecal suspensions were homogenized for min at rpm with a minilys homogenizer (bertin technologies, montigny-le-bretonneux, france) and filtered consecutively through μ m, μ m and . μ m membrane filters (merck millipore, massachusetts, usa) for s at g. the filtrate was then treated with a cocktail of benzonase (novagen, madison, usa) and micrococcal nuclease (new england biolabs, massachusetts, usa) at °c for h to digest free-floating nucleic acids. nucleic acids were extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions but without addition of carrier rna to the lysis buffer. first and second strand cdna synthesis was performed and random pcr amplification for cycles were performed using a whole transcriptome amplification (wta) kit procedure (sigma-aldrich), with a denaturation temperature of °c instead of °c to allow the denaturation of dsdna and dsrna. wta products were purified with msb spin pcrapace spin columns (stratec, berlin, germany) and the libraries were prepared for illumina sequencing using the nexteraxt library preparation kit (illumina, san diego, usa). a cleanup after library synthesis was performed using a . ratio of agencourt ampure xp beads (beckman coulter, inc., nyon, switzerland) . sequencing of the samples was performed on a hiseq platform (illumina) for cycles ( × bp paired ends). partial sequences were completed using rt-pcrs with specific primers (supplementary s ). for gene segments lacking the ′ and/or ′ ends of the orf the single primer amplification method (primers in supplementary s ) was used as described previously . sanger sequencing was done on an abi prism genetic analyzer (applied biosystems, massachusetts, usa). lysoka local clinic and kumba district hospital of the south west region of cameroon ( fig. ) after informed consent was obtained from patients or their parents or guardians. the patients were either diarrheic or came into contact with bats directly (by eating, hunting or handling) or indirectly (if family member is directly exposed to bats). the samples were put in utm containing tubes and stored the same way like the bat samples. screening primers (supplementary data s ) were designed from a consensus sequences of human and bat vp rvas and a total of samples from infants ( - years) who had diarrhea were screened by reverse transcriptase polymerase chain reaction (rt-pcr) using the onestep rt-pcr kit (qiagen). the products of positive samples were sequenced using sanger sequencing method. genomic and phylogenetic analysis. raw illumina reads were trimmed for quality and adapters using trimmomatic , and were de novo assembled into scaffold using spades . scaffolds were classified using diamond in sensitive mode . contigs assigned to rva were used to map the trimmed reads using the burrows-wheeler alignment tool (bwa) . open reading frames (orf) were identified with orf finder analysis tool (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) and the conserved motifs in the amino acid sequences were identified with hmmer . amino acid alignments of the viral sequences and maximum likelihood phylogenetic trees were constructed in mega . , using the gtr + g (vp , vp , nsp and nsp ), gtr + g + i (vp -vp , vp and nsp ), hky (nsp ) and t (nsp ) substitution models (after testing for the best dna/protein model), with bootstrap replicates. nucleotide similarities were also computed in mega by pairwise distance using p-distance model. phylogenetic analyses were performed using appropriate reference strains in addition to the rva discovered in this study. determinants of rotavirus host range restriction-a heterologous bovine nsp gene does not affect replication kinetics in the pig virus taxonomy: ninth report of the ictv candidate new rotavirus species in sheltered dogs exotic rotaviruses in animals and rotaviruses in exotic animals full genome-based classification of 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rotaviruses in rotarix and rotateq genotype constellation and evolution of group a rotaviruses infecting humans metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis g rotavirus strains isolated in the democratic republic of congo belong to the ds- -like genogroup trimmomatic: a flexible trimmer for illumina sequence data spades: a new genome assembly algorithm and its applications to single-cell sequencing fast and accurate short read alignment with burrows-wheeler transform hmmer web server: interactive sequence similarity searching molecular evolutionary genetics analysis version . . molecular biology and evolution r: a language and environment for statistical computing. r foundation for statistical computing geographic analysis and modeling with raster data this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . / key: cord- -ymhcfyxx authors: gromeier, matthias; lu, hui-hua; wimmer, eckard title: mouse neuropathogenic poliovirus strains cause damage in the central nervous system distinct from poliomyelitis date: - - journal: microb pathog doi: . /s - ( ) - sha: doc_id: cord_uid: ymhcfyxx poliomyelitis as a consequence of poliovirus infection is observed only in primates. despitea host range restricted to primates, experimental infection of rodents with certain genetically well defined poliovirus strains produces neurological disease. the outcome of infection of mice with mouse-adapted poliovirus strains has been described previously mainly in terms of paralysis and death, and it was generally assumed that these strains produce the same disease syndromes in normal mice and in mice transgenic for the human poliovirus receptor (hpvr-tg mice). we report a comparison of the clinical course and the histopathological features of neurological disease resulting from intracerebral virus inoculation in normal micewith those of murine poliomyelitis in hpvr-tg mice. the consistent pattern of clinical deficits in poliomyelitic transgenic mice contrasted with highly variable neurologic disease that developed in mice infected with different mouse-adapted polioviruses. histopathological analysis showed a diffuse encephalomyelitis induced by specific poliovirus serotype isolates in normal mice, that affected neuronal cell populations without discrimination, whereas in hpvr-tg animals, damage was restricted to spinal motor neurons. mouse neurovirulent strains of poliovirus type differed from mouse neurovirulent poliovirus type derivatives in their ability to induce cns lesions. our findings indicate that the characteristic clinical appearance and highly specific histopathological features of poliomyelitis are mediated by the hpvr. our data lead us to conclude that the tissue tropism of mouse-adapted poliovirus strains in normal mice is fundamentally different from that of poliovirus in hpvr-tg mice and primates, and that this is indicative of an as yet unknown mechanism of adsorption and uptake of the virus into cells of the murine cns. poliomyelitis is a rare neurological complication of primate infection with poliovirus (pv), a member of the picornavirus family, genus enterovirus. ingestion of virulent particles results in intestinal uptake, presumably through m-cells, initial viral replication in subjacent lymphatic tissue, spread to deeper cervical and mesenteric *author to whom correspondence should be addressed. - / / + $ . / o academic press limited lymph nodes (lymphatic phase), and ultimately entry of the virus into the bloodstream (viremic phase). the latter is a prerequisite for passage of the blood brain barrier and subsequent lytic infection of motor neurons. only a small proportion of pv infections lead to a distinctive neurological syndrome that ranges in severity from transient flaccid monoparesis to progressive flaccid paraplegia with respirato.ry impairment and sometimes bulbar involvement. the hallmark of poliomyelitis histopathology is selective damage to anterior horn motor neurons along the entire spinal cord. spinal neurons outside the motor neuron system are characteristically spared, in spite of their close anatomical relationship to the target of polioviral attack. the predominant molecular determinant of pv tropism is the hpvry the nucleotide sequence of hpvr identified it to be a member of the immunoglobulin superfamily, whose four mrna isoforms are the result of alternative splicing events, and give rise to different receptor molecules. ' hpvrc~ and hpvr& are integral membrane proteins with divergent cytoplasmatic domains, whereas hpvr/~ and hpvr are secreted molecules lacking the putative transmembrane domain. the hpvr is a highly glycosylated protein with an apparent molecular weight of kda the animal model for poliomyelitis in hpvr-tg mice showed pv-induced damage of comparable anatomical distribution as in primates, °' an observation confirming views of the hpvr as the critical determinant conferring pv susceptibility. unexpectedly, hpvr mrna and hpvr-related proteins were shown to be present in a wide variety of tissue homogenates not known to be sites of pv replication, ~' ° an observation suggesting additional limiting factors of pv susceptibility, mrnas specifying the simian and murine ~ homologues to hpvr have been isolated, but only the monkey-specific pvr can promote pv infection. ~ attempts to use rodents as possible models of pv-induced disease resulted in the isolation of mouse-neurovirulent (ran) strains. a pv serotype field isolate from lansing, michigan, [pv (l)], which caused a syndrome described as 'polioencephalitis' upon intracerebral injection in the cotton rat, tm provided the first system of pv encephalitogenesis in the wild type (wt) mouse. ~s histopathological analyses of murine infection with the rodent-passaged pv (l) isolate described a pattern of damage in accordance with concepts of primate poliomyelitis. ~ a structural element conferring mouse neurovirulence to pv (l) was determined to map in the capsid region. ~ sufficient in causing this effect was a segment within the bc-ioop of vp since mouse avirulent pv type (mahoney) [pvi(m)], after transposition of the lansing bc-ioop, caused neurological disease in mice. ' surprisingly, point mutations in distant regions of vp and vp could be shown to exert a mn phenotype. °. ~ the histopathological features of neurological disease caused by these genetically well defined mn pv strains have not been reported thus far. in addition to the selection of wt pv strains expressing a mn phenotype in mice, attenuated pv strains with a mouse host range phenotype but reduced neurovirulence have also been described [pv type , strain w ]. the attenuated (att) phenotype of pv (w ) is reminiscent of the sabin strains of pv that express an att phenotype for primates. the genetic determinants of viral neurotropism have been studied in several viral systems. most frequently, these determinants mapped to the envelope gene; examples are the alphaviruses sindbis virus we have infected swiss-webster-, and icr-mice with a variety of mn virus isolates of different serotypical origin. type strains pv (l), pv (mef- ), a recent pv isolate from india (ind), and pv (w ) caused a fatal diffuse encephalomyelitis in these mice with considerable differences in intensity between individual viral strains. pvl(lsa), a mouse-adapted derivative of pvl(m), caused a characteristic non-progressive panmyelitic syndrome. we have also studied the histopathology of murine infection with pvi(m), carrying a single mutation in position of capsid protein vp that we constructed according to a published report. ~ infections with each of these strains did not follow the stereotypic course of predictable progression seen in pv-induced poliomyelitis in hpvr-tg icr-mice. syndromes with distinct and consistent features followed intracerebral infection with each group of strains. these syndromes could be separately characterized and distinguished from hpvr-mediated poliomyelitis, on both clinical and histopathological grounds. poliovirus isolates and experimental animals. pv transgenic icr-mice expressing the hpvr ~° were a generous gift from a. nomoto (the university of tokyo, japan). swiss-webster-and normal icr-mice were obtained from taconic (germantown, ny, u.s.a). ultraviolet (uv) irradiation conditions. uv irradiation was performed in a uv-stratalinker (stratagene, lajolla, ca, u.s.a) a suspension of pv was irradiated at a distance of ca. cm with an intensity of ca. . j/m z. virus was exposed to the irradiation source in ice-cooled plastic dishes at a solution depth of ca. ram. the loss of infectivity was confirmed in a plaque assay. poliovirus intracerebral infections. virus preparations were used to infect tg or normal mice with the desired input titer by intracerebral inoculation. mice were anesthetized, and a gauge hypodermic needle was used to inject maximally pl of virus suspension in dulbecco's minimal essential medium (dmem). the point of injection was the middle on the median between the ear pinnacle and the eye. infected mice were regularly observed for symptoms. none of the treated animals showed any external signs of damage or neurological disturbances in h following injection. tissue processing, sectioning, and staining. affected animals in the final stage of their disease were sacrificed according to approved protocols, and their bodies were immediately perfused with ml of phosphate buffered saline (pbs), followed by ml of % neutral buffered paraformaldehyde. the brain and spinal cord were removed and fixed for h at room temperature in the fixative used for perfusion. tissue specimens were rinsed for rain in icecold pbs, then placed in % ethanol over night. dehydration was achieved through a scheme of gradually increasing ethanol concentration, followed by clearing in toluene for h and infiltration with paraffin at . °c for h. neural tissues were embedded and cut on a rotary microtome at a thickness of /~m. tissue sections were placed on microscopic slides treated transgenic mice expressing the pv receptor tm were infected intracerebrally with pvi(m) with an amount of virus ranging from to x plaque forming units (pfu). all infected mice developed a characteristic neurological syndrome displaying stereotypic clinical features irrespective of input titer of virus and differing only with regard to onset of symptoms (table ) . once visible signs of functional impairment were apparent, the progression of disease followed a predictable course. two to five days after intracerebral injection, initial signs were invariably a flaccid paralysis of the lower extremities and tail (fig. a) . the condition was rapidly progressive leading to complete immobilization and respiratory difficulty within h. respiratory distress caused visible signs of bobbing of the head, strenuous respiratory effort, insufficient thoracic excursions, and nasal flaring. animals in the final stage of the disease were killed and their cns tissues processed according to standard procedures. histopathological analysis of the cns of pvl(m)-infected hpvr-tg mice revealed the spinal pathology of poliomyelitis described in earlier reports. °' selective loss of anterior horn motor neurons along the entire spinal cord was invariably present (fig. b) . foci of virally-induced damage in the higher cervical cord extended into the brain stem and were accompanied by minor signs of inflammation in a predominantly perivascular distribution. characteristically, apart from a clearly defined lesion in the pyramidal cell layer of the hippocampal formation, lesions above the brain stem were absent in all cases analyzed. the appearance of initial lesions in the lumbar spine, distant from the injection site without evidence of damage to cortical motor neurons or descending tracts, indicated that virus had been disseminated via the hematogenous or cerebrospinal fluid route. twenty eight day old swiss-webster-and icr-mice were injected intracerebrally with pvi(m), pv (l), pv (mef- ), pv (ind), or pv (w ), with amounts of virus ranging from to x . three groups of four animals each with different genetic backgrounds, swiss-webster and icr, were injected with the same viral strain. it is important to note that we were unableto detect clinical or histological evidence for differences in susceptibility towards pv infection between both outbred mouse strains used. therefore, in the following text, the term 'normal mouse' will refer to swiss-webster mice. all mn pv strains assayed caused poliomyelitis when injected into hpvr-tg mice (clinical and histopathological details are described later). none of the normal mice injected with pvi(m) showed clinical signs of neurological damage, whereas inoculation of type pv strains produced signs of cns infection ( table ) . mice infected with pv (mef- ) and pv (ind) were most severely affected. initial symptoms appeared - days post infection (p.i.) in all animals but they were inconsistent regarding the sites of manifestation and quality of functional impairment. motor symptoms consisted predominantly of spastic weakness involving the lower or upper extremities in a random fashion ( table ) . paraplegic animals had a characteristic posture marked by kyphoscoliotic deformity (fig. b) . the progression of motor symptoms did not follow any apparent topographical scheme. pareses frequently were accompanied by gait ataxia or motor incoordination. clinical signs of respiratory involvement as described above usually did not appear during the course of the disease ( table ). the clinical course proceeded to a terminal stage within days after onset of symptoms. preterminal animals were severely emaciated once functional impairment supervened, and refused intake of fluid and food offered within their reach. compared to pv (mef- ) and pv (ind) a proportionally lower number of pv (l)infected animals died (table ) . their clinical syndrome was less variable, but also diverged from the regular pattern of disease localization and progression seen in poliomyelitic hpvr-tg mice. motor symptoms similar to those in pv (mef- ) infected littermates dominated the clinical picture. progression of motor involvement did not follow any recognizable pattern and it only rarely included respiratory function ( table ). eventually immobilization led to quick deterioration of the starving animals. pv (w ) was previously reported to be of attenuated neuropathogenicity with respect to pv (l) in mice. accordingly, the proportion of infected animals that developed disease, and the severity and pace of progression of symptoms, were less than in animals infected with other pv type isolates (table ). this observation confirms the attenuated nature of the strain." the spectrum of functional deficits observed was identical to pv (l)-induced disease. likewise, there was considerable variability in the sites involved in production of symptoms, course of progression, the clinical discrepancies between wt pv-induced poliomyelitis in hpvr-tg mice, and mouse-adapted pv infections in normal mice, were confirmed by histopathological differences. the severity and variability of clinical signs secondary to infection with the pv strains in normal mice was consistent with the histopathological findings. extent, morphology and localization of viral lesions in swiss-webster-and icr-mice were comparable. the range of vitally-induced damage encompassed a variety of lesions indiscriminately affecting structures within the cerebral hemispheres and the entire spinal cord. characteristically, spinal pathology was patchy and irregular, and it did not follow the consistent pattern of damage to the entire cord seen in pvinfected hpvr-tg mice. unexpectedly, anterior horn motor neurons remained frequently unaffected in normal mice infected with mn pv , although severe pathological changes within the spinal cord were apparent (fig. c,d) . in contrast, routine poliomyelitis in hpvr-tg mice invariably led to complete and exclusive elimination of the motor neuron population in the spinal cord with only minor infiltrative changes ( fig. b) . unlike the limitation of vitally-induced lesions in pv infections of hpvr-tg mice to the spinal cord, pv -induced disease involved the entire cns. microglial nodules accompanied by mixed lymphocytic and neutrophilic infiltrates were scattered throughout the brain and were found within the insular, piriform and temporal cortex (fig. f) , and the basal ganglia and thalamus (fig. h) . these structures were never affected in infected hpvr-tg mice (fig. e,g) . perivascular cuffing and dense infiltration of perivascular and periventricular parenchyma were distributed ubiquitously in the cns. the cerebral white matter was a frequent site of viral lesions. lymphocytic infiltrates within the cerebral or cerebellar peduncles, the internal capsule, or the long descending tracts and posterior columns within the upper cervical cord, occasionally caused rarefaction necrosis with secondary demyelination (fig. ) . immunohistochemical staining for viral proteins with a polyclonal anti-pv antiserum revealed positive signals in those structures frequently involved by virally- fig. . signs of widespread lesions and diffuse encephalomyelitis were most commonly associated with the isolates pv (mef- ) and pv (ind), whereas in pv (l) cases a myelitic pattern of disease predominated over encephalitis. parallel to the clinical findings, overall cns damage induced by pv (w ) was moderate in comparison with the other serotype isolates. neurological damage focused on the spinal cord with rare cerebral involvement. spinal pathology qualitatively resembled the pv panmyelitis described above but the extent and number of lesions were reduced (data not shown). to analyze the clinical course of murine infection with pv in mice expressing the hpvr, tg mice were inoculated intracerebrally with pv (mef- ) or pv (l). the resulting clinical picture featured signs typically seen in murine poliomyelitis. the onset of disease was marked by a flaccid paraparesis, followed by ascending progressive symptoms as in poliomyelitis. all affected animals developed a preterminal dyspneic stage before they were killed. associated histopathology showed elements characteristic of hpvr-mediated poliomyelitis, as well as pv -induced encephalomyelitis. anterior horn motor neurons had changes typical for poliomyelitis. in addition, there was almost always severe hemispheric involvement, never associated with hpvr-tg murine poliomyelitis resulting from infection with pvi(m). lesions equivalent to those seen in pv -induced encephalomyelitis were distributed in the same widespread indiscriminate manner throughout the cns. gray matter structures were affected as well as cerebral white matter (data not shown). two groups of ten -day-old swiss-webster-and icr-mice were each infected intracerebrally with pvi(ls-a) at a range of to x pfu. as in previous assays, there was no notable difference in clinical signs and histopathological features in mice with different genetic background. all infected animals developed a specific neurological syndrome with stereotypical onset, clinical course, functional deficits, and pattern of progression. four days p.i., mice showed the first signs of an insidious spastic paraparesis (fig. c) . no ascending motor deficits occurred, respiratory function remained unaffected, and there were no fatalities. intracerebral inoculation of pvl(ls-a) was required to produce symptoms, since intravenous injection of virus with concomitant intracerebral needle puncture produced no neurological impairment. intracerebral inoculation of equal particle numbers of pvi(ls-a) virus, inactivated by uv-irradiation, also caused no neurological signs of cns damage. this finding indicated viral replication to be a prerequisite for pvi(ls-a) neuropathogenicity. histopathology of the neurological syndrome caused by this variant centered exclusively on the spinal cord. extensive infiltrates, originating from spinal blood vessels, invaded both the gray and white matter. pathologic changes involved the entire spinal cord, but did increase in extent of damage caudally (fig. a,b) . there was no apparent predilection for any cell subtype. motor neuron damage was seen within areas of destructive necrotic tissue change secondary to the inflammation. at no time during the infection could damage to motor neurons within the thoracic or cervical cord be distinguished (fig. b) , despite prominent inflammation affecting these regions. spinal sections taken from animals which underwent partial reversal of neurological defects, weeks p.i., revealed complete regression of infiltrative changes and preservation of motor neuron populations (fig. c ). a mutant pvi(m) virus, which had previously been reported to exert neuropathogenic potential in normal mice was tested in a similar manner as the above mentioned strains. pvl(vp - ) carried a point mutation at position (p s) within capsid protein vp . intracerebral inoculation of each four swiss-webster-and icr-mice with at least i x pfu resulted in visible signs of neurological damage in % of affected animals in both groups. with the genetic variant constructed in this laboratory, this large inoculum was required to induce clinical signs. the nature of the neurological syndrome was partly obfuscated by the vigorous host response to the application of excess viral antigen, and the resulting clinical deterioration. sick mice showed prominent systemic signs of disease such as ruffled fur, reduced activity, and emaciation. symptoms of minor motor impairment were present in half of the diseased mice, but the limb pareses were difficult to assess in quality due to the general weakened state of the animal. mild limb weakness did not progress with any apparent disease pattern. severely weakened, emaciated animals succumbed to the effects of infection, without the clear causal relationship of neurological disease and fatal outcome seen in poliomyelitis, hpvr-tg mice injected with pv (vp - ) developed a neurological condition indistinguishable from pvi(m) related poliomyelitis (data not shown). similar to clinical findings, the histopathologic features induced by pvl(vp - ) were less defined than in the other described infections. there was no supraspinal involvement, and spinal cord sections at all levels displayed widespread parenchymal invasion and reactive migroglial proliferation. no targeted attack against specific cell populations was apparent (fig. d) . histopathologic evidence for a primary thoracic and cervical cord affection was not present. with the rare exception of certain serotype pv strains, e.g. pv (mef- ) and pv (l), naturally occurring pv strains can only infect primates, and even pv (mef- ) and pv (l) are mouse neurovirulent only when artificially inoculated by intracerebral injection. the narrow host range of pv results from its stringent dependence on the express exactly the same cell tropism in the murine cns as they do in the primate cns unlikely and, hence, that mn pvs would cause the same specific disease syndrome (poliomyelitis) in normal mice as they do in primates. to shed some light on these complex problems we have compared the disease syndromes caused by a variety of different pv strains in hpvr-tg and normal mice. we have come to the conclusion that mn pv strains do not produce poliomyelitis in normal mice. the occurrence of poliomyelitis in hpvr-tg mice corroborates the function of the hpvr as a major determining factor in the pathogenesis of the peculiar histopathological and clinical features of this disease. °' poliomyelitis in tg mice followed a stereotypic course: initial flaccid pareses of the lower limbs, followed by cranial progression, involving respiratory function, and fatal outcome. in contrast, the clinical symptoms resulting from mn pv infections in normal swiss-webster-and icr-mice could not be explained by classical terms of the syndrome poliomyelitis. infection of normal mice with mn pv strains caused a diffuse encephalomyelitis with highly variable neurological symptoms appearing in random order, at topographically unrelated sites. mouse neuropathogenicity was shared by several serotype field isolates, the mouse adapted attenuated strain pv (w ), the pvi(m) derivative pvi(ls-a), and pvi(m) with a mutation in residue of capsid protein vpi. interestingly, the type pv field isolates pv (mef- ) and pv (ind), have been reported to be mn without a lengthy process of adaptation, whereas other type [pv (l)] and type strains [pvi(ls-a)] were passaged in the rodent cns before they acquired the mn phenotype. mn type or type pv field isolates have not been described. the molecular basis underlying this peculiar property of type pv strains is not understood. earlier reports stressed the similarity between primate poliomyelitis and encephalomyelitis caused by pv (l) in normal mice. comparative studies at that time were impeded by the lack of a murine model of hpvr-mediated poliomyelitis. viral antigen was shown to be expressed in spinal anterior horn motor neurons, ~ but in situ hybridization revealed the presence of viral rna in cells in posterior portions of the spinal cord as well as the white matter. "s previous analyses of pv neurovirulence using mn strains were based on the assumption of a similar pathogenesis of mn pv infections in wt mice and in primates. one parameter to assess the potential of viral neurovirulence is the ldso value, and in earlier studies, animals showing evidence of neurological functional impairment were generally killed without further histopathological analysis (see for example, ref. ) . our results suggest that the expression of the mn phenotype of pv strains in mice should not be compared to the expression of pv neurovirulence in primates, the normal host. indeed, it appears that the expression of a mn phenotype in wt mice can result from very different genetic changes in pv strains, and that each genetic change can produce different syndromes. this is apparent not only from various type mn strains, but also from the type mn strain pvi(ls-a) [a derivative of pvi(m) whose genotype has been recently elucidated ]. histopathological changes within the cns should therefore be monitored when murine infections with mn pv strains are studied. a major determinant for the mn phenotype of pv (l) is the bc-ioop of vp ~ a surface protrusion of about amino acids located at the apex of the poliovirion that functions as a neutralization antigenic site. exchange of the vp bc-ioop of the mouse-inert pvi(m) with the bc-ioop of pv (l) produces the mn phenotype, ~ ' an observation that prompted speculation that the vp bc-ioop is involved in the recognition of a putative mouse receptor. no evidence for this hypothesis has been obtained as yet. strain pvi(ls-a), a mn derivative of pvi(m), accumulated point mutations during passage in non-human tissue, of which mutations led to amino acid exchanges in the capsid region these capsid mutations, however, were insufficient to produce the mn phenotype. instead, the mutations in capsid protein vp plus, surprisingly, five mutations in the coding region of the non-structural protein a "r°, a proteinase, produced mouse neurovirulence. the syndrome pv (ls-a) ir~duced in normal mice, however is distinct from the encephalomyelitis caused by type pv strains. a pathological entity different from those observed with all other mn poliovirus strains, wes seen with pv (vp - ), a virus that we constructed according to a previous report. pv (vp - ) is a mn derivative of pvi(m) that carries only a single amino acid exchange in capsid protein vp (p s). the mutation, located at an interpentameric interface inside the capsid, was suggested to destabilize the virion in the mouse cns, but the neurological syndrome caused by infection with pv (vp - ) was not histopathologically characterized. infection of mice with pv (vp - ) led to neurological damage only after intracerebral inoculation of excess viral antigen. the poorly defined histopathological features of cns involvement lacked characteristics of specificity observed in hpvr-mediated poliomyelitis. the genotypic differences between mn pv strains and the clinical syndromes they cause, which differ from hpvr-mediated poliomyelitis, challenge the hypothesis of common determinants of a mn phenotype. rather, it appears that a multitude of non-related genetic elements affect this phenotypic marker in various ways. as indicated before, the nature of the receptor(s) used by the mn pv strains in the mouse cns remains to be determined. available data suggest that the mouse homologue to the hpvr does not serve as surrogate for the mn pv strains. it is more likely that the mn pv strains enter neuronal tissue via cell-surface protein(s) unrelated to hpvr. if so, it is not surprising that the syndromes produced by these viral strains are distinct from poliomyelitis. in recent years picornaviruses other than poliovirus have been implicated as causative agents of neurological disease. these include enterovirus (ev) , the etiologic agent of acute hemorrhagic conjunctivitis, or ev . however, it has also been reported that the encephalomyelitis caused by the newly emerging human pathogen ev did not always resemble poliomyelitis. s° the evolution of enterovirus strains with new neuropathogenic properties distinct from previously non-neuropathogenic ancestors might be due to adaptation to new receptor entities. the mouse-neurotropic pv variants with altered cell tropism constitute a precedent for the emergence of non-poliomyelitic picornaviral cns disease. we thank a. nomoto for the generous gift of the hpvr-tg mouse strain used in this study and b. jubelt and o. kew for kindly providing pv type strains. we are grateful to p. coyle for critical review of this manuscript. we thank n. peress for helpful discussion and d. colflesh for help with microscopic imaging. this work was supported in part by nci grant ca , and nih grants ai and ai . m.g. is a recipient of a grant from the stipendienprogramm infektionsforschung, heidelberg, germany. poliovirus type enters the human host through intestinal m cells emerging concept of poliomyelitis 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analysis of the vp and rna-dependent rna polymerase regions of human norovirus gii.p -gii. in – date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: wbawzbhz human norovirus (hunov) gii.p -gii. (kawasaki variant) reportedly emerged in and caused gastroenteritis outbreaks worldwide. to clarify the evolution of both vp and rna-dependent rna polymerase (rdrp) regions of gii.p -gii. , we analyzed both global and novel japanese strains detected during – . time-scaled phylogenetic trees revealed that the ancestral gii. vp region diverged around , while the ancestral gii.p rdrp region diverged around . the evolutionary rates of the vp and rdrp regions were estimated at ~ . × (− ) and ~ . × (− ) substitutions/site/year, respectively. the phylogenetic distances of the vp region exhibited no overlaps between intra-cluster and inter-cluster peaks in the gii. strains, whereas those of the rdrp region exhibited a unimodal distribution in the gii.p strains. conformational epitope positions in the vp protein of the gii.p -gii. strains were similar, although some substitutions, insertions and deletions had occurred. strains belonging to the same cluster also harbored substitutions around the binding sites for the histo-blood group antigens of the vp protein. moreover, some amino acid substitutions were estimated to be near the interface between monomers and the active site of the rdrp protein. these results suggest that the gii.p -gii. virus has produced variants with the potential to alter viral antigenicity, host-binding capability, and replication property over the past years. human norovirus (hunov) is a major causative pathogen of acute viral gastroenteritis (de graaf et al., ) . although hunov is most prevalent during autumn and winter in the northern hemisphere, the virus can be detected throughout the year (ahmed et al., ) . a previous report suggested that approximately million people annually suffer from gastroenteritis due to hunov infection worldwide (kirk et al., ) . molecular epidemiological studies have indicated that distinct gii. variants have recurrently emerged every - years in the past decade to cause pandemics of gastroenteritis in all aged individuals (bull et al., ) . furthermore, gii.p -gii. (kawasaki variant) has suddenly been prevalent since in various countries including japan, china, south korea, italy, romania, argentina, brazil, and the usa (chan et al., ; fu et al., ; matsushima et al., ; medici et al., ; dang thanh et al., ; dinu et al., ; cannon et al., ; degiuseppe et al., ; silva et al., ) . however, the epidemiology of this virus over a long period of time is unclear at present. hunov belongs to the genus norovirus of the family caliciviridae, and it is further genetically classified into three genogroups, gi, gii, and giv. genetic analyses have shown that hunov gi and gii can be further classified into and genotypes, respectively (kroneman et al., ) . of these, many genotypes have been associated with gastroenteritis outbreaks throughout the world (hoa tran et al., ) . the genomes of noroviruses contain three open reading frames and encodes six non-structural proteins, including the rna-dependent rna polymerase and the two capsid proteins vp and vp (de graaf et al., ) . molecular evolutionary analysis based on advanced bioinformatics technologies is a powerful tool to better understand not only the phylogeny of pathogens, but also their antigenicity (bok et al., ; siebenga et al., ; boon et al., ; lu et al., ; parra et al., ; tohma et al., ; nagasawa et al., ) . furthermore, next-generation sequencing technologies are also useful for the comprehensive analysis of various virus genomes (quiñones-mateu et al., ) . in this study, we used both these methodologies to conduct a detailed molecular evolutionary analysis of the vp and rdrp regions of gii.p -gii. strains detected in various countries. a total of strains of gii.p -gii. detected in miyagi ( strains), kanagawa ( samples), saitama ( samples), ibaraki ( strains), gunma ( strains), aichi ( strains), hiroshima ( strains), tochigi ( strains), fukuoka ( strains), yamaguchi ( strains), and aomori ( strain) prefectures from to were sequenced in this study. fecal samples were collected from patients with acute gastroenteritis associated with hunov infection under compliance with the food sanitation law and the law concerning the prevention of infections and medical care for patients of infections of japan. informed consent was obtained from all participants, which was acquired from the subjects or their legally acceptable representatives for sample donation. the personal data of the patients was anonymized. to perform extraneous study (this study) and due to the lack of written informed consent, this study obtained ethical approval from the research and ethical committees for the use of human subjects of the national institute of infectious diseases, tokyo, japan (no. ). all methods were conducted in accordance with the approved guidelines. information on the samples is given in table s . rna was extracted from % suspensions of fecal samples in phosphate buffered saline using a qiaamp viral rna mini kit (qiagen, hilden, germany). the extracted rna was subjected to sequencing as described below. sequencing was performed with sanger and next-generation sequencers. for sanger sequencing, a reverse transcriptionpolymerase chain reaction (rt-pcr) was first performed for min at • c and then min at • c, followed by a total of cycles of s at • c, s at • c and s at • c, and then a final extension of min at • c using specific primers for the vp and rdrp regions and a primescript ii high fidelity one step rt-pcr kit (takara, shiga, japan; table s ). cycle sequencing was performed for min at • c, followed by a total of cycles of s at • c, s at • c and min at • c using a bigdye terminator v . cycle sequencing kit (applied biosystems, carlsbad, california, usa). the dna sequences were analyzed using a genetic analyser (applied biosystems). full-length nucleotide sequences of the vp and rdrp regions were acquired using the primer walking method. next-generation sequencing was conducted as described previously (dennis et al., ; ide et al., ) . data analysis was performed using clc genomics workbench v . . (qiagen). contigs were assembled from the obtained sequence reads by de novo assembly. hunov genotypes were determined using the norovirus genotyping tool (version . ) and the human calicivirus typing tool (kroneman et al., ) . all full-length nucleotide sequences of the vp and rdrp regions of gii. , including information on sample collection years and no mixed nucleotides, were obtained from genbank (accessed on august, ). for the gii.p -gii. genotype, only sequences with information on sample collection years and months were used in this study. moreover, nine sequences associated with some recent outbreaks of gii.p -gii. were combined with those of the new japanese strains above. to construct time-scaled phylogenetic tree, we added representative vp sequences of all gii genotypes, including porcine nov gii (gii. , gii. , and gii. ) and other hunov gii genotypes ( strains), as well as an outgroup strain of hunov gi genotype (gi. ) to the dataset of the vp region (resulting in a total of strains). the representative rdrp sequences of the gii genotypes, including porcine nov (gii.p and gii.p ) and other hunov gii genotypes ( strains), as well as an outgroup strain of hunov gi genotype (gi.p ), were appended to produce an rdrp dataset consisting of strains ( table s ). the constructed datasets were aligned with mafft software (katoh and standley, ) . phylogenetic trees with molecular clocks were generated by the bayesian markov chain monte carlo (mcmc) method using the beast package v . . (bouckaert et al., ) . best substitution models (gtr+Γ +i for both vp and rdrp) were determined for the constructed datasets ( strains for vp and strains for the rdrp) by comparison of the bayesian information criterion (bic) values using jmodeltest software (guindon and gascuel, ; darriba et al., ) . appropriate clock and tree prior models (relaxed clock exponential and coalescent exponential population tree prior for both vp and rdrp) were selected by path-sampling/stepping stone-sampling marginal-likelihood estimation using the beast package (baele et al., ) . the mcmc runs were conducted with chain lengths of , , steps with sampling every , steps for vp , and with chain lengths of , , steps with sampling every , steps for rdrp. the analyzed data was evaluated by the effective sample sizes (ess) values using tracer software, and values of or more were accepted. maximum clade credibility trees were generated by discarding the first % of trees (burn-in) using treeannotator v . . in the beast package. the time-scaled phylogenetic trees were visualized by figtree v . . software. branch reliability was supported by highest posterior densities (hpds) of %. moreover, the evolutionary rates of gii.p -gii. strains were estimated as described above. the analyzed parameters with substitution, clock, and tree prior models are shown in table . in this study, the evolutionary rates of overall clusters in the gii.p -gii. strains were calculated since the rate of a cluster could transition of effective population sizes was estimated by a bayesian skyline plot using the beast package v . . . appropriate substitution models were selected based on comparison of the bic values in the dataset, including strains of the kawasaki variant in the vp region. the clock models were appropriately selected on the basis of path-sampling/stepping stone-sampling marginal-likelihood estimation. the mcmc was run on chain lengths of , , steps with sampling every , steps. after evaluation based on the ess values, the bayesian skyline plot was generated by tracer software. phylogenetic trees were created using the maximum likelihood method in mega software (kumar et al., ) . best substitution models (gtr+Γ +i for vp and k +Γ for rdrp) were selected using the jmodeltest . branch reliability was supported by , replications of bootstrap values. phylogenetic distances between gii. strains were calculated by patristic software (fourment and gibbs, ) . were generated based on homology modeling using the modeller software v . (webb and sali, ) . three crystal structures for vp (pdbid: ihm, f m and lkc) and one for rdrp (pdbid: sh ) were used as the templates based on basic local alignment search tool (blast) analyses of the proteins. amino acid sequences of the templates and the target strains were aligned using mafftash software (standley et al., ; katoh et al., ). the constructed structures were minimized using the gromos , implemented by swiss pdb viewer v . and evaluated by ramachandran plots via the rampage server, which showed the favored regions of . % ± . (vp ) and . % ± . (rdrp), the allowed regions of . % ± . (vp ) and . % ± . (rdrp), and the outlier regions of . % ± . (vp ) and . % ± . (rdrp) (mean ± sd) of all residues in each structure (guex and peitsch, ; lovell et al., ) . the final models were modified and colored using chimera v . (pettersen et al., ) . conformational epitopes on the vp dimer structures of the gii. strains were predicted using discotope . , epces, and epsvr programmes (liang et al., (liang et al., , kringelum et al., ) . cut-off values were set at − . for the discotope . and for the epces and epsvr in order to encompass epitopes numbers of strains substitution models clock models tree prior models evolutionary rates ( % hpd intervals) on the vp of gii. identified by a previous in vitro study (lindesmith et al., ) . consensus sites identified by more than one of the three tools and regions with two or more closely apposed residues of the sites on the vp dimer structures were determined to be conformational epitopes. positive selection sites in the vp region of the gii. strains were predicted using the fast, unconstrained bayesian approximation (fubar) and mixed effects model of evolution (meme) algorithms on the datamonkey server (pond and frost, ; delport et al., ; murrell et al., murrell et al., , weaver et al., ) . generally, fubar hypothesizes constant selection pressure at each site for the entire phylogeny and utilizes a bayesian approach to estimate non-synonymous (dn) and synonymous (ds) substitution rates at each site. meme is used to detect positive selection sites under a proportion of branches. significance levels were set at posterior probabilities of > . for fubar and p < . for meme. transmission links between gii.p -gii. strains were analyzed using a median joining network with popart software (bandelt et al., ; leigh and bryant, ) . we constructed a dataset that covered nucleotide sequence lengths from the start position of rdrp to the terminal position of vp (resulting in a total of strains). the dataset was then processed to detect recombination between the rdrp and vp sequences based on seven primary exploratory recombination signal detection methods (rdp, geneconv, bootscan, maxchi, chimera, siscan, and seq) using rdp software (martin et al., ) and the threshold of the p-value for significance was set at . . recombination regions were assigned when they were identified by more than four of the seven methods; however, this criterion resulted in the identification of no recombinant sequences in the dataset. the median joining networks were analyzed using an epsilon value of zero. time-scaled phylogeny of the vp and rdrp regions in the gii.p -gii. strains we constructed time-scaled phylogenetic trees using the mcmc method based on the full-length of the vp ( strains) and rdrp ( strains) regions in the gii. strains detected in the various countries (figures , ) . the mcmc tree for the vp region estimated that the common ancestor of the gii. , gii. , and gii. diverged in september, ( % hpd january, -february, . a common ancestor of the gii. strains diverged in november, ( % hpd september, -november, ) and further diverged into seven clusters by around october, . of these, the gii.p -gii. strains belonged to clusters and (figure ) . the gii. strains in clusters , , , and (no information on the rdrp sequence in cluster ) were composed of the distinct rdrp genotypes, including gii.p , gii.p , gii.p , and gii.pe, respectively (figure and table s ). most strains of the gii.p -gii. belonged to the cluster (the kawasaki type). the common ancestor of the cluster diverged in october, ( % hpd may, -august, , while that of cluster (the kawasaki type) emerged in july, ( % hpd july, -september, ) (figure ) . moreover, analyses of phylogenetic distances exhibited no overlap between intra-and inter-cluster peaks at a value of . ( figure a ).with respect to the rdrp region (figure ) , the common ancestor of the gii.p and gii.p diverged in january, ( % hpd december, -may, ). subsequently, the common ancestor of the gii.p diverged in october, ( % hpd may, -april, and then further into two clusters by around august, . the gii.p -gii. strains were contained in clusters and in the rdrp region, which was compatible with the classification in the vp region. most strains of the gii.p -gii. also belonged to cluster (the kawasaki type). the common ancestor of cluster diverged in august, ( % hpd december, -january, ), while that of cluster (the kawasaki type) did so in april, ( % hpd september, -october, . furthermore, phylogenetic distances overlapped between the intra-and inter-cluster peaks ( figure b) . these results suggest that the gii.p -gii. strains emerged and formed two variants in approximately the past years. phylodynamics of gii.p -gii. strains in the vp region we estimated the transition of population sizes of gii.p -gii. in the vp region using a bayesian skyline plot. the population of this region increased sharply in around and remained constant from after an immediate reduction (figure ) . these results suggest that this was compatible between the fluctuation of the plot and actually epidemiological gii. prevalence. conformational epitopes and selective pressures in the vp protein were analyzed in order to assess the possibility of antigenicity changes at pre and post emergence of gii.p -gii. based on the time-scaled phylogeny for the vp region in figure . as a result, common epitopes among strains of clusters , (gii.p -gii. clusters) and [a closely related cluster (gii.p -gii. strain) to the gii.p -gii. clusters] were found in the shell domain and the protruding (p ) domain. although the amino acid epitope positions were similar among the strains, many amino acid substitutions were identified ( figure and table ). substitutions in epitopes were also identified in strains belonging to the same cluster. of these substitution residues, amino acids (aa) , aa , aa , aa , and aa are located close to the histo-blood group antigen figure | that comprise the dimer structures is colored gray (chain a) and dim gray (chain b). the predicted epitope regions of the strains are circled in black and the amino acids of the epitopes with no substitutions are colored blue for the shell domain and cyan for the p domain. positive selection sites are colored yellow with the positions under the order of alignments. orange circles represent the hbga binding sites on the structures. amino acid substitutions on the epitopes, non-epitopes, and positive selection sites within the clusters are colored green, magenta, and red, respectively. and arg leu/glu/pro). of these sites, aa and aa were at predicted epitopes ( figure ) . these results indicate that gii.p -gii. could evolve with changes in antigenicity and binding affinity to hbgas. to assess the possibility of changes of rna replication properties at pre and post emergence of gii.p -gii. based on the timescaled phylogeny for the rdrp region in figure , we mapped the amino acid substitutions between gii.p strains and other rdrp genotypes onto three-dimensional structures of the rdrp protein (figure ) . a total of amino acid substitutions were identified among the gii.p and gii.p strains (accession number; fj ). of these, the substitutions at aa , aa , aa , and aa were located close to the interface between monomers, while substitutions at aa , aa , aa , aa , and aa were located close to the active site ( figure a) . moreover, a total of amino acid substitutions were estimated among the gii.p and gii.p (a closely related genotype to gii.p ) strains (accession number; kj ). the substitution at aa was located adjacent to the interface between monomers, whereas the substitutions at aa , aa , aa , and aa were located proximal to the active site ( figure s ). for intra-gii.p genotype, phe leu and lys arg substitutions were found around the active site in the strains of cluster ( strains) ( figure b ). thirty one substitutions were also found in the strains belonging to the cluster ( strains), and a ser asn substitution was located proximally at the interface between monomers. ile val, ile val, asp glu, ile val, thr ala, val ile, asn ser, val met, gly ser, and phe leu substitutions were located close to the active sites ( figure c ). links in circulation of prevalent gii.p -gii. (cluster ) in japanese regions and in other countries were analyzed using a median joining network. the strains analyzed in this study were divided into four clusters. strains belonging to cluster were mainly found from japan, while those belonging to clusters and were found in various countries including japan, china, hong kong, taiwan, and australia. the strains in cluster were detected only in miyagi prefecture in japan. notably, the network contained the key strains that were linked to the many other strains (figure ) . these results suggest that the gii.p -gii. strains form a broad network and that some strains are associated with the prevalence of the virus. in this study, we demonstrated the molecular evolution of both the vp and rdrp regions of gii.p -gii. since the emergence of the virus. first, a mcmc time-scaled evolutionary phylogenetic tree based on gii. vp nucleotide sequences showed that all the present strains grouped into a total of seven clusters (figure ) . trees also indicated that the recently emerged gii.p -gii. strains uniquely formed clusters (clusters and ) based on both the vp and rdrp regions. the common ancestor of the vp region of the gii.p -gii. viruses diverged first, followed by divergence of the rdrp region after a severalyear delay (figures , ) . previous reports have also suggested a gap in the divergence of gii.p -gii. strains (mizukoshi et al., ; nagasawa et al., ) . to resolve this issue, we analyzed the phylogenetic distances of the vp and rdrp regions and found that the distance of the vp region was longer than that of rdrp, indicating that the genetic diversity of vp in gii.p -gii. is larger than that of rdrp (figure ) . however, the evolutionary rates for the overall cluster in gii.p -gii. were similar between the vp and rdrp regions (table ) . thus, we also calculated the rates of the strains belonging to the cluster , which resulted in reduction of the value in the rdrp region, but not in the vp region (data not shown). these results suggest that the differences of divergent times and genetic distances between these regions may be associated with those in the changes of the evolutionary rates. furthermore, we analyzed the time-scaled mcmc phylogenetic trees by adding sequences of gii. for the vp , sequences of gii.p for the rdrp and data of not only collection years but also months, which produced confidence intervals ( % hpd values) that were smaller in our analyses than in an earlier report (sang and yang, ) . thus, more precise collection data, as well as the use of a large number of virus strains, may facilitate the construction of more precise mcmc phylogeny. the evolutionary rates of the vp and rdrp regions in the gii.p -gii. were ∼ . and ∼ . × − substitutions/site/year, respectively ( table ) . the rate for gii. is likely similar to that for gii. in the vp region, but lower than that of gii. (bok et al., ; siebenga et al., ; mizukoshi et al., ; motoya et al., ) . furthermore, the rate for gii.p is likely analogous to that for gii.p , but lower than that of gii.p ozaki et al., ) . interestingly, it has been suggested that gii.p -gii. virus prevalence is associated with the rapid evolution of the vp regions with a high ratio of non-synonymous amino acid substitutions to synonymous substitutions (bull et al., ; parra et al., ) . previous reports have also suggested that the rates of non-synonymous amino substitutions in the vp protein differ between gii. and gii. , with gii. being greater than gii. (mori et al., ) ; therefore, the rates of antigenicity change between gii. and gii. viruses may be distinct. in contrast, it has been estimated that the number of negative selection sites in gii.p rdrp are larger than that in gii.p (ozaki et al., ) , and thus variations in nucleotide substitution in gii.p rdrp may be restricted compared to gii.p . however, despite such restrictions, we observed a lower evolutionary rate in gii.p than gii.p . this may be due to differences in replication activities and/or in replication errors of the rdrp protein between gii.p and gii.p . additional in vitro studies may be needed to address these possibilities. we also assessed the phylodynamics of gii.p -gii. and found that the genome population sizes of gii.p -gii. increased rapidly during - and then decreased rapidly thereafter (figure ) . these fluctuations may be associated with epidemics of the newly emerged hunov and the acquisition of herd immunity. for example, the fluctuations in the phylodynamics of the gii. virus could be associated with the emergence of a variant virus with altered antigenicity and the acquisition of herd immunity to the vp capsid protein of that virus lindesmith et al., ; motoya et al., ) . thus, it is possible that the phylodynamic fluctuations in gii.p -gii. identified in this study are associated with epidemics in various regions and subsequent acquisition of herd immunity (chan et al., ; fu et al., ; matsushima et al., ; dang thanh et al., ) . these results suggest that continuous phylodynamic analysis may provide early notice of other hunov epidemics and repeat prevalence of hunov, including gii. . we additionally found amino acid substitutions in the conformational epitopes of the vp capsid protein of gii.p -gii. ( figure and table ). such substitutions were also identified around the hbga binding sites. such evolutionary substitutions may induce both changes in antigenicity and in hbga binding ability (tan et al., ; chen et al., ; de rougemont et al., ; lindesmith et al., ; debbink et al., ; jin et al., ) . indeed, lindesmith et al. showed that some amino acid substitutions (corresponding to aa -aa in our alignments) are located adjacent of the hbga binding sites and result in changes in the antigenicity of the gii. capsid protein (lindesmith et al., ) . jin et al. also reported changes to the antigenicities and hbga binding affinities of gii. capsid proteins belonging to different phylogenetic clusters (jin et al., ) . moreover, an amino acid substitution (aa ) located adjacent the hbga binding sites was identified as a possible positive selection site. previous reports have suggested that aa and aa are associated with the hbga binding ability (he et al., ; koromyslova et al., ) ; however, to the best of our knowledge, there have been no reports regarding possible roles of the aa substitution in antigenicity and the hbga binding capability, which is a subject for possible further study. we also showed that gii.p -gii. contains amino acid substitutions in residues adjacent to the rdrp active sites and the contact surfaces between rdrp monomers (figure ) . ng et al. showed that the amino acids around the active sites regulate viral genome replication (ng et al., ) . moreover, substitutions around the monomer the residues of active sites for rna replication are colored red. the purple quadrilaterals highlight the region of the active site cleft. figure | a median joining network for the major epidemic cluster of gii.p -gii. strains based on nucleotide sequences from the rdrp to vp ( bp). the detected areas for japanese strains used in the analysis are shown with the number of strains on a japanese map. the filled circles represent viral haplotypes, including collection years and months for the strains. the sizes of the circles represent the number of strains within the haplotype. red arrows indicate strains associated with the production of many haplotypes. contact surfaces of rdrp affect the stability of the dimer and its rna binding abilities . thus, the similar substitutions found in the present study warrant additional investigation into changes in the properties of the rdrp protein. previous studies have also revealed key amino acids associated with the efficiency of hunov genome replication (bull et al., ; eden et al., ) . of these, the threonine residue at aa is phosphorylated by a host factor, akt, and is involved in producing high replication rates (eden et al., ) . this residue is located around the interface between monomers in the rdrp structure. the rdrp protein of gii.p contains a threonine residue at the position, while that of gii.p contains an asparagine (data not shown). this may suggest that the enzyme activity of rdrp differs between the gii.p and gii.p genotypes. we constructed a genome network of the gii.p -gii. strains examined in this study and found four major clusters (figure ) . notably, the strains belonging to clusters and were detected exclusively in japan, while the strains belonging to clusters and were detected from various countries. moreover, the two haplotype strains found in japan might give rise to many variant types. however, we could not determine specific amino acid substitutions in the strains belonging to clusters and , which contained the haplotype strains (data not shown). thus, these haplotype strains may exhibit no phenotypes acquiring high infectivity due to the mutations, although the reason for this remains unclear at present. moreover, the strains belonging to cluster had unique amino acid substitutions in p , p , protease, rdrp, and vp proteins (data not shown). these viruses were found only in miyagi prefecture in japan, during short periods and was never detected in other areas, perhaps because these mutations are deleterious to the propagations of the virus. as a limitation, the results of the present analyses may partially be affected by selection bias introduced in the collection of the strains. in conclusion, the gii.p -gii. virus has evolved differently to gii.p -gii. . however, this virus may have the potential to alter its antigenicity, host-binding capability (i.e., hbga) and genome replication efficiency. such changes could recurrently generate variants of gii. with the potential to produce pandemics such as those caused by gii. variant strains. thus, additional and continuous evolutionary analyses of this genotype should be needed in the future. the datasets generated for this study can be found in the genbank and the accession numbers are as follow; lc -lc , lc , lc , lc -lc , lc -lc , lc -lc . the studies involving human participants were reviewed and approved by the research and ethical committees for the use of human subjects of the national institute of infectious diseases, tokyo, japan (no. ). informed consent was obtained from all participants, which was acquired from the subjects or their legally acceptable representatives for sample donation. ym, kk, and hk designed this study. ym conducted all bioinformatic analyses and experiments. yd and mk performed the next-generation sequencing. ym, fm, nsa, yu, yo, tm, ht, nn, nsh, hy, ro-n, rs, rt, ft, tt, ks, hs, kn, ja, and hi collected samples and conducted the sanger sequencing. ym and hk wrote the manuscript. hs, no, ar, kk, and hk supervised this study. all authors read and approved the manuscript. this work was partly supported by a commissioned project for research on emerging and re-emerging infectious diseases from japan agency for medical research and development, amed (grant numbers; fk h and fk h ) . a systematic review and meta-analysis of the global seasonality of norovirus improving the accuracy of demographic and molecular clock model comparison while 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conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited we would like to thank enago for the english language review. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material key: cord- -jh fdks authors: jiang, yi; cheng, xu; zhao, xiumei; yu, yan; gao, mingyan; zhou, sheng title: recombinant infectious bronchitis coronavirus h with the spike protein s gene of the nephropathogenic ibyz strain remains attenuated but induces protective immunity date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: jh fdks infectious bronchitis (ib) is a highly infectious viral disease responsible for major economic losses in the poultry industry. a reverse genetic vaccine is a safe, rapid, and effective method of achieving ib prevention and control. in this study, we constructed the recombinant strain, rh -s /yz, using a reverse genetic system, based on the backbone of the h vaccine strain, with the s gene replaced with that of the qx-like nephropathogenic strain, ck/ch/ibyz/ , isolated in china. the results of dwarf chicken embryos, growth kinetics, and viral titration in the embryos demonstrated that the biological characteristics of the recombinant virus remained unchanged. like the rh -infected group and in contrast to the ribyz-infected group, no mortality, clinical signs, or lesions were observed in the lungs or kidneys of young chickens inoculated with rh -s /yz. the viral loads in various tissues, cloacal, and oral swabs was lower in most types of samples, indicating that the rh -s /yz strain was highly safe in chicks. compared to rh vaccination group, when the efficacy of this strain was evaluated against the qx-like ibv strain, better protection, with % survival rate and no disease symptom or gross lesion was observed in the chickens vaccinated with rh -s /yz. increased levels of ibv-specific antibodies were detected in the serum of the rh -s /yz-vaccinated animals days post-vaccination. collectively, our results suggest that the recombinant strain, rh -s /yz, may represent a promising vaccine candidate against qx-like ibvs. infectious bronchitis (ib) was first described as a respiratory disease affecting chicks in the us in as an acute and highly contagious viral disease, and continues to cause major economic loss within the poultry industry worldwide [ ] [ ] [ ] . as a result of the damage to the respiratory system, the clinical signs of this disease include depression, coughing, head-shaking, as well as nasal and ocular discharge [ ] . since the virus causing ib replicates in many non-respiratory epithelial surfaces (e.g., kidney and gonads), nephritis, reduced egg production, and egg quality is also observed [ , ] . the causative agent of ib is infectious bronchitis virus (ibv), a non-segmented, positive-sense, single-stranded rna virus, which belongs to the genus gammacoronavirus, family coronaviridae, in the order nidovirales [ ] . the full length rna genome of ibv is . kb in length and encodes non-structural proteins, four accessory proteins, and four structural proteins (spike [s], matrix [m], nucleocapsid [n] , and envelope [e] ). the s protein is cleaved into s and s as two subunits at the s /s cleavage site. the s protein contains the receptor-binding domain and mediates viral attachment to host cells, whereas s is responsible for membrane fusion [ , ] . errors generated during the genomic replication of ibv and the selective pressure following the use of live attenuated vaccines or multiple infections with different ibv serotypes has led to the emergence of numerous ibv variants [ , ] . in particular, small change as little as % in the amino acid composition of s gene may lead to alteration in cross protection among closely related serotypes [ ] . in china, ibv was first observed during the early s, and outbreaks have since been frequently reported [ ] . the qx-like genotype is thought to have originated during the mid- s and continues to be the predominant strain, whereas the ldt , / , and taiwan subtypes have also recently been frequently isolated in china [ ] [ ] [ ] [ ] . severe nephritis, false layer syndrome, and high mortality are observed in chickens infected with this viral genotype, which represents a major problem in the poultry industry, particularly in china. using a live attenuated or inactivated vaccine is typically considered to be the most cost-effective means of controlling viral infections [ ] . unfortunately, inactivated ib vaccines are not effective if used alone, which could induce little or no protection against egg loss [ , ] , as well as impaired ciliary activity in the trachea [ ] ; thus, birds must continue to be given one or a series of vaccinations with live attenuated ib vaccines to provide broad heterologous protection [ ] . commercial attenuated live vaccines used against ibv in china include the h , ldt , and / strains [ ] ; however, phylogenetic analysis indicates that the qx-like genotype is genetically distant from the strains described above, which may explain the poor cross-protectivity against infection in chickens immunized with these classical vaccines [ , ] . recently, there has been increasing research on live attenuated vaccines against the qx-like ibv strains. different strains of qxlike ibvs were serially passaged in embryonated eggs or primary chicken kidney cells (ck) to obtain vaccines that are less virulent and exhibit high immunogenicity against the qx-like epidemic ibv strains [ ] [ ] [ ] [ ] [ ] . however, in addition to causing tissue damage or secondary bacterial infections in young vaccinated chicks [ , ] , live attenuated vaccines of rna viruses may not be genetically stable or they may be associated with the tendency to revert back to a virulent form [ , ] . moreover, the potential for recombination between vaccine and virulent strains can lead to the creation of new virulent virus [ ] . furthermore, it takes a substantial amount of time to prepare a extensively passaged attenuated vaccine strain, which may not be able to effectively solved the loss problem caused by the rapid variation exhibited by prevalent strains on the flocks. in considering the limitations of the live attenuated and inactivated ibv vaccines, reverse genetic ibv vaccines have been developed in recent years as they display increased safety and efficacy [ ] [ ] [ ] . as reverse genetic technology is primarily based on the h and beaudette strains [ ] [ ] [ ] , both of which are passagegenerated attenuated strains, the mechanism of virus virulence attenuation remains unclear, thereby posing difficulties in the development of attenuated vaccines using this technology. in addition to the function of host invasion and increasing genetic diversity, the ibv s subunit can induce virus neutralizing and cross-reactive antibodies [ ] . therefore, some researchers have created an attenuated ibv vaccine by replacing the s or s genes from other virulent strains using reverse genetic technology to generate protection against strains of the corresponding serotype [ , , ] . in the present study, we constructed a recombinant strain, using the reverse genetic system. rh -s /yz is based on the backbone of the vaccine strain h , and the s gene was replaced with that of the qx-like nephropathogenic strain, ck/ch/ibyz/ , which was isolated in china. our results demonstrate that this recombinant strain is safe and provides effective protection in young chickens against qx-like ibv challenge. the h vaccine strain is currently a widely used vaccine strain, created from serial passages of the h strain isolated in . the ck/ch/ibyz/ strain (referred to as ibyz, genbank kf . ) used in this study is classified as a qx-like ibv isolated from a flock presenting with ib symptoms by our research group in in jiangsu province, china. both of these strains were recovered from a full-length clone using reverse genetics, as previously described [ , ] . fertilized specific pathogen free (spf) chicken embryos were obtained from beijing merial vital laboratory animal technology co., ltd. (beijing, china) and hatched in our lab (lab of chicken infectious disease protection and control, poultry institute, chinese academic of agriculture sciences, yangzhou, china). for hatching, one-day-old spf chickens from the spf chicken embryos as described previously were incubated at °c in a relative humidity of - %. all chickens were maintained in isolators under negative pressure, and food and water were provided ad libitum. the ribv rh -s /yz strain used in this study is described in the schematic illustration presented in fig. . the genome rna was synthesized in vitro by t rna polymerase and transfected into bhk- cells, as previously described [ , ] . the cell supernatants were harvested h following transfection and propagated in -day-old spf chicken embryos. the allantoic fluid was harvested for rt-pcr, whole genome sequencing, and was then stored at À °c until further use. to determine the recombinants' pathogenicity in chicken embryos, . ml of rh , ribyz, or rh -s /yz virus (diluted to : in normal saline) was inoculated into the allantoic cavities of five -old-day embryonated spf chicken eggs, respectively. chick embryo lesions were examined for lesions at h postinoculation at °c. to examine the viral growth ability in chicken embryos, a realtime reverse transcription quantitative polymerase chain reaction (rt-qpcr) method was established. according to the sequences of ibv from genbank, primers were designed based on conservative area in the -utr. the upstream primer was -ccgttgctt gggctacctagt- , and the downstream primer was -cgcctac cgctagatgaacc- . the amplification product was cloned to pmd -t vector (takara) as a positive plasmid and its concentration was measured. a gradient dilution of  -  copies/ll of the plasmid was used as template for quantitation test. by plotting the cycle threshold (ct) values against the copies of the plasmid, the standard curve was generated. -day-old embryonated spf chicken eggs (six eggs/group) were inoculated with rh , ribyz, and rh -s /yz at a dose of viral rna copies/ ll, and used for growth curve experiments. the allantoic fluids were collected separately by syringe from the six inoculated embryonated eggs of each group at , , , , , , h per inoculation. rna was extracted from the allantoic fluids using trizol reagent (invitrogen, carlsbad, ca, usa) following the manufacturer's instructions. cdna was obtained by reverse transcription using a primescript rt master mix perfect real time kit (takara, otsu, shiga, japan). the viral copies were measured by absolute quantitative method of realtime pcr, which was performed using sybr Ò premix ex taq tm ii (takara, otsu, shiga, japan) on an applied biosystems fast real-time pcr system. the standard curve was plotted against the log of the template copy number. all of the assays were run in triplicate and the copy number of each virus was calculated according to the standard curve. serial -fold dilutions from copies to copies per . ml of virus of the recombinant strain, rh -s /yz, and its parental strains (rh and ribyz) were inoculated into the allantoic cavity of -day-old embryonated spf chicken eggs. for each dilution, . ml of the virus solution was injected into each egg and eggs were used for each dilution. pbs was used as a negative control. after a h incubation, the dead embryos were considered nonspecific deaths and discarded. the embryos were examined for the presence of specific lesions caused by the virus after a -h incubation. dead and live embryos that displayed ibv infectious signs (e.g., dwarfing, curling, and stunting) were considered positive samples. the % embryo infectious dose (eid ) of these ribvs was calculated using the reed-muench method [ ] . the chickens were separately housed in isolators under consistent conditions, and food and water was provided ad libitum. a total of one-day-old spf chickens were randomly divided into four groups (n = per group). the chickens in the experimental groups were intranasally inoculated with ll allantoic fluid per chick containing eid of the rh , ribyz, and rh -s / yz strains. the control group (n = ) was inoculated with ll sterile pbs via the same route. the morbidity and mortality was schematic diagram for the construction of the chimeric s gene and production of a full-length cdna of rh -s /yz. (a) replacement of the h s fragment by the corresponding sequence of ibyz for construction of pyzs h s . the plasmids pibyzs and ph s contained the s gene of ibyz strain and h strain, respectively, were constructed during the establishment of reverse genetic system. primers ps f and ps r were used to amplify the s fragment of ibyz and vector fragment, while primers ps f and ps r were used to amplify the s fragment of h strain. by overlapping pcr, the pyzs h s was constructed which contained a chimeric s gene. (b) strategy for the construction of full-length cdna clones of rh -s /yz. ten cdna fragments covering the entire genome of h strain was amplified by rt-pcr. unique bsa i sites were inserted at the junctions between each clone, a unique t start site was inserted at the end of clone tm , and a -nucleotide t tail was inserted at the end of clone tm . the s fragment in the pmdtm plasmid of h strain was replaced by chimeric s gene. by using appropriate ligation strategy, the genomic cdna of rh -s /yz was assembled by in vitro ligation using appropriate restriction sites as indicated. followed-up for days. all experimental groups were monitored daily for clinical signs related to ib infection for days postinfection (dpi), including coughing, sneezing, and tracheal rales. dead chickens were examined for gross tracheal, lung, and kidney lesions. a total of -day-old spf chickens were assigned to four groups (n = per group). the birds were inoculated with ll allantoic fluid containing eid of the rh , ribyz, or rh -s /yz strains, respectively via eye drop and the intranasal route and ll pbs per chick was administered to the control group (n = ) via the same route. the tracheas, lungs, kidneys, and bursa from the inoculated birds per group were harvested at , , , and dpi, weighed, and collected into ml pbs per sample and frozen at À °c. after grinding the samples, the viral rna was extracted using trizol, and the cdna was obtained by reverse transcription using a primescript rt master mix perfect real time kit (takara, otsu, shiga, japan). the viral rna copies from each of the different samples were detected by real-time pcr as described above. all assays were run in triplicate and the copy number for each virus was calculated according to the standard curve. after inoculation with eid dose of the rh , ribyz, and rh -s /yz strains, three infected chickens per group were randomly selected to be euthanized by bleeding, and the tissue pathology was examined in the different groups at , , , dpi. the tracheas, lungs, kidneys, and bursas of three dead or other randomly-selected chickens from the experimental and control groups (inoculated with pbs) were collected at or dpi and further processed for histopathology. the samples were fixed in formalin, embedded in paraffin, cut into lm sections, and stained with hematoxylin and eosin. the slides were examined under light microscopy for the presence of lesions. to determine the level of viral shedding of the different recombinants, birds per group were inoculated with ll allantoic fluid containing eid of the rh , ribyz, or rh -s /yz strains, respectively. ll pbs per chick was administered to the control group via eye drop and the intranasal route. . oral and cloacal swabs were collected from each bird into ml pbs at , , , , , and dpi and stored at À °c. after freezing and thawing three times and centrifuging at ,  g for min at °c, relative amount of virus present in ll of supernatants of each sample was quantified by rna extraction, reverse transcription, and real-time pcr as described previously. . . efficacy of rh -s /yz vaccination . . . viral challenge in total, one-day-old spf chickens were divided into three groups of chickens. the three groups were intranasally vaccinated with the rh and rh -s /yz strains at a dose of eid / ll, and the control group received pbs. two weeks post-vaccination, the chickens in each group were challenged with the qx-like strain, ribyz, at eid / ll via eye drop and the intranasal route. any clinical signs, as well as the percentage of morbidity and mortality were recorded for days. the dead chickens were also examined for gross tracheal, lung, and kidney lesions. -day-old spf chickens were divided into three groups (n = per group). after vaccinating with the rh and rh -s /yz strains at a dose of eid / ll, the sera of birds from each group were randomly collected and antibodies were tested using an elisa created in our lab at , , , and days post-vaccination (dpv). elisa plates were coated with antigen of culture supernatants of inactivated ibv rmj , a vero cells adaption strain domesticated from its parental strain of ibyz in our lab. individual chicken sera collected from different groups diluted to : were loaded and the plates were incubated at °c for min. ibv-specific igg was detected with anti-chicken igy (igg) (whole molecule)-peroxidase antibodies produced in rabbits (sigma-aldrich, germany). after incubated with , , , -tetrame thylbenzidine(tmb) substrate for min and terminated with mol/l h so solution, the absorbance at nm was measured using a model microplate reader (bio-rad, usa). graphpad prism software (graphpad software inc., la jolla, ca, usa) was used for the statistical analyses. for the antibody test, growth kinetics, tissue tropism, and viral shedding test, the collected data were analyzed using a two-way anova to determine whether there was a significant difference between the different groups. the significance was considered as follows: significant at p . (*); highly significant at p . ( ** ); and extremely significant at p . ( *** ). characterization of the rh -s /yz strain after being inoculated with the recombinant strains, typical ib lesions in the chick embryos from each group were observed, including amniotic membrane thickening, dwarfing, stunted growth, curling, or death of the embryo (fig. a) . the eid of the virus was calculated according to the reed-muench method. the titers of the rh , ribyz, and rh -s /yz strains were determined to be . eid /ml, . eid /ml, and . eid /ml, respectively (fig. b) . the growth kinetics of the ribvs were assessed by inoculation with copies of virus per egg in eggs from each group. the relative viral load was determined at , , , , , , and h post-inoculation by quantitative real-time rt-pcr. the growth curves of all three strains reached a relatively stable plateau at - h after infection; the level of viral rna for the rh -s / yz strain was significantly higher than that of the ribyz strain for the majority of the time points (fig. c ). the earliest death was observed at dpi in the qx-like strain ribyz group, which virus was highly pathogenic to one-day-old spf chickens; the final mortality of this group was . % (fig. a) . the morbidity of the infected chickens in the ribyz group reached higher than %, and the diseased chickens exhibited respiratory symptoms (e.g., sneezing, coughing, as well as tracheal and bronchiolar rales), and the severe cases presented with additional signs of listlessness, huddling, and ruffled feathers. at necropsy, lesions were detected both in the respiratory and urinary system, including mucus, hyperemia, and hemorrhage in the trachea, as well as swelling and urate deposition in the kidney. edema and congestion were observed in the lungs in % of cases (fig. b) . however, following inoculation with the rh or rh -s /yz strains, the morbidity rates of the chickens was less than %, with moderate respiratory signs of coughing in some of the chickens. all of the infected chickens presented no visible lesions in the kidney and the survival rate was % through dpi in both the rh and rh -s /yz groups ( fig. a and b) . no obvious clinical signs, gross lesions, or death attributable to ibv was observed in the control group. for the chickens inoculated with the qx-like strain ribyz, lesions in the trachea at dpi were characterized as cilia necrosis and loss, epithelial cell congestion, hemorrhage, and lymphoid infiltration (fig. c ). in the bronchial and capillary lumen of ribyz-infected lungs, congestion, hemorrhage, as well as erythrocyte and inflammatory cell infiltration was observed in some severe cases from to dpi (fig. g) . moreover, erythrocyte and lymphocyte infiltration, as well as tubular dilation was observed in the kidney of ribyz-inoculated chickens (fig. k) . similar to the rh group, chickens inoculated with rh -s /yz presented with mild pathology in the trachea at the early infection time points of , dpi. lesions in the trachea consisted of the loss of epithelial cells and cilia, decreased number of secretory cells, and thickening of the mucosa (fig. b and d) . in contrast, there were no lesions observed in the lungs and kidneys of rh and rh -s /yz-infected chickens during the infection period (fig. f , h, j, and l). no histopathological evidence of damage was detected in the bursa of all the groups and no ibv-associated lesions were found in the tissue samples of the control chickens (fig. a , e, i, m-p). the time-dependent levels of viral replication in the different tissues determined by real-time rt-pcr presented in fig. are consistent with the tissue histopathology results. compared to rh and rh -s /yz groups, one day after inoculation with the qxlike strain, ribyz, the viral load in all organs began to rise rapidly, except at dpi in the trachea. the viral load in tracheal samples in the rh -s /yz group increased and peaked at dpi. the level of viral rna was significantly lower than the rh group both at dpi and dpi (fig. a) . the viral load in lung samples from all infection groups increased starting at dpi, and the viral rna in the lungs of the rh -s /yz group was significantly lower compared with both the rh and ribyz groups (fig. b) . the viral load in the kidney of all ibv-inoculated groups was detected at dpi. in contrast to the rh and rh -s /yz groups, the viral growth in kidney of ribyz-infected chickens exhibited an extremely significant increase at dpi and peaked at dpi. the viral of the recombinant ibvs, rh , ribyz, and rh -s /yz in spf chicken embryonated eggs. (c) the multicycle growth kinetics of the recombinant ibvs, rh , ribyz, and rh -s /yz in spf chicken embryonated eggs. all data are presented as the mean ± standard deviation (sd). some of the error bars are too small to be seen. the qrt-pcr was carried out with three replicates. markers of statistical difference were acquired by comparing between rh group and rh -s /yz group. *** indicates an extremely significant difference at p . ; ** indicates a highly significant difference at . < p . ; *indicates a significant difference at . < p . . rna copies of the rh -s /yz group peaked at dpi, after which they gradually decreased (fig. c) . no viral load was detected in the control samples at any of the time points. in all ibv infection groups, the viral load in both the oral and cloacal swabs generally showed a gradual decline from dpi to dpi. compared with the rh group, the reduction in viral load between or dpi was significantly greater for chickens infected with rh -s /yz than for chickens infected with rh , and the viral rna of the qx-like strain ribyz was lower than that of the rh group at , , and dpi but with no statistically significance (fig. a) . in contrast, the viral load in the cloacal swab of the ribyz group was significantly higher than that of the rh and rh -s /yz groups at dpi. compared with other groups, the viral rna in the cloacal swab of the rh -s / yz group was significantly lower at , , and dpi (fig. b) . no virus was detected in the control group at any time point. the rh -s /yz group exhibited effective protection against challenge with the qx-like strain ribyz. the mortality rate in the rh -s /yz group was %, and no clinical signs or lesions were observed during the ribyz challenge period (fig. a) . in contrast, chickens vaccinated with the rh strain began to exhibit clinical signs (e.g., coughing and nasal discharge) at days post-challenge (dpc), and some chickens showed severe signs of listlessness, hud- fig. . pathogenicity of the ribv rh , ribyz, and rh -s /yz strains. (a) percent survival of spf chickens infected at -day-of age during the -day observation period. no mortality was observed in rh , rh -s /yz, or the control groups. (b) gross lesions on the lungs and kidneys of the infected chickens at or dpi. fig. . histopathological changes in the trachea, lungs, kidneys, and bursa of spf chickens infected with the recombinant rh , ribyz, and rh -s /yz strains at -day-ofage. black arrows in the trachea indicate cilia and necrosis loss. black arrows in the lungs indicate congestion and hemorrhage. trachea samples were collected at , dpi (a-d); lung and bursa samples were collected at , dpi (e-h; m-p); and kidney samples were collected at , dpi (i-l). black arrows in kidneys indicate tubular dilation, congestion, and inflammation. scale bar = lm or lm. fig. . viral load in the different organs at , , , and dpi of spf chickens infected with the recombinant strains rh , ribyz, and rh -s /yz at -day-of age. the bars indicate means ± standard deviations. *** indicates an extremely significant difference at p . ; ** indicates a highly significant difference at . < p . ; *indicates a significant difference at . < p . . dling, and ruffled feathers, with the mortality rate reaching % during the observation period. at necropsy, the dead animals displayed typical kidney lesions characterized by swelling and urate deposition in the tubules and ureters. in contrast to chickens vaccinated with rh -s /yz, the chickens in the unvaccinated control group exhibited clinical signs at dpc, and a % mortality rate; the gross lesions of the dead birds in this group were similar to that of the rh group (fig. b ). antibodies against the ibvs were measured using an elisa (fig. c) . none of the birds in the rh or rh -s /yz groups showed a positive antibody response at dpv, whereas % of the birds had an antibody response detectable at dpv, with the antibody level gradually increasing during the observation period. however, there was no significant difference in the level of serum antibodies between rh -vaccinated chickens and rh -s /yz-vaccinated chickens. no positive serum antibody response was detected in the non-vaccinated controls. vaccines, particularly live-attenuated vaccines, remain the most effective means of protection against ibv challenge (e.g., the mass serotype strains h , h , ma , and w , which are still widely used in the poultry industry) [ ] [ ] [ ] . however, due to the poor cross-protection provided between the different serotypes, these attenuated serotype-specific vaccines cannot provide complete protection, and the ib endemic in china cannot be controlled [ ] . genetic mutations and immune pressure during the replication process of the single stranded rna virus often results in the emergence of ibv variants [ , ] . to date, at least six genotypes of ibv strains have been identified in china, of which the fig. . viral loads at , , , , and dpi in the oral and cloacal swabs of chickens infected with the recombinant rh , ribyz, and rh -s /yz strains. all data are presented as the mean ± standard error of the mean (sem); *** indicates an extremely significant difference at p . ; ** indicates a highly significant difference at . < p . ; *indicates significant difference at . < p . . fig. . efficacy of the rh and rh -s /yz vaccines. (a) percent survival of the vaccinated chickens challenged with the qx-like ibv ribyz strain during the -day observation period. no mortality was observed in the chickens vaccinated with the rh -s /yz strain. (b) gross lesions observed on the kidneys of the vaccinated chickens challenged with the ribyz strain at or dpc. (c) the mean antibody od value at , , , and dpv. all data are presented as the mean ± standard error of the mean (sem). qx-type (first isolated in ) remains the most prevalent genotype [ , , ] . the poor relationship between the qx-type strains, which are abundant and display variable virulence in various parts of china, and the mass-type vaccine strains could explain the failure of the mass-type vaccination programs to control ibv in these flocks [ , ] . consequently, the development of novel vaccines against circulating ibv strains in china using local ibv strains is required [ , , ] . optimal vaccination against circulating ibv strains in china requires the development of attenuated vaccines designed from local strains [ ] [ ] [ ] ] . however, due to the particularity of rna virus replication, attenuated vaccine strains generated by continuous passage remain associated with the risk of reversion to virulence or potential recombination between vaccine strains and virulent field strains [ , , ] , which represents a substantial hidden concern to the poultry industry. to reduce the problems associated with vaccine reversion, researchers have explored the option of creating vaccine viruses using reverse genetic technology (e.g., beaudette strains carrying the s gene of the h vaccine strain, virulent m strain, or qx-like strain) [ , ] . however, the titer of the beaudette strains carrying the s gene of the vaccine h strain only reaches . ± . eid [ ] , which is lower than that of the h backbone strain. therefore, we aimed to develop a recombinant rh -s /yz strain based on the h vaccine strain that carries the s gene of the ck/ch/ibyz/ strain (a chinese qx-like nephoropathogenic strain) using reverse genetic technology [ , ] . in the present study, we isolated a nephoropathogenic strain (ibyz) in . this strain was isolated, identified, and preserved from a large-scale chicken farm where an outbreak of ibv emerged in jiangsu province. chickens infected with the ibyz strain showed severe kidney damage and a high mortality rate (fig. b) . the complete sequence has been deposited in the genbank database under the accession number of kf . the s gene sequencing data indicated that the ibyz strain belonged to the qx-like genotype (fig. ) , with a nucleotide homology with the attenuated vaccine h strain of only . %, and amino acid homology of . %. moreover, the s /s cleavage site of ibyz is hrrrr/s, which differed from rrfrr/s of the h vaccine strain. after wholegenome sequencing, we constructed a molecular clone strain using reverse genetic technology, termed ribyz. in addition, another strain used in this study is the live attenuated ibv vaccine, h , and the molecular clone strain, rh , which was constructed using the same method. the spike protein is the coronavirus structural proteins, which plays an important role on pathogenicity in coronaviruses [ , ] . however, replacing the s gene of beaudette strain by the virulent strains does not make it virulent [ , ] . the s protein can be cleaved into s and s subunits at the s /s cleavage site [ ] , the s subunit is responsible for receptor binding to the host cells [ ] . whether the s gene has an effect on the virulence of the virus is unknown. in this study, the recombinant strain, rh -s /yz, expressed the s gene of the qx-like strain, which retains the s /s cleavage site of h , maintained the ability to replicate and exhibited pathogenicity in embryos. however, providing ibyz s sequences to h did not increase its virulence to that of ribyz. ibvs replicate in many epithelial cells, including respiratory tissues, the alimentary canal, kidney, gonads, and bursa [ , ] . the qx-like ribyz strain is capable of considerable growth in the trachea, lung, and kidney, which leads to severe tissue damage. in addition, this strain caused cilia necrosis and loss, lung congestion and hemorrhage, nephritis, as well as inflammatory cell infiltration in these tissues in infected chickens at different time points postinfection. the ability of ibv to replicate within many respiratory, enteric, and other epithelial surfaces may be partially related to the fact that the attachment of ibv to host cells is dependent on sialic acid on the cell surface being recognized by the s subunit [ , , ] . however, the binding of virus to the host cell is the first step in determining tropism and s is responsible for membrane fusion [ ] . thus, to further explore the different tissue tropism between rh -s /yz and ribyz, we examined the viral loads and histopathology in the tissues of the rh -s /yz-infected group. it was found that rh -s /yz remains moderately pathogenic in the trachea (e.g., some cilia necrosis and loss and decreased secretory cells) but no lesions were observed in the lungs and kidneys of infected chickens, in contrast to chickens infected with the ribyz strain. this finding suggests that both the s and s genes might play an important role in viral tissue tropism as also suggested by others [ ] . another possibility is that the lack of viral replication and damage in kidney following rh -s / yz is due to lower replication ability of backbone except s gene from its parental rh strain. the viral load of the respiratorytype strain, rh in the different tissues indicated that the viral levels in the trachea, early following infection were significantly higher than those of ribyz, which indicates that the ability of the virus to attached to trachea epithelial cells of the rh strain was stronger than that of the ribyz strain. however, during the later time points post-infection, the level of the rh strain was significantly lower than ribyz, which suggests that the virulence of ibv depends both on tissue invasion and viral replication ability, as well as other functions mediated by other viral proteins (e.g., non-structural, structural, and accessory proteins) [ , , [ ] [ ] [ ] . the reduction of the viral load at dpi in the group infected with the ribyz strain may be associated with the necrosis and shedding of the trachea epithelium. moreover, the ability to replication in the different organs of chickens infected with rh -s /yz as reflected by viral loads at multiple time points was lower than that exhibited in the chickens infected with the ribyz strain, which demonstrated that the s subunit was not the only determinant of viral tropism that was consistent with results by others [ , , ] , and the significant difference in viral load was related to differences in the viral backbone. however, when comparing the rh -s /yz and rh strains which carry the same backbone, it was found that the viral rna of rh -s /yz in the lungs and kidneys was significantly lower than that of rh , which may due to the replacement of the ibyz s gene in the recombinant virus, whose structure on the surface contains some degree of changes, which we speculated that may affects the functionality of the s protein of coronaviruses. although cryo-em structure of ibv spike protein has been determined, the relation between structure and function of different ibv s genes is unknown. [ ] . in addition to the growth in epithelial tissues observed during the early infection period, ibv can establish long-term persistent infections in chicken flocks via oral and cloacal shedding, which represents a substantial challenge to ibv control [ , ] . in this study, we detected viral rna in the oral and cloacal swabs isolated from infected chickens at , , , , and dpi by real-time pcr. compared with rh and ribyz, the viral load in both the oral and cloacal swabs of the rh -s /yz group at different time points after infection was significantly decreased. however, while it has been reported that the persistent virus will be re-excreted at the point of lay [ ] , the persistence of rh -s /yz strain in the egg-laying chickens needs to be further verified. in a word, due to its low virulence i presented in virus loading and lesion in different tissue, oral and cloacal shedding of host animals, rh -s /yz can be considered a safe attenuated vaccine candidate for young chickens. in addition to its role in tissue tropism, the s glycoprotein also has an important function in inducing a neutralizing antibody response [ , ] , and small differences in the s contribute to poor cross protectionby neutralization test [ , ] . to evaluate the protective efficacy of this candidate vaccine, we selected the qx-like virulent ribyz strain to infect chickens at dpv that were vaccinated with either the rh -s /yz strain or rh strain. we found that little protection against ribyz virulent strain challenge in the rh vaccinated group was induced, with a % survival rate and severe clinical symptoms associated with ib. this finding could be explained by differences in the antigenicity between rh and ribyz. no death, clinical signs, or lesions were observed in the rh -s /yz-vaccinated group, indicating that this strain could provide effective protection against ribyz challenge in young chickens. since high humoral antibody titers can contribute to reduced viral replication in various organs and disease recovery [ , ] , the level of specific igg antibodies remains an important standard for evaluating the immune response to an ibv vaccine. in the present study, the humoral antibody level of chickens vaccinated with the rh -s /yz strain was observed to gradually increase compared to the non-vaccinated group. however, although the rh strain was able to induce similar levels of humoral antibodies, it provided poor protection against ribyz, suggesting that protection against ibv infection is not only with humoral immunity, but also with both mucosal immunity combined with the local tracheal and cellular immune response [ ] [ ] [ ] [ ] . thus, the detection of mucosal and cellular immunity in rh -s /yz-vaccinated animals requires further exploration. nevertheless, some studies have shown that a peptide located near the amino terminal end of s can be recognized by neutralizing monoclonal antibodies [ ] , and the s domain also plays an important role in inducing protective immunity [ , , ] . because the s domain plays an important role in cell tropism [ ] , we also considered that expressing the s subunit would provide greater safety than the entire s gene, which could lead to further destruction in some organs of the vaccinated chickens; however, further experiments are required. in conclusion, we constructed the recombinant strain, rh -s /yz, which was based on the backbone of the h vaccine strain, and replaced the s gene with that of the qx-like nephropathogenic strain, ck/ch/ibyz/ , isolated from china using the reverse genetic system. the safety results showed that this recombinant strain was not lethal to one-day-old chicks and it had not gained the ability to tissue invasion or replication in the kidneys and lungs of infected animals. moreover, the viral load of the chickens was significantly decreased compared with the rh and ribyz strains. furthermore, rh -s /yz was found to provide effective protection against challenge with the qx-like nephropathogenic strain in young chickens. the level of antibody production in the serum of the vaccinated chickens detected by elisa continued to increase after dpi. thus, rh -s /yz may be considered a potential live vaccine candidate for protection against chinese qx-like nephropathogenic ibv infection. all of the animals were cared for in accordance with the animal ethics guidelines and approved protocols, and the experimental protocols were performed with the approval of the animal welfare conceptualization, methodology, software, investigation, validation, visualization, resources, writing -original draft, writing -review & editing, visualization, funding acquisition. xu cheng: investigation, data curation. xiumei zhao: visualization, investigation. yan yu: investigation, software. mingyan gao: investigation. sheng zhou: conceptualization, validation, resources, writing -original draft the long view: years of infectious bronchitis research coronaviruses in poultry and other birds 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avian coronavirus infectious bronchitis virus induces an attenuated phenotype both in vitro and in vivo the hydrophobic domain of infectious bronchitis virus e protein alters the host secretory pathway and is important for release of infectious virus the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity protection against infectious bronchitis virus by spike ectodomain subunit vaccine effects of hypervariable regions in spike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins amino acids within hypervariable region of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes identification of amino acids involved in a serotype and neutralization specific epitope within the s subunit of avian infectious bronchitis virus location of antigenic sites defined by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide a long-term study of the pathogenesis of infection of fowls with three strains of avian infectious bronchitis virus systemic and local antibody responses to infectious bronchitis virus in chickens inoculated with infectious bursal disease virus and control chickens specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus protectotypic differentiation of avian infectious bronchitis viruses using an in vitro challenge model kinetics of lymphocytic subsets in chicken tracheal lesions infected with infectious bronchitis virus cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry analysis of an immunodominant region of infectious bronchitis virus identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus we would like to thank all members of the poultry institute, chinese academy of agricultural sciences (yangzhou, china) for their contribution to this study. yi jiang designed the study and conducted all the experiments, analysis and interpretation of the data, and wrote the manuscript. sheng zhou helped perform the recombinant ibv construction. xu cheng, xiumei zhao, yan yu, and mingyan gao participated in the whole animal experiment in different groups. xu cheng conducted the experiments involving viral shedding. all authors read and approved the final manuscript. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/ . /j.vaccine. . . . key: cord- - oc ykds authors: gendy, sherif; chauhan, ashvini; agarwal, meenakshi; pathak, ashish; rathore, rajesh singh; jaswal, rajneesh title: is long-term heavy metal exposure driving carriage of antibiotic resistance in environmental opportunistic pathogens: a comprehensive phenomic and genomic assessment using serratia sp. srs- -s- date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: oc ykds the carriage of both, heavy metal and antibiotic resistance appears to be a common trait in bacterial communities native to long-term contaminated habitats, including the savannah river site (srs). there is widespread soil contamination at the srs; a united states department of energy (doe) facility with long-term contamination from past industrial and nuclear weapons production activities. to further evaluate the genomic and metabolic traits that underpin metal and antibiotic resistance, a robust mercury (hg) and uranium (u)-resistant strain- srs- -s- , was isolated. minimum inhibitory concentration of this strain revealed resistance to hg ( μg/ml) and u ( mm), the two main heavy metal contaminants at the srs. metabolic assessment of strain srs- -s- using biolog metabolic fingerprinting analysis revealed preference for carbohydrate utilization followed by polymers, amino acids, carboxy acids, and esters; this physiological activity diminished when hg stress was provided at and μg/ml and completely ceased at μg/ml hg, indicating that continued release of hg will have negative metabolic impacts to even those microorganisms that possess high resistance ability. development of antibiotic resistance in strain srs- -s- was evaluated at a functional level using phenomics, which confirmed broad resistance against . % of the antibiotics tested. evolutionary and adaptive traits of strain srs- -s- were further assessed using genomics, which revealed the strain to taxonomically affiliate with serratia marcescens species, possessing a genome size of , , bp, , proteins (cds), genes for transfer rna (trna), and an average g + c content of . . comparative genomics with closest taxonomic relatives revealed distinct genes in srs- -s- , with multiple functions related to both, antibiotic and heavy metal resistance, which likely facilitates the strain’s survival in a metalliferous soil habitat. comparisons drawn between the environmentally isolated serratia srs- -s- with other strains revealed a closer functional association with medically relevant isolates suggesting that propensity of environmental serratia isolates in acquiring virulence traits, as a function of long-term exposure to heavy metals, which is facilitating development, recruitment and proliferation of not only metal resistant genes (mrgs) but antibiotic resistant genes (args), which can potentially trigger future bacterial pathogen outbreaks emanating from contaminated environmental habitats. the carriage of both, heavy metal and antibiotic resistance appears to be a common trait in bacterial communities native to long-term contaminated habitats, including the savannah river site (srs). there is widespread soil contamination at the srs; a united states department of energy (doe) facility with long-term contamination from past industrial and nuclear weapons production activities. to further evaluate the genomic and metabolic traits that underpin metal and antibiotic resistance, a robust mercury (hg) and uranium (u)-resistant strain-srs- -s- , was isolated. minimum inhibitory concentration of this strain revealed resistance to hg ( µg/ml) and u ( mm), the two main heavy metal contaminants at the srs. metabolic assessment of strain srs- -s- using biolog metabolic fingerprinting analysis revealed preference for carbohydrate utilization followed by polymers, amino acids, carboxy acids, and esters; this physiological activity diminished when hg stress was provided at and µg/ml and completely ceased at µg/ml hg, indicating that continued release of hg will have negative metabolic impacts to even those microorganisms that possess high resistance ability. development of antibiotic resistance in strain srs- -s- was evaluated at a functional level using phenomics, which confirmed broad resistance against . % of the antibiotics tested. evolutionary and adaptive traits of strain srs- -s- were further assessed using genomics, which revealed the strain to taxonomically affiliate with serratia marcescens species, possessing a genome size of , , bp, , proteins (cds), genes for transfer rna (trna), and an average g + c content of . . comparative genomics with closest taxonomic relatives revealed distinct genes in srs- -s- , with multiple functions related to both, antibiotic and heavy metal resistance, which likely facilitates the strain's survival in a metalliferous soil habitat. heavy metal contamination is a global environmental problem shown to cause both ecological and public health problems (tchounwou et al., ) . however, ecosystems with pervasive and long-term heavy metal contamination, such as the savannah river site (srs), located in aiken, sc, united states, are known to harbor metal-resistant microorganisms, which have acquired genomic features to resist and circumvent metal toxicity (ye, ; agarwal et al., ; pathak et al., pathak et al., , gendy et al., a,b) . the two main heavy metal contaminants within the impacted srs soils include mercury (hg) and uranium (u) (sowder et al., ) . our ongoing culture-dependent and culture-independent work on the srs metalliferous soils has demonstrated several different bacterial groups involved in metal cycling processes, such as arthrobacter spp., burkholderia spp., bacillus spp., bradyrhizobium spp., pseudomonas spp., lysinibacillus spp., paenibacillus spp., stenotrophomonas spp., and serratia spp. (agarwal et al., a,b; gendy et al., b) . many of these groups have been demonstrated to cycle hg by activity of their mer operon, which consists of regulatory proteins-merr and merd, transport proteins-merp, t and e, as well as protein(s) with reductase activity-mera pathak et al., ) . in reference to u cycling processes, several different genes have been shown to render u resistance and biomineralization, such as the gene encoding uranium binding complex (ubc), uranium response in caulobacter (urca), phosphatase genes (phoy; phok; and phon) and pib-type atpase (martinez et al., ; hillson et al., ; nilgiriwala et al., ; nongkhlaw et al., ; yung and jiao, ; kulkarni et al., ; thorgersen et al., ) . however, no specific bacterial operon has been shown to be active under u stress to date. despite this advantage of microbially based heavy metal bioremediation, studies have also unequivocally shown that bacteria can acquire antibiotic resistance when exposed to longterm contamination, especially with hg and u (benyehuda et al., ) , including our previous work gendy et al., a,b; pathak et al., ) . this builds upon significant evidence that exists on the srs soils which unequivocally shows higher levels of antibiotic resistance in contaminated srs soils relative to reference soils wright et al., ; thomas et al., ) . furthermore, both, empirical and anecdotal evidence from srs soils strongly suggest recruitment of antibiotic resistances in many human pathogens that are native to these soils [e.g., burkholderia, ralstonia, massilia, acinetobacter, and pseudomonas ]. despite the fact that many of these srs native soil bacteria are bioremediative being armed with an arsenal of metal resistance genes (mrgs), but they can also pose public health risks by virtue of their acquired antibiotic resistance(s), via the activity of antibiotic resistant genes (args), thus strongly negating their beneficial properties. thus, heavy-metal induced development and carriage of args, or the 'resistome' , within the native soil microbiota, can lead to the emergence and rapid spread of multidrug-resistant (mdr) bacteria, also referred to as "superbugs" (davies and davies, ) , leading to potential future pathogenic outbreaks. in fact, antimicrobial resistance is projected to cause approximately million deaths, annually by year (o'neill, ) , outpacing cancer, which is the current leading cause of human mortality. it is also pertinent to mention here that secondary infections by nosocomial, or hospital acquired pathogens is on the rise; at least % of the deaths reported from the ongoing covid- outbreak were caused from secondary infections (antibiotic resistance: the hidden threat lurking behind covid- , ). specifically, respiratory disease caused initially by the covid- viral agent has been shown to rapidly ensue in pneumonia, thus predisposing patients to secondary bacterial infections, and exacerbating underlying cardiac and pulmonary conditions leading to death. therefore, it is critical to obtain a better understanding on the state of antimicrobial resistance prevalent in environmental representatives of bacterial pathogens, because the environment is a well-known reservoir of pathogens. a central question in our ongoing work is focused on assessing those genomic features that underpin antimicrobial resistances in opportunistic bacterial pathogens in the environment, so that appropriate mitigation strategies to combat these pathogens can be obtained. among the bacteria genera that are listed above and relevant to the srs ecosystem, one genus that deserves further study because of its double life strategy is serratia species, from the enterobacteria family. notably, recent genomics analysis has resulted in the division of serratia spp. into the following broad strain categories based on their functional activities: pathogenic, environmental and symbiotic strains (sandner-miranda et al., ) . furthermore, the environmental strains are divided into three sub-groups: environmental strains that are symbiotic and associated with soil and plants, environmental strains that have been isolated from water and environmental strains that were isolated from food sources. overall, it was demonstrated that pathogenic and environmental serratia isolates presented a high number of antibiotic resistant genes (args); especially for efflux systems (levy, ; nelson et al., ; armalytė et al., ; ben maamar et al., ) . the nosocomial pathogenic serratia strains possessed a higher degree of acquired resistance genes relative to environmental isolates. however, stress posed by long-term heavy metal contamination is known to exacerbate recruitment and proliferation of args in the native soil microbiota (cycoń et al., ; kraemer et al., ) , leading to the main question for this study: are environmental serratia species developing broad antimicrobial resistance activities? to address this overarching question, we performed this case study on environmental representatives of serratia spp., specifically on a newly isolated representative strain (gendy et al., b) , compared to a plethora of other serratia spp., having originated from both environmental and clinical settings. note that the environmental serratia species perform a variety of ecosystem services in the environment, including bioremediation of pentachlorophenol (singh et al., ) , dichlorodiphenyltrichloroethane (bidlan and manonmani, ) , diesel (rajasekar et al., ) , chlorpyrifos (cycoń et al., ) , as well as heavy metals to include, nickel (kannan and ramteke, ) , chromium (mondaca et al., ; campos et al., ) , molybdate (yunus et al., ; shukor et al., ) , lead, cadmium (cristani et al., ) , manganese (ii) and hg (françois et al., ) . some serratia spp., are also well-known plant growth promoting bacteria (pgpb), exhibiting positive impacts by increasing nutrition to the plants, reducing stress and enhancing overall plant productivity (singh and jha, ) . as stated above, despite these attractive environmental and agricultural traits of serratia spp., they are also known to possess pathogenicity, accounting for nosocomial infections of the respiratory tract, urinary tract, surgical wounds and even meningitis (khanna et al., ; abreo and altier, ; cristina et al., ) , specifically caused by serratia marcescens. therefore, this potential duallife strategy of serratia marcescens needs to be carefully assessed, specifically for the presence and activities of args, in context to their environmental applications, such as for u and hg bioremediation. with this backdrop, we pursued this study keeping in mind the following objectives which are summarized as follows: ( ) obtain an environmental serratia isolate resistant to hg and u; ( ) evaluate metabolic potential of the isolated strain and screen how metabolic activity is affected by stress posed by hg; ( ) evaluate the presence and functional activity of antimicrobial resistance using phenomics; and finally, ( ) survey the genomic potential of the isolated strain in context to both, metal resistant genes (mrgs) and antibiotic resistant genes (args). overall, this study provides a broader and comprehensive understanding on the myriad of evolutionary and adaptive traits possessed by an environmental isolate of serratia species, which can be opportunistic pathogens, with special emphasis on carriage of heavy metal and antibiotic resistances. acquired resistance to multiple antibiotics has been recognized as a global crisis, which needs urgent attention so that a major outbreak, similar to the ongoing global crisis brought about by covid- pandemic, can be avoided. studies such as this, suggests immediate remedial measures be adopted to obtain mitigation strategies against the development of multidrug resistant bacteria, such as the environmental serratia species, and promulgate strategies in the event a bacterial pathogen outbreak occurs and causes large-scale human fatalities, akin to the covid- , which is ongoing, as of this writing. as part of an ongoing study, soil samples were obtained from the srs mills branch (area ) which is found cocontaminated with total hg concentrations in the range of - ng/g dry weight (xu et al., ) as well as u. to isolate heavy metal resistant bacteria, soils were serially diluted and plated onto lysogeny broth agar (lba) containing µg/ml of mercuric chloride; the resulting colonies were picked and further isolated based on their distinct morphologies and pigmentation agarwal et al., agarwal et al., , a . plates were incubated aerobically at • c in the incubator, and resulting colonies were further isolated, yielding a robust hg resistant (hgr) strain, labeled as srs- -s- . strain identification was performed using s rrna sequencing, as recently reported (gendy et al., b) . hg resistance of strain srs- -s- was evaluated by dilution of overnight grown strain to od of . . this inoculum ( µl) was then added to µl of lb amended with incremental concentrations ( - µg/ml) of mercuric chloride. od was then evaluated every h for up to h at • c using the bioscreen c microbial growth analysis system (growth curves usa, piscataway, nj, united states), as shown previously . this minimum inhibitory concentration (mic) assay was performed three times, and the average values are reported. controls were also run on plates that did not contain hg and compared under the same growth conditions. to evaluate resistance against uranium, our recently developed plate mic method was used because u is known to cause precipitation in liquid media causing turbidity which interferes with monitoring of bacterial growth . briefly, strain srs- -s- was prepared taking a single colony inoculated into lb broth overnight and then diluted to an od of . . further, a -fold dilution series was prepared up to − dilution range and plated onto lb plates containing u in concentrations of , , , and mm, using uranyl nitrate hexahydrate. plates were incubated at • c and photographs were taken after hours of growth to identify mic against u. inoculum was prepared in lb broth with a single colony of strain srs- -s- , incubated at • c until od reached . . then the cells were washed five times with . % nacl to eliminate any nutrients originating from previous growth in lb broth. this cell suspension ( µl) was used as inoculum for establishing biolog r ecoplates tm , as suggested by the manufacturer (biolog inc., hayward, ca, united states), such that, each well contained a cell concentration of − , which was checked by microscopic evaluation of cell density. the biolog r ecoplates tm consists of -well microtiter plates with three replicates of common substrates utilized by soil microbiota, thus are ecologically relevant for microbial physiological and metabolic activity assessment (preston-mafham et al., ) . a separate set of ecoplates was established, containing hg at different concentrations to assess the strain's physiological response; these concentrations were , , and µg/ml mercuric chloride, respectively. the resulting absorbance was measured via plate reader at nm (spectrophotometer-spectramax-m ), every h over a -day period. obtained data was then plotted using microsoft excel and further analyzed. average well color development obtained from the biolog r ecoplates tm was estimated as shown before (gamo and shoji, ; deng et al., ; gryta et al., ) . phenotype microplate tm (pm) from biolog is an array of well microtiter plate, where each set of wells contain the same antibiotic in increasing increments along with the needed minimal medium components and specific dye. the arrays can provide resistance patterns against antibiotics belonging to different chemical classes, e.g., aminoglycosides, β-lactams, lincosamides, synthetic antibiotics, glycopeptides, tetracyclines, amphenicols, macrolides, sulfonamides, and rifamycins. the antibiotic resistance of bacteria was assessed using biolog pm c and pm b microplate tm plate assays, as suggested by the manufacturer; layout of antibiotics is as detailed by the manufacturer for the biolog r ecoplates tm (biolog inc., hayward, ca, united states) . to initiate the pm assay, strain srs- -s- was grown overnight at • c on an lb agar medium. a single colony was then picked and inoculated into ml of the inoculating fluid [if- a gn base inoculating fluid ( . ×), biolog inc.]. resulting cell density was measured by a plate reader according to the biolog protocol (biolog inc., hayward, ca, united states), followed by inoculation of the cell suspension into plates ( µl/well) and incubated at • c for hours. od was collected every min to determine color shifts resulting from microbial growth in the wells reflected of phenotypic differences and sensitivity/resistance to different antibiotics. collected data was organized and plotted in microsoft excel. data obtained from biolog r ecoplates tm over the -day period was analyzed using primer v (primer-e ltd.). statistical relationships were evaluated by running non-metric multidimensional scaling (nmds) analysis, where the different concentrations of substrates utilized in the presence and absence of hg amendments were log transformed [(log (x + )], hg treatments were used as variables, as appropriate and scaled by maximum data point. the whole genome shotgun project of serratia sp. srs- -s- reported in this study has been deposited at ddbj/ena/genbank under the accession vhnf ; bioproject: prjna ; biosample: samn . the version described in this research is version vhnf . . to understand the genomic characteristics of strain srs- -s- in reference to mrgs and args, a single colony of the bacterium growing on lb plate, amended with hg, was inoculated into liquid lb media and incubated overnight at • c in a shaker at rpm. after overnight growth, the media was centrifuged at , rpm for min to obtain a pellet, which was then used for dna extraction with zr fungal/bacterial dna kit (zymo research, irvine, ca, united states) and sequenced using an illumina hiseq instrument. assembly and annotation on the obtained contigs were performed, as reported in our recent study (gendy et al., b) . default settings were used in all the genomic analysis, unless otherwise stated. taxonomic affiliation was conducted using the onecodex workflow (minot et al., ) , which is based on the identification of typically - bp short sequences, that are found unique to a specific taxa within the inputted sequence reads. based on the collection of k-mers found in a given read, the whole genome sequence is then assigned to a taxon. phylogenomic analysis was run using the default value of k = for the taxonomic assessment of strain srs- -s- using the onecodex database. to obtain a physical map of the genome, cgview comparison tool was used with default settings (grant et al., ) . the genome, with a × coverage was annotated and the genes were predicted using rapid annotations using subsystems technology-rast (aziz et al., ) , prokaryotic genomes automatic annotation pipeline (pgaap), version . (tatusova et al., ) , the pathosystems resource integration center (patric, version . . ) (wattam et al., ) or integrated microbial genomes (img) system (chen et al., ) . average nucleotide identity (ani), and average amino acid identity (aai) was also obtained via edgar (blom et al., ) and ezbiocloud pipelines (yoon et al., ) . ani is a measure of the genomic resemblance of two different bacterial species and typically, ani values between genomes of the same species are % or beyond (goris et al., ) . the merr gene taxonomy in strain srs- -s- was evaluated using comparative functions embedded within the patric pipeline. venn diagram and phylogenomic comparisons were obtained using the edgar pipeline, version . (blom et al., ) . after edgar analysis, the newick phylogenomic tree file was downloaded and an amino acid based tree was constructed using megax (wattam et al., ; kumar et al., ) . island viewer (bertelli et al., ) was used to identify genomic islands (geis) (yung et al., ) , within the genome of srs- -s- . remnants of phage dna, also called prophage, present in the whole genome sequence of srs- -s- were located using the prophage hunter (song et al., ) . our recent studies suggest that the metalliferous srs soils may be serving as a reservoir for the recruitment and proliferation of metal and antimicrobial resistances in the native microbial communities gendy et al., a,b; pathak et al., ) , thus presenting risks to the ecological processes and public health. overall, this study further builds upon evidence to show that the srs contaminated soils inherently harbor higher antibiotic resistance, relative to the reference soils, and thus driving antimicrobial resistance to the native microbiota, including several known pathogens (e.g., burkholderia, ralstonia, massilia, acinetobacter, and pseudomonas wright et al., ; thomas et al., ) . to assess if carriage of metal resistant genes (mrgs), and antibiotic resistant genes (args) is a rampantly widespread trait, especially within opportunistic bacteria native to the srs soils, this study was conducted on soils collected from a different site ( ), relative to that reported by our group recently (pathak et al., ) . specifically, soils from a highly contaminated site-srs were serially diluted and spread plated onto lb agar plates supplemented with µg/ml mercuric chloride. site has been shown to be co-contaminated with a variety of different heavy metals, such as hg and u (edwards et al., ) . plates were incubated at • c, for hours and colonies that appeared on the plates were further purified resulting in a vigorous hg-resistant (hgr) strain, labeled as srs- -s- . identification of the isolated strain using s rrna gene sequencing revealed closest taxonomic affiliation with s. marcescens (gendy et al., b) . specifically, phylogenomic analysis, as stated later in this study, revealed . % of obtained genome reads affiliated with s. marcescens ( figure a) ; at the species level, % of the reads belonged to s. marcescens strain ww , followed by % of s. marcescens strain egd-hp ( figure b) . to determine the resistance potential of strain srs- -s- against hg, the bacteria was grown in lb broth, supplemented with different concentrations of hg; these results were presented in our recent study (gendy et al., b) . it was observed that strain srs- -s- could resist up to µg/ml hg, which revealed a strong hg resistance (hgr) phenotype of strain srs- -s- . to test for resistance against uranium (u), a plate mic method was used, which showed resistance of strain srs- -s- at , , and mm but strain was sensitive at mm u, respectively (figure ) . note that s. marcescens has previously been shown to be resistant to u under both aerobic and anaerobic environments (kumar et al., ; choudhary et al., ; nongkhlaw et al., ; newsome et al., ) . to further understand the hg and u-resistant ability of this strain, physiological and genomic studies were then performed. biolog r ecoplates tm , containing distinct nutrient sources, was used to evaluate the metabolic and physiological response of strain srs- -s- in the absence and presence of hg amendments, as a representative heavy metal contaminant. the cellular response in this assay was monitored as a function of color change of the tetrazolium salt when substrates in each well are utilized by cellular respiration, resulting in the production of a purple formazan dye. higher the substrate utilization, the more intense the color of the formazan produced and by inference, metabolic activity of the cell. kinetic growth response curves were obtained for each growth substrate and plotted to assess cellular phenotype comparisons by calculation of the average well color development (awcd), which revealed that serratia sp. srs- -s- utilized different sources of carbon in the following order carbohydrates > polymers > amino acids > carboxy acids > esters (figure a) , respectively. esters initially were preferred over carboxy acids and amino acids but after days, the above stated trend held up until the experiment ended at days. specific to different carbon sources, it was found that strain srs- -s- preferred the following carbon substrates: specific to different carbon sources, it was found that strain srs- -s- preferred the following carbon substrates: d-mannitol, i-erythritol, γ-amino butyric acid, l-asparagine, n-acetyl-d-glucosamine, and tween (data not shown). to understand how hg stress may impact metabolic potential, growth of strain srs- -s- was evaluated in the presence of increasing concentrations of hg, which showed that at both, and µg/ml hg concentrations, the strain's metabolic potential (awcd) became inhibited; the strain started to use substrates after a lag of almost days ( figure b) . however, at an hg concentration of µg/ml, the strain was unable to utilize any of the carbon substrates, despite the strain's potential to resist up to µg/ml hg based on mic values (gendy et al., b) . this difference in the mic and physiological activity response in the presence of hg amendments can be explained on the basis that the higher mic in lb broth, which is a richer nutrient source relative to those present in the biolog r ecoplates tm . regardless, this extensive physiological characterization demonstrated that serratia sp. srs- -s- used an average of . % of the different nutrient sources tested without hg stress (table ) ; conversely, in the presence of µg/ml hg, this response became inhibited at . % of total substrates; at µg/ml hg even fewer substrates were utilized-at an average of . %, while at a concentration of µg/ml hg, total physiological activity of the hg-resistant strain ceased ( figure b and table ) , despite the robust hg resistance ability of srs- -s- . interestingly, the utilization rates of amino acids, such as l-asparagine, l-alanine, and l-serine, relative to other substrates significantly decreased upon exposure across all treatments (data not shown), most likely due to binding of hg to amino acids which reduces their availability and hence physiological activity. it could also be that metabolic pathways for the utilization of these amino acids are hampered by hg toxicity. it has been well-established that mercury, the most toxic heavy metal toxicity, is mainly owed to its high degree of affinity to the sulfhydryl ligands in amino acids, binding to which causes alteration in protein structure along with loss of protein function. overall, this part of the study suggests that hg can interfere with the bacterial physiological response, even on an organism that is resistant to hg. therefore, continued addition of hg can have detrimental impacts to microbially mediated ecosystem services, such as biogeochemical cycling of nutrients. this is a significant finding from an environmental standpoint, which has not been demonstrated before, to our knowledge. statistical analysis of the strain's physiological response to hg amendment was assessed using primer v , which is shown in figure c . cluster analysis clearly showed that in the absence of hg, the strain's metabolic activity was a positive sign (+) indicates strain utilization of carbon source and the negative sign (−) indicates inability of the strain to utilize the substrate source. statistically different relative to hg treatments. specifically, the metabolic activity profiles at µg/ml hg and µg/ml hg clustered together and away from the highest tested concentration of µg/ml hg, mirroring the trends shown in figures a,b . this confirms the observation that hg can interfere with the physiological and metabolic response, even in an organism that harbors hg resistance. therefore, continued addition of hg will likely have significant detrimental impacts to microbially mediated nutrient cycling processes; even to those microorganisms that have developed tolerance/resistance against heavy metals, such as hg. arguably, microbiota that are unsuccessful in the recruitment of resistant gene determinants against heavy metals will have likely been removed from a contaminated soil habitat and ecosystem services rendered by the outcompeted bacteria are either lost, diminished or taken over by the surviving metal resistant bacteria. to further understand the gene determinants for heavy metal resistance, genomic studies were performed on strain srs- -s- . genomic sequences of strain srs- -s- , with a coverage of × were obtained (gendy et al., b) , and characterized by the following parameters: contig count ( ); total length ( , , bp); n ( bases); l ( ); an average gc content of . %; a coding sequence of , proteins (cds) and genes for transfer rna (trna) and genes for rrna, respectively. figure shows a circular genomic map of strain srs- -s- constructed with cgview comparison tool. gene prediction performed using patric, rast and ncbi pipelines revealed that strain srs- -s- harbored a total of protein coding genes; . % of these genes were annotated as protein coding genes with predicted functions; . % genes were annotated as protein coding genes with enzyme production and . % genes were associated with kegg pathways, respectively. moreover, . % of the protein coding genes were annotated with cogs, i.e., clusters of orthologous groups of protein; cogs represent an ortholog or direct evolutionary counterpart among bacterial genomes as they evolve over time. as stated before, phylogenomic analysis of strain srs- -s- relative to other sequenced serratia species revealed closest taxonomic affiliation with serratia species and other genera, such as salmonella ( figure a) . specifically, onecodex analysis resulted in the assignment of % contigs from strain srs- -s- to s. marcescens ww , followed by % to s. marcescens egd-hp and % with s. marcescens subsp. sakuensis, respectively ( figure b) ; these are all environmental strains not directly related to their hospital or nosocomial counterparts. similar result was demonstrated by edgar analysis (data not shown), in which a phylogenomic tree of strain srs- -s- was built with serratia genome sequences, build out of a core of genes per genome, in total. the core had amino acid residues/bp per genome, in total. furthermore, average nucleotide identity (ani) analysis mirrored the previous results such that s. marcescens sp. ww was the closest strain to serratia sp. srs- -s- with a . % value followed by s. marcescens sp. egd hp ( . %) and serratia sp. fs ( . %), respectively (supplementary figure s ) . note that the ani calculator estimates the average nucleotide identity using both best hits (one-way ani) and reciprocal best hits (two-way ani) between two genomic datasets, as calculated by goris et al. ( ) . typically, the ani values between genomes of the same species are above % and therefore, strain srs- -s- is assigned to s. marcescens. after the taxonomic assessment of strain srs- -s- , further functional characterization was performed using rast, which revealed the presence of subsystems with the following top six categories, genes in parenthesis: amino acid metabolism ( ); carbohydrate metabolism ( ); vitamin and cofactor metabolism ( ); protein metabolism ( ); membrane transport ( ) and fatty acids and lipids ( ), respectively. similar results were shown by patric based annotation also revealed the presence of genes (in parenthesis) for metabolism ( ); energy production ( ); protein processing ( ); stress response/defense/virulence ( ); membrane transport ( ); cellular processes ( ); dna processing ( ); genes were also found for resistance to antibiotics and toxic compounds. also identified are genes shown homologous with prophages, transposable elements and plasmids, indicating the receptivity or interactions of strain srs- -s- with mobile genetic elements. of particular relevance are several gene homologs that have previously been shown to be involved in the resistance against heavy metals/radionuclides (genes in parenthesis), including efflux systems ( ) and membrane transporters ( ), which likely facilitates survival of strain srs- -s- in a metalliferous soil habitat. these findings are similar to our previous genomic analysis of another serratia species-strain s b, isolated from the savannah river swamp system soils . note that the srss site is located at the confluence of four mile creek and the savannah river, which received liquid hg (hg) effluents from a chloralkali facility near augusta, ga, united states, until the s. strain s b, native to these hg-contaminated soils, was found to harbor a genomic size of approximately , , bases with , coding sequences and a gc content of . %. a total of subsystems were annotated from strain s b with genes (number in parentheses) for membrane transport ( ), stress response ( ), metabolism of aromatic compounds ( ), motility and chemotaxis ( ), among gene homologs shown to be involved in the resistance against heavy metals, similar to results presented here for strain srs- -s- . collectively, these analyses of serratia species strain srs- -s- revealed the presence of several genome-enabled metabolic and catabolic processes, which likely play a significant role in the colonization and strain's survival within the metalliferous srs soil habitat; similar to previous studies that we have conducted on long-term contaminated sites (olaniran et al., ; ojuederie and babalola, ; chauhan et al., ) . further analysis was performed using comparative genomics to evaluate evolutionary and adaptive traits of strain srs- -s- with the closest taxonomic strains-serratia species ww , egd_hp , vgh , and fs , as shown by edgar analysis (data not shown). this revealed the presence of distinct genes present only in strain srs- -s- relative to the other closely associated serratia strains (figure ) . similarly, distinctive genes were also identified in the other four strains analyzed, shown in parenthesis: serratia marcescens egd_hp ( ); s. marcescens vgh ( ); s. marcescens ww ( ), and s. species fs ( ), respectively. even though genes present in srs- -s- (# sector in figure ) , made up only about % of the total genome size of the strain, but this set of genes are representative of "distinct" determinants for efflux, metal resistance, universal stress proteins, cytochromes, drug resistance, transport proteins as well as transposases. moreover, many of the unique genes in srs- -s- were classified as hypothetical proteins, functions of which are unknown at this time. it is also noteworthy that the strain harbored several genes that have been implicated in diverse roles pertaining to plant promoting activities, such as: phosphate metabolism (phou, pstb, psta, pstc, and psts); spermidine synthase and n acetyltransferase genes (data not shown). these traits are similar to other serratia species (matteoli et al., ) , and may render a plant growth promoting role of strain srs- -s- in its native soil habitat, simultaneously reducing the metal toxicity. overall, this comparative genomic analysis confirmed a strong genomeenabled bioremediative as well as plant growth promoting potential of strain srs- -s- . using the integrated prediction method to identify geis, strain srs- -s- was shown to harbor different geis, ranging in size from , to , bp (figure ) , when s. marcescens ww was used as the reference genome. note that genomic islands is a universal trait in many environmental isolates we have studied to date pathak et al., ) ; geis are beneficial gene segments recruited by host bacteria from their external environment via horizontal gene transfer (hgt) mechanisms (pérez-pantoja et al., ) . typically, geis have been shown to be associated with host-beneficial adaptive traits such as bioremediation, virulence, antibiotic resistance and metabolism. overall, these gei-encoded traits can be binned under the following four major categories (bertelli et al., ) : ( ) metabolic islands (mis), which are a set of genes for secondary metabolite biosynthesis; symbiotic islands (sis), which are those set of genes that facilitate symbiotic associations with other micro and/or macroorganisms; pathogenicity islands (pais), coding for virulence or disease causing genes; and resistance islands (ris), rendering resistance for antibiotics and other bacteriostatic/bactericidal agents. overall, this analysis led to the identification of several genomic islands in strain srs- -s- , with gene functions previously identified for metal resistance, efflux and transposons. this strongly indicates a likelihood that geis are probably recruited via hgt to promote survival in a heavy metal contaminated soil habitat (bertelli et al., ) . these findings are similar to our previous reports from serratia strain b , which also revealed a repertoire of biodegradative genes, several occurring on genomic islands . the presence of these geis in srs- -s- also provides clues into the strain's genome plasticity, introduced by mobile genetic elements including integrases or transposases, which were also identified. notably, one mechanism for geis to be incorporated into bacterial genomes is through bacteriophage attack, which can leave phage remnant dna, or prophage, integrated into the bacterial genome. thus, bacteriophage attacks is a major mechanism that influences bacterial evolution, resulting in the recruitment of genomic traits beneficial to the host species, such as virulence factors, antibiotic resistance mechanisms and bioremediative functions (bernheim and sorek, ) . when presence of prophage was evaluated in strain srs- -s- , candidate genes were identified in this category, as shown in supplementary table s . specific details on the top phageassociated genes are as follows (sizes in parenthesis): salmonella phage fsl sp- ( , bp); salmonella phage ( , bp); nocardia phage nbr ( , bp); edwardsiella phage pei ( , bp) and klebsiella phage lv- ( , bp), respectively. in fact, different salmonella-specific prophages were found associated in the genome of strain srs- -s- followed by from klebsiella spp. conversely, only phage x was identified in the genome of strain srs- -s- that was related to serratia spp., which indicates strain srs- -s- was more responsive to salmonella-specific phage interactions relative to its own genus. because salmonella possess pathogenic traits, it is likely that pathogenicity of serratia spp. are transmitted from salmonella into serratia via hgt mechanisms, but this observation is based only on genomic analysis with no rigorous functional proof available as of this writing. this observation, however, also goes well in line with the taxonomic similarity between the strain's genome with salmonella spp., albeit at only . % (data not shown). in this context, genome-wide analysis of serratia species have recently established that the environmental serratia representatives have commonalities pertaining to the high number of antibiotic resistant genes relative to the hospital or nosocomial strains (sandner-miranda et al., ) and it appears that the environmental isolates are also equally potent in their antimicrobial activities. genomic mining of serratia sp. srs- -s- also revealed several genes that likely enable bacterial adaptability and survival to survive in a contaminated environment. for example, it was found that the strain contained several gene homologs for heavy metal and biocide resistance such as efflux proteins [e.g., resistance, nodulation, and proteinfamily (rnds); outer membrane channel tolc], and heavy metal-responding transcription regulators. furthermore, the srs- -s- strain genome had significant numbers of abc-type transport and heavy metal detoxication proteins, which could maintain homeostasis of metals -all of these adaptations likely contribute for environmental and evolutionary response of strain srs- -s- and survival in a metalliferous soil habitat. one major mechanism underpinning microbial resistance against both heavy metals and antibiotics is by efflux of these compounds outside of the cellular environment, as soon as the cells sense their presence (levy, ; nies, ; blanco et al., ; buffet-bataillon et al., ) . strain srs- -s- possessed at least genes related to different efflux groups such as abc-type efflux, multidrug efflux, rnd efflux system etc. (data not shown). therefore, efflux mechanisms may be one major response of strain srs- -s- to survive in a highly metalliferous soil habitat. toward this end, hgresistance is also likely efflux based in the strain. genome mining revealed that the strain did not support a complete mer operon but only harbored the transcriptional regulator, merr family (fig| . .peg. ) (supplementary figure s a) , along with the heavy metal sensor histidine kinase (hmhk) and the dna-binding heavy metal response regulator (hmrr). the merr gene in strain srs- -s- was taxonomically closest to s. marcescens ww and s. marcescens egd-hp (supplementary figure s b) ; these strains were also identified closest relatives in the phylogenomic based analysis shown in figures a,b . therefore, one central question arises from the lack of a complete mer operon in strain srs- -s- -what mechanism(s) render hg resistance to serratia marcescens strain srs- -s- ? to this end, previous body of information revealed that not all hgr bacteria support a complete mer operon. the mer operon has origins in geothermal environments (boyd and barkay, ), and has evolved from being a constitutively expressed system made up of simple genetic elements to a highly regulated and complex operonic system-the complete mer operon. we are tempted to speculate that hg resistance in strain srs- -s- is likely mediated via efflux mechanisms, as proposed elsewhere. in fact, studies have found energy-dependent efflux systems to render resistance to many heavy metals, including cadmium, zinc, nickel, cobalt, and copper (reyes et al., ) . in a seminal report, several mer negative but hgr bacteria exhibited decreased resistance to hg when amended with the protonophore-carbonyl cyanide m-chlorophenylhydrazone (cccp) (reyes et al., ) . it may also be that strain srs- -s- harbors genes which are not homologous to recognized mer genes and hgr is a mechanism driven by potentially novel genes; we are currently conducting more work to resolve these possibilities in strain srs- -s- as it relates to hg resistance. regardless of these possibilities, the presence of abundant mrgs in strain srs- -s- provides for a deeper understanding on soil survival strategies. to validate the phenomic response indicating carriage of broad antibiotic resistance in srs- -s- , a comprehensive survey of antibiotic resistance genes (args) was performed employing patric, card, and rast pipelines. this bioinformatic analysis revealed a plethora of args in serratia sp. srs- -s- . specifically, patric analysis revealed the presence of several genes classified under speciality category, that included genes for transporters, genes for drug targets, genes for virulence factors, and genes for antibiotic resistance, respectively ( figure a ). this points to the repertoire of speciality genes, many related to virulence and args, that are recruited by this soil-borne isolate as its arsenal againts multiple antimicrobials, likely shaped by long-term exposure to heavy metals, such as uranium and mercury. similar results were obtained by using card, which revealed resistomes for the following drugs (percent identity with the corresponding region is shown in parenthesis): fluoroquinolone ( . %), tetracycline's ( . %), cephalosporin ( %), penam ( %), carbapenem ( . %), cephamycins ( . %), fosfomycin ( . %), macrolide ( . %), monobactam ( . %), and aminoglycoside ( . %) (jia et al., ) , as shown in figure b . several other genes that possibly confer antibiotic resistance were also recognized, including antibiotic target protein (penicillin-binding protein mutations), antibiotic target alteration, antibiotic self-resistance gene, resistance-nodulationcell division (rnd), major facilitator superfamily (mfs), efflux pump complex etc. this overall arg evaluation of serratia sp. srs- -s- indicates that the strain has acquired resistance to multiple antibiotics, most likely exacerbated by exposure to hg, u and possibly other heavy metal contamination in the srs soils. these findings are in line with previous body of information on srs soils, which unequivocally show that the legacy contaminated srs soils serve as a repository for acquisition of both metal and antibiotic resistances wright et al., ; thomas et al., ) . however, this study is focused on serratia spp., which are opportunistic pathogens and brings out the necessity for further focused studies on this aspect rather than a more generalized assessment of the proliferation and abundance of mrgs and args within the srs soil habitat, as has been the case with most previous studies. to validate the functional activity of args in serratia sp. srs- -s- , phenotype microarray (pm) technique was used, which is a microtiter-plate-based substrate utilization assay (bochner and savageau, ; bochner, ; bochner et al., ) , similar to the biolog ecoplates (borglin et al., ) . note that the pm technique, also referred to as phenomics, offers the ability to obtain cellular response of axenic microbiota or environmental communities, as well as screening antimicrobial resistances (blanco et al., ) . the results obtained from the pm microplate tm analysis are shown in figures a,b . this data is compared in table , showing that only out of antibiotics tested, inhibited or ceased growth, indicating high antibiotic resistance. in fact, . % of the antibiotic/concentration tested did not affect growth, suggesting the broad arg potential of this strain. the antibiotics and concentrations (as provided by the manufacturer), that affected growth included the following: cloxacillin ( µg/ml), lomefloxcin ( . µg/ml), minocycline ( µg/ml), nafcillin ( µg/ml), enoxacin ( . µg/ml), nalidixic acid ( µg/ml), potassium tellurite ( . µg/ml), f- neomycin ( . µg/ml), ofloxacin ( . µg/ml), carbenicillin ( µg/ml), oxacillin ( , µg/ml), d,l-serine hydroxamate ( µg/ml), novobiocin ( µg/ml) d,l-serine hydroxamate ( µg/ml), dodecyltrimethyl ammonium bromide ( µg/ml), respectively. after correlating the findings from the antimicrobial resistance genes (args) analysis and phenotype microarray (pm) antibiotic profile of serratia sp. srs- -s- , it can be concluded that the serratia sp. srs- -s- harbors a suite of args against many antibiotics as well as metal resistant genes (mrgs). the antibiotic profile of serratia sp. srs- -s- was also mirrored in the genomic analysis (figures a,b) . biolog pm microplate tm array assay on serratia sp. srs- -s- yielded an extensive resistance pattern to antibiotics. the development of a broad spectrum of args in strain srs- -s- are very likely due to the stress imposed by the long-term contamination of srs soils with hg and u, thus presenting with serious public health and ecological risks. note that hg resistance is almost invariably found to co-occur with antibiotic resistance, specifically for ampicillin, tetracycline, erythromycin, and penicillin (ready et al., ) . therefore, presence of hg and u stressors present in the srs soils pose public health and ecological concerns from the metal driven antibiotic resistome or vice versa. serratia marcescens represents a recently recognized pathogenic species and hence remains largely understudied. furthermore, whole genome sequence projects on this pathogen have also only recently become available to the scientific community (abreo and altier, ) , thus limited understanding is available on the genome-wide adaptive traits of s. marcescens as a pathogenic agent. toward this end, this study reveals evolutionary and adaptive traits possessed by an environmental serratia species, and in particular, carriage of heavy metal and antibiotic resistance gene determinants. it is very likely that broad antibiotic resistances are being acquired by microbiomes, such as serratia, in sites that remain historically contaminated with heavy metals, which is a major public health concern. note that resistance of opportunistic pathogens to one or more antibiotics has been recognized as a global crisis, which needs immediate attention such that a major pathogenic outbreak such as the covid- , can be avoided. even though covid- is a viral disease but some preliminary estimates suggest that % of mortalities are occurring from nosocomial secondary infections. therefore, one broader recommendation from this study is to take immediate action against development of multidrug resistant bacteria and develop strategies should a bacterial pathogen outbreak occur that is similar in magnitude to the ongoing covid- , as of this writing. it is noteworthy that outbreaks related to s. marcescens and other serratia species have occurred sporadically since s (grohskopf et al., ; mahlen, ; yao et al., ) , mainly in neonatal and cardiac icus, orthopedic clinics and dialysis centers. there is a large body of information available on serratia epidemiology and resistance patterns and s. marcescens strains are typically resistant to all penicillins and susceptible to all carbapenems; a recent study, however, was found resistant to all tested antibiotics (bertrand and dowzicky, ) . however, not much is known on the antimicrobial patterns of environmental serratia members, and this study addresses this gap. more recently, pangenome-based analysis of s. marcescens strains from nosocomial and environmental origins as well as different countries, revealed consistently presence of high number of args (abreo and altier, ), likely due to stress imposed by environmental contaminants, as also suggested in this study on strain srs- -s- . to obtain a better understanding on the relationship between the environmentally relevant s. marcescens strain srs- -s- with some other medical/clinically relevant isolates, comparative whole genome analysis was run using the integrated microbial genomes (img) system, and results are shown in figure . specifically, we selected the closest taxonomic relatives of strain srs- -s- identified in previous analysis, such as s. marcescens ww , s. marcescens egd-hp , along with several strains isolated from bacteremia or clinical settings, such as s. marcescens umh , s. marcescens ; a total of s. marcescens strains were included in this analysis. remarkably, strain srs- -s- clustered closest to the strains umh and umh in a pca drawn on cog profiles for the selected genomes ( figure a) . conversely, strains ww and egd-hp -the closest taxonomic relatives of strain srs- -s- , clustered away from the cohort with which strain srs- -s- clustered, which were all medically relevant strains. as stated before, cogs represent families of orthologous protein-coding genes and is a reliable assessment of comparing protein phylogeny from microbial genomes (galperin et al., ) , and hence, is a functional assessment of a bacteria of interest. this observation was again confirmed when pca clustering was based on the presence of kegg pathway (ec) profiles (figure b) , which is based on the enzymatically catalyzed network of metabolic pathways and reactions, constructed upon the entirety of all available biochemical information and is not organism-specific. similar to the cog-based analysis, the closest association of strain srs- -s- was with strains umh and -many of these strains were active in bacteremia (anderson et al., ) . overall, this is strong evidence that strain srs- -s- , isolated from a metalliferous contaminated soil environment, is functionally more similar to medically relevant isolates. overall, this is succinct evidence that the environment is potentially serving as a niche for the development, recruitment and proliferation of pathogenic traits within serratia species, which needs to be evaluated to obtain mitigation strategies and avoidance of possible future pathogenic outbreaks emanating from ecological systems; as is indicative for the origin of the covid- viral pathogen from bats, which is likely the main reservoir for this pathogen (banerjee et al., ; andersen et al., ) . the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the section titled "nucleotide sequence accession number". sg, ma, ap, and rr designed the experiments. sg, ap, and ac performed the genomic analysis. sg, ma, and rr performed the biolog and phenomic tests and analyzed the results. ac, sg, ap, ma, rr, and rj drafted the manuscript. 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interest.copyright © gendy, chauhan, agarwal, pathak, rathore and jaswal. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -pz htccp authors: kohn, dennis f.; barthold, stephen w. title: biology and diseases of rats date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: pz htccp nan the diversity of research for which the laboratory rat is used is probably greater than that associated with any other animal. the laboratory rat is a descendent of the wild rat, rattus norvégiens, which originated in asia and reached europe in the early s. wild and albino mutants were first used for ex perimental purposes in europe in the mid- s and in the united states shortly before . the wistar institute in phil adelphia was prominent in the development of the rat as a labo ratory animal, for here originated many of the rat strains now used worldwide. henry donaldson and his colleagues at the wistar institute used these early rat strains for a variety of stud ies dealing with neuroanatomy, nutrition, endocrinology, ge netics, and behavior. the history and evolution of the many rat strains used today have been recently summarized (lindsey, ) . the most commonly used outbred rat stocks in north amer ica are the wistar, sprague-dawley, long-evans, and holtzman. all are albino except the long-evans stock, which is usually marked with a black or gray hair coat over the shoul ders and is sometimes referred to as a "hooded rat." there are numerous inbred and mutant rat strains, although the number is less than that in the mouse. table i lists the more commonly used strains. there are a rather large number of commercial vendors of laboratory rats in the united states. most of the stocks and strains mentioned above can be obtained from more than one source. although the origin of an outbred stock, such as the sprague-dawley, may have been the same for a number of vendors, in many cases it has been to years since such a stock has been removed from its original breeding colony. ac cordingly, the genotype of outbred stocks and inbred strains may vary among sources and be reflected by differences in data when multiple sources of rats are used. a standardized scheme of identifying stocks and strains of rats has been devel oped and is now used by nearly all commercial vendors. more over, it is important that authors correctly identify stocks and strains that are used in their research since the success in re peating the work in another laboratory may be dependent upon the genotype (source of the rat). table ii summarizes the stan dardized nomenclature for outbred stocks as developed by the table i commonly used strains "national institutes of health ( ) . . letters preceding the colon designate the supplier/breeder code consist ing of a capital and two or three lowercase letters . capital letters following the colon are used by a breeder to identify his stock . letters in parentheses denote origin of stock . subscript symbols indicate rearing by means other than natural mother (f, fostered; fh, fostered by hand) international committee on laboratory animals (icla). table iii contains the scheme for designating inbred strains of rats (national institutes of health, ) . "animals for re search" (national academy of sciences, ), a directory of sources for laboratory animals sold in the united states and canada, lists all rodents according to standard nomenclature, and is a valuable aid in purchasing laboratory animals. commercial production of rats has markedly changed since the s due to the development of hysterectomy-derived and barrier-maintained breeding colonies. prior to the application of this technology to production colonies, infectious diseases were ubiquitous in rats from most sources. today, vendors can be selected who offer pathogen-defined animals for most stocks and strains. concomitant with changes in commercial sources of rats are the major advances made in the design and construction of institutional animal resources and husbandry practices within them. optimum housing of rats today includes provisions for quarantining and isolation of animals according to vendor subpopulations that have a similar microbial flora. there are various levels of sophistication to provide barriers to the spread of infections in rat colonies. since many rat pathogens are spread by aerosol, ventilation control is very important. nonrecirculating room air or high-efficiency panic ulate air (hepa)-filtered air has become a design standard in modern animal facilities. as discussed in chapter , clean/contaminated corridor-designed facilities aid in contain ment against the spread of pathogens by aerosol, personnel, table iii nomenclature for inbred rats . the strain designation is given in capital letters followed by a slash . the substrain designation follows the slash and is given as numbers or as individual or company codes. numbers are used to denote substrains that were derived from a common strain but separated before the completion of inbreeding . subscript symbols indicate rearing by means other than natural mother and contaminated equipment. a more complete barrier system may include an entry area in which incoming supplies and equipment are sterilized and in which personnel shower and don sterile clothing and filter masks before entering animal rooms. more recently, laminar-flow (mass air displacement) rooms and mobile units have become popular because they can be incorporated in existing buildings that lack design charac teristics mentioned above. environmental control in rat rooms is important to the com fort and health of the animals, as well as to the consistency of data derived from the rats. room temperatures between and °f are desirable, and the relative humidity should range be tween and %. daily fluctuations in temperature and hu midity act as significant stressors. these fluctuations may be associated with the environmental control system of a building or may be induced by procedures such as cleaning floors with a water hose or high pressure sprayer. twenty-four-hour tem perature/humidity recorders are useful in detecting changes in environmental conditions. light intensity should be evenly distributed to all animals within a room. seventy-five to fc have often been suggested as an optimal range for light inten sity. however, recent evidence indicates that this intensity can induce retinal degeneration in albino rats (anver and cohen, ) . light-timing devices are a convenient means to provide desired day/night cycles such as - or - hr. caution should be exercised in the use of insecticides and air-deodorizing chemicals, since some have been shown to in duce hepatic microsomal enzymes in rats. accordingly, their use in animal rooms is not usually recommended (baker et al., a) . rats can be housed in either wire-or solid-bottom cages. wire-bottom cages are more frequently used since they are less labor-intensive. frequency of cage and litter pan changing is a function of animal density. solid-bottom cages should be sani tized two to three times per week, while wire-bottom cages should be sanitized on a weekly or biweekly schedule with litter pans changed two or three times per week. feed should be contained in hoppers. either automatic systems or bottles are satisfactory for providing water to rats. some caution is necessary when using automatic systems, since weanling and newly arrived rats may not drink initially from such devices. to avoid undesirable microbial contamination, water bottles should be sanitized before they are refilled and automatic sys tems should be drained and flushed when racks are sanitized. acidification of water to a ph of . to . or chlorination at to ppm will control pseudomonas aeruginosa contamina tion of water (weisbroth, ) . however, this treatment is not necessary for immunocompetent animals. wood shavings or chips are the most commonly used contact bedding mate rials. hardwoods are preferred to softwoods, since the latter are capable of inducing hepatic microsomal enzymes (baker et al, a) . this section summarizes some of the anatomical characteris tics of the rat with emphasis on characteristics that are unique. the reader is advised to refer elsewhere in the literature for comprehensive descriptions (bivin et al., ; caster et al., ; hebel and stromberg, ; smith and calhoun, ; zeman and innés, ) . the rat dental formula is ( / , c / , pm / , m / ) = . the incisors are well developed and grow continuously. the rat lacks tonsils and water taste receptors. the major pairs of salivary glands are the parotid, submandibular (submaxillary), and sublingual. the parotid gland is a serous gland consisting of three to four lobes and is located ventrolaterally from the caudal border of the mandible to the clavicle. the submandibular glands are mixed glands located ventrally between the caudal border of the mandibles and the thoracic inlet. the sublingual glands are mucous glands and are much smaller than the parotid and submandibular glands. they are located at the rostral pole of the submandibular glands to which they are closely associated. brown fat deposits are present in the ventral cervical region. these multilocular deposits are well demarcated and can be confused with salivary glands or lymph nodes. the stomach of the rat is divided into two parts; the forestomach (nonglandular) and the corpus (glandular). the two portions are separated by a limiting ridge. the esophagus en ters at the lesser curvature of the stomach through a fold of the limiting ridge. this fold is responsible for the inability of the rat to vomit. the forestomach, which is thinner than the cor pus, is linked with an epithelium similar to that of the esopha gus and extends from the cardia to a narrow band of cardiac glands at the junction of the glandular portion. the small intestine is composed of the duodenum ( cm), jejunum ( cm), and ileum ( cm). the cecum is a thinwalled, comma-shaped pouch that has a prominent lymphoid mass in its apical portion. the colon is composed of the as cending colon, with prominent oblique mucosal ridges, trans verse and descending colons, with longitudinal mucosal folds; followed by a short rectum that is confined to the pelvic canal. the liver has four major lobes (median, right lateral, left, and caudate) and is capable of regeneration subsequent to par tial hepatectomy. the rat has no gallbladder. the bile ducts from each lobe form the common bile duct that enters the duo denum mm from the pyloric sphincter. the pancreas is a lobulated, diffuse organ that extends from the duodenal loop to the gastrosplenic omentum. it can be dif ferentiated from adjacent adipose tissue by its darker color and firmer consistency. up to excretory ducts fuse into - large ducts, which empty into the common bile duct. the nasal cavity is not markedly different from that of other mammals. the rat has a maxillary recess (sinus) located be tween the maxillary bone and the lateral lamina of the ethmoid bone. the recess contains the lateral iiasal gland (steno's gland) that secretes a watery product that is discharged at the rostral end of the nasal turbinate. it has been postulated that the nonviscous secretion contributes to the humidification of in spired air and acts to regulate the viscosity of the mucous layer overlying the nasal epithelium. the left lung has one large lobe, and the right lung is divided into four lobes (cranial, middle, accessory, and caudal). the pulmonary vein in the rat has cardiac striated muscle fibers within its wall that are contiguous with those in the heart. the rat does not have an adrenergic nerve supply to the bronchial musculature, and bronchoconstriction is controlled by vagai tone. unlike the guinea pig, the rat lung has a low concentra tion of histamine (bivin et al., ) . the heart and peripheral circulation in the rat differ little from that of other mammals. the blood supply to the heart is derived from both coronary and extracoronary arteries. the latter arise from the internal and subclavian arteries. the right kidney, which is more craniad than the left, has its cranial pole at the l, vertebra and its caudal pole at the level of l . the rat kidney is unipapillate as are kidneys of other ro dents, lagomorphs, and insectivores. having only one papillus and calyx makes the rat useful for studies in which cannulization of the kidney is done. the presence of superficial nephrons in the renal cortex has made the rat widely used as a model for studying nephron transport in an in vivo micropuncture system. the male reproductive system has a number of highly devel oped accessory sex glands. these include large seminal vesi cles, a bulbourethral gland, and a prostate gland composed of the coagulation gland (dorsocranial lobe) and ventral and dorsolateral lobes. the inguinal canal remains open throughout the life of a rat and testes descend initially by days of age. the female rat has a bicornate uterus that is classified as the duplex-type because the lumina of the uterine horns are com pletely separate with paired ossa uteri and cervices. the female urethra does not communicate with the vagina or vulva, but rather exits at the base of the clitoris. the brain of the rat has very large olfactory bulbs and a nonconvoluted cerebrum. the hypophysis is behind the optic chiasma and is attached to the base of the brain by a thin hol low stalk, the infundibulum. the blood supply to the brain is from the internal carotid and vertebral arteries. blood leaves the brain via a system of sinuses that are enclosed in the dura mater. the ventricular system is similar to that of other ani mals, but the rat lacks a foramen of magendie. it must be recognized that many of the normal values deter mined for a specific group of rats may be accurate for only that rat stock/strain, source, and conditions under which they are held. selected physiological, hematological, and clinical bio chemical parameters are listed in tables iv-vii. more com plete information on biological values is available (mitruka and rawnsley, ; ringler and dabich, ) . nutritionally adequate diets are readily available from com mercial sources. these standard rations are quite satisfactory for most applications. however, for some types of experimen tation there are factors, other than nutritional adequacy, which must be considered. the nutrient composition of diets and the contamination of feed components by mycotoxins, antibiotics, synthetic estrogens, heavy metals, and insecticides may have a profound impact on many studies. for instance, caloric intake and the percent of fat and protein in the diet of rats influence the incidence of neoplasia (altman and goodman, ) . sim ilarly, various contaminants have an adverse effect on data from toxicologie, gerontological, and reproductive studies. standard commercial diets are formulated from natural ingre dients and will vary in nutrient composition on a batch-tobatch basis due to differences in type and quality of ingredients used. commercial makers of rodent feeds take precautions to preclude the presence of contaminants in feeds, but only a few products have a defined profile of maximal levels of heavy metals, aflatoxins, chlorinated hydrocarbons, and organophosphates. for some investigative purposes, feeds formulated with re fined ingredients (purified diets) or with chemically defined compounds are useful when control of nutrient concentrations is essential (national research council, ) . these diets are, however, too expensive for general use. baker et al. ( b) and bivin et al. ( ). rats are commonly fed ad libitum, and food intake will vary according to requirements for growth, gestation, and lactation. the nutritive requirements for the rat are listed in table viii. the duration of storage and the temperature at which feeds are stored prior to use effect the nutritive quality of diets. com mercial diets are formulated to have a shelf life of up to months. however, storage in a hot or damp environment will reduce this shelf-life. to help assure that only fresh diets are used, products should be used which have milling dates identi fied on their containers (see chapter ). sexual maturity occurs between and weeks for both sexes, although the onset of first estrus in females occurs at about weeks. the vagina opens between and days, and the testes descend between and days, although they remain fully retractable in adults. rats ovulate spontaneously, but ovulation can also be induced by forced coitus during nonestrous intervals. vaginal stimulation during mating is impor tant in rat reproductive physiology. the more often a male inserts his penis into the vagina prior to ejaculation, the greater the probability of a resulting pregnancy. however, natural or artificial stimulation of the vagina within min of a first mat ing will abrogate pregnancy from the first mating by inhibition of sperm transport. a -hr estrous period recurs every to days and after parturition, without seasonal variation. estrus can be suppressed when females are housed in groups and syn chronized in the presence of a male or its excreta (whitten effect), but this effect is not as pronounced as in the mouse. female fertility wanes at to days, but estrous cycles may continue through months. male fertility is lost between and months. fertility of both sexes is generally regarded as maximal between and days of age (adler and zoluth, ; baker, ; farris, ; lane-petter, ; leathern, ) . males will mount estrous females numerous times with one or two rapid ejaculations in the course of to minutes. ejaculated semen coagulates, forming a copulatory plug that remains in the distal vagina for a few hours, after which time it dissolves or is extruded. copulation is usually nocturnal. du ration of gestation varies with strain, age, litter size, and other variables, and ranges from to days, with an average of or days. primiparous females tend to have a slightly longer gestation than multiparous females (farris, ) . estrus can be detected in a number of ways. females in es trus are hyperactive and brace themselves when touched. their ears quiver when they are stroked on the head or back, and stimulation of the pelvic region induces lordosis (farris, ) . the vulva becomes swollen, and the vagina becomes dry in contrast to the moist pink wall during metestrus or diestrus. as proestrus occurs (approximately hr), smears of va ginal cells contain nucleated epithelium, leukocytes, and occa sional cornified cells. estrus (approximately hr) begins with about % nucleated and % cornified cells, with cornified cells predominating as estrus continues. metestrus follows (ap proximately hr) with large numbers of leukocytes and corn ified cells, which form abundant caseous vaginal detritus. metestrus is characterized by the presence of large flat nucle ated (pavement) cells. diestrus persists for approximately hr (baker, ; farris, ) . breeding dates can be established by examination of vaginal swabs for spermatozoa or examining the distal vagina or cage pan for copulatory plugs. timed pregnancies are best achieved by placing the female in the male's cage in the afternoon and examining her for a plug or spermatozoa the following morning. abdominal enlargement becomes evident at about weeks. pseudopregnancy is rare (lane-petter, ) . rats reproduce successfully under a variety of conditions, but husbandry practices can significantly influence fecundity. rats can be bred as monogamous pairs, taking advantage of postpartem estrus for maximal breeding efficiency. polyg amous breeding is more economical, since only one male can be kept with to females. females are often removed to a separate cage prior to whelping, since they may not tolerate other females in the cage while nursing. they will tolerate their mates, however. females with litters do best on clean dust-free wood shavings in solid-bottom cages. due to heat regulation, pups neither thrive in overly spacious cages with wide flutuations in ambient temperature, nor in overly crowded cages where they cannot dissipate heat. the recom mended cage floor area for a female and her litter is in. . ambient room temperature and humidity should be within the acceptable range with minimal fluctuation. high ambient tem perature can cause male infertility (baker, ; baker et al., a; lane-petter, ) . the rat estrous cycle is particularly sensitive to variations in light. daily lighting at an average of fc with a spectrum approximating natural light for to hr is best for breeding. constant light for as few as days may induce persistent es trus, hyperestrogenism, polycystic ovaries and endometrial hy pertrophy or metaplasia (baker et ai, a; gralla, ) . nutrition may also affect reproductive performance. re quirements for certain components are increased during preg nancy and growth, but overfeeding is deleterious. caloric re striction may actually improve fertility and possibly reproduc tive life of the female (leathern, ) . excess dietary protein can adversely affect female sexual development. vitamin defi ciencies can cause infertility, particularly those vitamins (a, e, riboflavin, and thiamin) that are most labile to autoclaving or deterioration (baker, ) . it is not necessary to add nesting material to bedding for successful breeding, but rats will utilize it if offered. shredded paper or cotton nesting material will be readily accepted and used by prepartem and nursing dams. parturition is heralded by pronounced postural stretching and rear leg extension. a vagi nal discharge may be noted li- hr prepartum. parturition is usually complete in or hr, but can range from a few minutes to several hours depending on litter size. dystocia is exceed ingly rare. litters average between and pups, with highest fecundity through the sixth litter. inbred rats tend to produce smaller litters. although infrequent, cannibalism is most apt to occur with nervous or primiparous females subjected to stress (farris, ; lane-petter, ; leathern, ) . the neonate weighs about i gm, depending on litter size, sex, strain, and physical condition of the dam. pups are born hairless, blind, with closed ears, undeveloped limbs, and short they are fully haired between and days (baker, ; farris, ; lane-petter, ) . maternal antibody is trans ferred in utero, via the yolk sac and by intestinal absorption of colostrum by the neonate for up to days after birth (chev ille, ) . optimal weaning age is - days, although pups can be weaned as early as days. differentiation of sex in adult rats is relatively easy after the testes descend. the adult testes can be readily retracted through large inguinal canals. male neonates have a larger genital papillus and the anogenital space is greater in males than females. from national research council ( ) . h adequate to support growth, gestation, and lactation; based on % dry matter. ( linoleic acid, . %, is required. ^one-third to one-half can be supplied by l-cystine. ^one-third to one-half can be supplied by l-tyrosine. ^mixture of glycine, l-alanine, and l-serine. ^vitamin a, iu = . ìg retinol, . ìg retinyl acetate, . ìg retinyl palmitate. vitamin d, iu = . ì£ ergocalciferol. vitamin e, iu = mg dl-a-tocopheryl acetate. artificial insemination can be achieved in rats, but the major obstacle is the coagulative properties of their semen. sperm can be obtained by maceration of the epididymis and vasa or by electroejaculation, although the latter method is unreliable and the semen often rapidly coagulates. coagulation can be eliminated by prior surgical extirpation of the seminal vesicles and coagulating glands without significant effect on fertility. semen can be diluted with a number of media but frozen stor age of rodent semen has met with little success. insemination can be achieved surgically by direct injection of seminal fluid into the uterus and by nonsurgical means. successful concep tion seems to require not only insemination during estrus but also induction of pseudopregnancy by mating with a vasectomized male or mechanical stimulation within a few hours (before or after) insemination. egg harvest for transfer can be accomplished by excision of the preovulatory ovaries and teas ing from gravid follicles or recovery from the oviduct or uterus by flushing with transfer medium. superovulation by injection of gonadotropisms may enhance yield, but is usually not nec essary. eggs are generally injected directly into the uterus but the recipient uterus must be at the same stage of the uterine cycle (bennet and vickery, ) . synchronization of estrus can be achieved by vaginal inser tion of polyurethane sponges containing . mg medroxyprogesterone for days. females are then put in a cage previously occupied by male rats, sponges are removed, and the rats are injected with iu of pregnant mare's serum. within hr, % will be in estrus. this can also be attained by administer ing mg medroxyprogesterone in ml ethanol/liter drink ing water, prepared fresh daily for days, then intramuscular injection of iu of pregnant mare's serum (bennet and vick ery, ). the rat has been utilized extensively in a variety of research fields, including behavioral science. rats are docile, adapt to new surroundings, tend to explore, and are easily trained to a variety of sensory cues by positive or negative reinforcement. rats sleep during daylight hours and activity, including feed ing, is greater during the night and early morning. laboratory rats are easily handled, but strain differences exist. sprague-dawley and lew rats tend to be less fractious than long evans or f rats. docility is improved with routine and proper handling. rats become nervous and refractory to han dling when they hear others squeal. nutritional deficiency, particularly hypovitaminosis a, and mishandling can make rats vicious. rats seek entry into small openings, a trait that is utilized for coaxing them into restrait apparatus. like other rodents, rats are coprophagic, which must be taken into con sideration when administering drugs, measuring fecal output, or performing nutritional studies. unlike mice, rats are less apt to fight, and males can be housed together. in addition, rats are not gregarious like mice, and seem to tolerate single caging well. experimental studies indicate significant changes in plasma corticosteroid levels, depending on cage cohort size. levels tend to be least in rats housed singly, to increase in groups up to , to decrease in larger groups up to - , then rise again in groups up to (lane-petter, ). infectious agents constitute a significant environmental vari able that impacts on research data derived from laboratory rats. as is the case with other species, infectious agents induce a wide range of diseases in the rat that vary from inapparent to overt clinical disease. most investigations use large numbers of rats in which a specific group or colony consists of several to hundreds of rats. accordingly, emphasis on disease is one of prevention and placed at the colony level rather than on a sin gle or a few animals. curative use of antibiotics, which is important in the treatment of bacterial diseases of nonrodent species, is rarely useful in the laboratory rat. administration of drugs to obtain therapeutic blood levels is difficult to achieve in a colony; also some animals may improve clinically but re main colonized by the pathogen and serve as carriers, reinfecting other animals. rats seldom show clinical signs of disease upon arrival to the laboratory from commercial sources. however, these rats may harbor pathogens that are of low to moderate virulence and that are capable of severely compromising the health of animals when the rats are exposed to various types of experimental stress. moreover, some of these pathogens may never cause clinical disease, yet induce microscopic lesions or biochemical aberrations that can have profound effects on research data. for these reasons, investigators and clinicians should be aware of the pathogen status of the animals used in studies, both ini tially and throughout the course of the studies. this section on infectious diseases contains those agents that are of principal importance to the investigative use of the rat. a. streptococcosis. the causative organism, streptococcus pneumoniae, is a gram-positive coccus that is rather ubiq uitous among humans and animals. streptococcus pneumoniae is frequently recovered from respiratory tract lesions in guinea pigs, nonhuman primates, and some domestic animals. in hu mans, it is often present in the nasopharynx in the absence of clinical symptoms of infection. upper respiratory tract infec tion of conventionally raised rats has been reported to be com mon. however, it is seldom present in barrier-maintained, commercial rat sources. as in pneumococcal disease in hu mans, a number of serological types have been associated with respiratory disease in rats. streptococcus pneumoniae infection in rats often remains lo calized in the nasopharynx without the development of overt disease. a shift in the host-parasite balance due to stress or concurrent infection with another pathogen may result in bronchopneumonia and bacteremia. the most common signs of respiratory disease are serous to mucopurulent nasal discharge and "red tears" due to porphyrin pigments secreted from the harderian glands, dyspnea, rales, and depressed activity. ani mals will often die within a few days after the onset of pneu monic signs. the severity and prevalence of clinical disease within an infected colony are associated with environmental conditions that induce stress (e.g., experimental manipulation, overcrowding, fluctuations in ambient temperature and humid ity, and copathogens). although all age groups are susceptible to infection and clinical disease, young animals are more apt to be clinically affected. transmission between rats is by aerosol droplet. although both humans and rats can carry the same serotypes of s. pneumoniae, the authors are unaware of evi dence indicating zoonotic or human-to-animal transmission. the most characteristic gross lesions are pulmonary consol idation and fibrinopurulent pleuritis and pericarditis ( fig. ). an extensive fibrinopurulent peritonitis, orchitis, or meningitis may occur as well. if a bacteremia occurs early, the disease may be acute with few gross lesions. streptococcus pneu moniae induces an outpouring of exudate rich in fibrin, neutrophilic leukocytes, and erythrocytes into the alveoli. bron chioles are filled with neutrophilic leukocytes. embolie lesions may occur in multiple tissues which include the spleen, liver, kidneys, joints, and brain. streptococcosis is diagnosed by clinical signs, characteristic lesions, and isolation of s. pneumoniae from lesions. the per icarditis, pleuritis, and pleural effusion noted above differenti ate pneumococcal disease from pneumonia due to mycoplasma, although the two pathogens often are superimposed. this organism produces an á-hemolysis on blood agar plates similar to that of the streptococcus viridans group. streptococ cus pneumoniae isolates are most commonly differentiated from nonpathogenic s. viridans by the sensitivity of the former organism to optochin (hydrocuprein hydrochloride). optochin-impregnated discs are placed on a blood agar plate which has been inoculated with a pure culture of the clinical isolate. if the isolate is s. pneumoniae, a distinct zone of growth inhibition will be present around the disc. although typing of s. pneumoniae isolates is seldom done today, one can type an isolate by reacting known specific s. pneumoniae antisera with s. pneumoniae isolates. this serological test is the neufeld-quellung reaction and is based on the capsular swelling that is induced by specific antiserum. there is no effective means to control s. pneumoniae infec tion once it is enzootic in a colony. benzathine penicillin ( , units/ gm body weight) may be helpful in reducing the severity of the disease and as an aid in limiting infections to a subclinical mode in some animals. however, antibiotics will not eliminate the organism from rat colonies. hysterectomy rederivation of breeding stock from infected colonies is an ef fective method of initiating new stock free from pneumococcal infection (weisbroth, ) . b. pseudotuberculosis (corynebacteriosis). the causative agent of pseudotuberculosis is the gram-positive bacillus, corynebacterium kutscheri. on occasion, other corynebacterium species can cause similar syndromes in rats. typically, the or ganism causes inapparent infections in rats, with exacerbation of respiratory disease under conditions of stress. when clinically ill, the most commonly seen signs include serous oculonasal discharge, dyspnea, anorexia, and loss of weight or retarded growth. animals with severe pulmonary signs usually succumb within several weeks, while rats with less severe signs often survive much longer. most rats will have inapparent infections in which c. kutscheri cannot be isolated from internal organs. little is known concerning how c. kutscheri is carried or transmitted within a colony. it has been suggested that the organism is transmitted via aerosol droplet or direct contact. once rats are infected, a hematogenous spread may be involved, since lung lesions are initially interstitial and not bronchial. gross lesions are characterized by a variable number of grayish-yellow foci surrounded by red zones, particularly in the lung (fig. ) . in longer-standing cases, individual foci co alesce into raised lesions cm or larger in diameter. occasion ally, fibrous adhesions occur between the lungs and thoracic walls. similar lesions may be seen in other organs, including the liver, brain, and kidneys. the hepatic lesions resemble tu bercles and have caseous centers and fibrous capsules. prepucial adenitis, arthritis, and otitis media may also be caused by c. kutscheri. the lesions in various target organs appear to be due to septic emboli. pulmonary lesions initially consist of a polymorphonuclear cell and macrophage infiltrate of the bronchioles and interstitial tissue with a round cell infiltrate occurring later. bronchi become impacted with polymorphonuclear cells and necrotic leukocytes. giemsa or gram staining of infected tissues will reveal the rod-shaped c. kutscheri organisms. diagnosis of c. kutscheri infection is made on clinical signs, gross and microscopic lesions, and isolation of the bacterium from infected tissues. although the respiratory signs are simi lar to those present with mycoplasmosis, the rapidity with which c. kutscheri clinically affected rats succumb helps dif ferentiate it from mycoplasma pulmonis-'mduced disease. un like streptococcosis, fibrinopurulent pericarditis, peritonitis, and pleural effusion are not seen. whereas peribronchial lymphoid hyperplasia is a dominant lesion in mycoplasmosis, it is unremarkable in c. kutscheri infections. corynebacterium kutscheri is easily recovered from lesions and upper respiratory tract exudates by culturing on blood agar plates incubated aerobically at °c. epizootics of pseudotuberculosis may occur in conven tionally raised breeding colonies, but rarely occur in barrierraised colonies. epizootics often can be retrospectively associ ated with an environmental stress (e.g., fluctuation in ambient temperature or ventilation). culling of ill animals will not eliminate c. kutscheri from animals remaining in a colony. isolation of the organism from animals with subclinical infec tions is not usually successful. for this reason, cortisone ad ministration has been advocated as a means for surveillance of infection in colonies prior to necropsy and culturing for c. kutscheri. in the past, most serological methods have been un satisfactory in detecting antibody in animals with inapparent infections (weisbroth, ) . recently, however, enzymelinked immunoabsorbant assay (elisa) has been shown to be capable of detecting antibody in animals without clinical signs of infection (ackerman et al., ) . hysterectomy derivation is an effective means to establish a c. kutscheri-free colony. antibiotic therapy will not eliminate c. kutscheri from a colo ny, but a -day regimen of penicillin has been reported to be effective in curtailing an epidemic of c. kutscheri-'mductd pneumonia (fox et al., ) . since c. kutscheri infection is, in most cases, inapparent and manifests itself whenever the host is sufficiently stressed, it can be a significant problem in experimentally stressed rats. c. tyzzer's disease. tyzzer's disease is caused by the gram-negative, spore-forming rod, bacillus piliformis. this organism, which is not a true bacillus, is an intracellular pathogen that has not been cultivated on artificial media, and is, as yet, taxonomically undefined. in the laboratory, b. piliformis is propagated in the yolk sac of embryonated chick eggs. this disease occurs in other rodent species and appears to be widely distributed in many nonrodent species, but there ap pears to be a degree of species specificity among b. piliformis strains. it occurs occasionally in conventionally raised rat colo nies. the vegetative form of b. piliformis is unstable in the environment. however, spores of the organism are relatively stable and are believed to be the source of transmission among animals. clinical signs associated with tyzzer's disease are not partic ularly distinctive and, accordingly, only suggestive in making a diagnosis. typically, affected rats are apt to be adolescents with signs such as lethargy, weight loss, and distended abdo mens. diarrhea is not a common sign in rats with b. piliformis infection. animals displaying clinical signs generally die with in several weeks. clinically inapparent infections occur and are most probably responsible for transmission of the organism within a colony. clinically evident tyzzer's disease is usually associated with experimentation that compromises the immunocompetence of rats. the most remarkable gross lesions involve the liver, ileum, and myocardium. hepatic lesions consist of numerous small, pale foci on the surface and within the parenchyma. the intes tinal lesion has been termed "megaloileitis" due to a segmen tai dilatation and inflammation of the ileum (fig. ) (jonas et ai, ) . heal distension is not always present. in some rats, circumscribed gray foci also occur in the myocardium. the pathogenesis of the disease is believed to involve a pri mary intestinal infection with spread to the liver via the portal circulation. bacillus piliformis invades enterocytes, resulting in villus shortening, inflammation, necrosis, and hemorrhage. intracellular organisms are demonstratable in epithelium of crypts and villi. the necrotic foci in the liver are most often present near vessels. surrounding these foci are varying num bers of leukocytes, macrophages, and fibroblasts. intracytoplasmic bacteria may be seen in hepatocytes at the periph ery of the lesions, but may be present in very small numbers and thus be hard to find. organisms are also found in myocar dium around foci of necrosis (weisbroth, ) . a presumptive diagnosis can be made by the gross lesions, but a definitive diagnosis is dependent upon observation of the organism within hepatocytes, intestinal epithelium, or myocar dium. impression smears of liver taken at necropsy and stained with gram, giemsa, or méthylène blue stains may be useful for a rapid diagnosis. however, formalin-fixed specimens stained by giemsa or warthin-starry methods are usually per formed to confirm a diagnosis. the ileal distension seen in rat tyzzer's disease must be differentrated from other causes of adynamic ileus, particularly chloral hydrate-induced lesions. prevention of tyzzer's disease in a colony is dependent upon a barrier that excludes entry of the agent by contaminated cages, equipment, and infected animals. routine cage sanita tion probably is ineffective in killing the spores of b. piliformis, but exposure of spores to °c for min has been shown to inactivate them. sodium hypochlorite ( . %) is an effective disinfectant (ganaway, ) . although antibiotics have been shown to be effective under experimental conditions in mice, there is no evidence to indicate that antibiotic therapy can be of value under natural conditions within a colony of rats (weisbroth, ) . d. pasteur elio sis. pasteur ella pneumotropica frequently infects conventionally raised rats and has been recovered occa sionally in rats from barrier-and axenic-maintained colonies. it is a pathogen of very low virulence, and most infections remain clinically inapparent. only a relatively few reports doc ument p. pneumotropica as a primary pathogen in cases of penumonia, otitis media, and conjunctivitis. as a copathogen with either m'ycoplasma pulmonis or sendai virus, it has a con tributory role in the resultant respiratory lesions and otitis. its localization is not limited to the respiratory tract, since it is frequently isolated from the oral cavity, intestinal tract, and uterus. it also has been associated with mastitis and furunculosis in rats. it has been suggested that p. pneumotropica is essentially an enterotropic rather than a pneumotropic orga nism. the intestinal tract is probably the primary site for local ization of the organism in subclinical infections. horizontal transmission is by the oral-fecal route and direct contact. since p. pneumotropica is frequently carried in the uterus, vertical transmission can occur, and, accordingly, this can compromise the microbial status of axenic and gnotobiotic colonies. distinctive clinical signs and lesions do not occur with p. pneumotropica-induoed disease. accordingly, a diagnosis must be based upon its isolation as the sole pathogen or, as in many cases, as a copathogen within lesions. blood agar medi um is satisfactory for primary isolation from nonenteric sites. however, for recovery from the intestinal tract, enrichment in a medium such as gn broth is recommended before isolation is attempted on blood agar plates (weisbroth, ) . hysterectomy derivation and barrier maintenance are the only means to control infection. however, particular attention must be made to ensure that hysterectomy-derived young came from dams that had culturally negative uteri. antibiotic thera py is not effective in eliminating the organism from a colony. e. salmonellosis. salmonella species that infect rats in clude salmonella enteritidis, s. typhimurium, s. dublin, and s. meleagridis. salmonellosis, which was once a major cause of disease in laboratory rat and mouse colonies, is rarely reported in either species today. however, it still exists in wild popula tions of rodents and, therefore, remains a potential threat to laboratory rodents. infection in an immunologically naive colony typically re sults in an epizootic of clinically affected rats and a varying proportion of animals with inapparent infection. these latter animals act as subclinical carriers to render the infection as enzootic in a colony. acute outbreaks will occur intermittently whenever immunological and other host defense mechanisms are altered. signs associated with salmonellosis in the rat are anorexia, depressed activity, starry hair coats, and soft to formless feces. affected animals die in to weeks. lesions that occur in salmonellosis differ depending on the stage of the disease. salmonellae penetrate the intestinal mucosa at the level of the ileum and cecum. the earliest le sions occur in this locale and consist of a mild dilatation, thick ened intestinal walls, and a granular mucosal surface. involve ment of the reticuloendothelial system is reflected by enlarged peyer's patches, mesenteric lymph nodes, and spleen. in some infected animals, a bacteremic state occurs that results in the demise of the host before the development of further lesions. however, in animals not succumbing to septicemia, ulcération of the ileal, colonie, and cecal mucosa occurs. histologically, the villus epithelium of the ileum is markedly degenerated, and the lamina propria is infiltrated with neutrophils and mac rophages. concomitant with intestinal lesions is the develop ment of focal necrosis and granulomas in the spleen and liver due to hematogenous spread of the organism (buchbinder et al, ; maenzae/fl/., ) . in rats who are intermittent or chronic shedders of salmonel la, the most remarkable lesions are lymphadenitis of the mes enteric lymph nodes and ulcération of the cecal mucosa. rats from which salmonella is chronically shed have more ad vanced lesions than do intermittent shedders of the organism. a diagnosis of salmonellosis relies upon identification of an isolate as a salmonella sp. recovery of salmonella from the intestines, spleen, and liver is readily accomplished in rats clinically affected during an epizootic. however, this is not true for asymptomatic carriers, since some will shed the orga nism intermittently in the feces, and recovery from tissues is difficult. recovery in carrier animals is best accomplished by initial incubation of fecal pellets in an enrichment broth, such as selenite f plus cystine broth, followed by streaking onto brilliant green agar (weisbroth, ) . from this medium, possible salmonella colonies are inoculated into triple-sugariron slants. final identification is then made by biochemical tests and serotyping. prevention of this disease is based upon the exclusion of wild rodents from laboratory animal facilities and the use of only feed and bedding that has been properly processed and pack aged to ensure against salmonella contamination. /. pseudomoniasis. pseudomonas aeruginosa, a ubiq uitous gram-negative bacterium found in soil and water, colo nizes plants, insects, animals, and humans. it often colonizes the oropharynx and can be isolated from the intestinal tract of rodents. infection with this organism in immunocompetent rats is nearly always inapparent. however, when rats are immunosuppressed, p. aeruginosa invades the upper respiratory mucosa and cervical lymph nodes, becomes bacteremic and induces an acute, lethal disease. in some cases, rats develop facial edema, conjunctivitis, and nasal discharge. in genet ically thymic-deficient rats (nude), retro-orbital abscesses may occur prior to bacteremia. transmission in laboratory rodents occurs primarily by direct contact and contaminated water bottles and automatic watering systems. phenolics are usually effective disinfectants, but quaternary ammonium compounds may actually support its growth. diagnosis of pseudomoniasis is based upon a history of immunosuppression associated with an epizootic of acute disease and isolation of p. aeruginosa from the blood and organs of affected rats. facial edema in affected rats must be differenti ated from viral sialodacryoadenitis. pseudomonas aeruginosa grows well on blood agar and most other standard laboratory media. most strains are ß-hemolytic and produce a bluish-green pigment, pyocyanin, as well as fluorescein. the use of specialized media (pseudomonas p agar) enhances pigment production. the organism derives energy from carbohydrates via oxidation rather than fermentative me tabolism. identification of isolates as p. aeruginosa is easily made by the above characteristics and appropriate biochemical reactions (weisbroth, ) . in most research applications, p. aeruginosa-free rats are not necessary for the conduct of the work. it is a major problem, however, in rats used for burn research and in studies in which drugs or radiation induce immunosuppression. infection can be relatively well controlled in a colony by hyperchlorinating drinking water at ppm or by acidification of water to a ph of . - . . in a closed colony, it is also advisable to remove rats that remain culturally positive after water treatment has been instituted. in studies requiring pseudomonas-free rats, isolators are useful in which a gnotobiotic environment can be achieved. alternatively, laminar flow units may suffice if supplies and equipment are sterilized and personnel wear sterile garments. g. streptobacillosis. streptobacillus moniliformis is a commensal bacterium often present in the nasopharynx of con ventionally raised rats. although it may be involved occasion ally as a secondary invader within inflammatory lesions of the rat, the chief importance of s. moniliformis is that it is the principal agent causing rat-bite fever in humans (anderson et ai, ) . the other bacterium associated with this clinical syndrome is spirillum minus. clinical signs in humans usually occur within days of a rat bite and consist of headache, weakness, fever, a generalized rash, and arthritis. often clini cal signs subside in several days but then recur at irregular intervals for weeks or months (see chapter ). a. murine respiratory mycoplasmosis. murine respirato ry mycoplasmosis (mrm) is the term now accepted for a dis ease which, for many years, had an undefined etiology and a number of synonyms [i.e., infectious catarrh, enzootic bronchiectasis, chronic respiratory disease (crd), and chronic murine pneumonia]. since , the causal relationship of mycoplasma pulmonis with this disease has become well estab lished (kohn and kirk, ; lindsey et al., ; whittlestone et al., ) . of all the pathogens occurring in laboratory rats, m. pulmonis has had the greatest negative im pact on studies. this has been primarily due to the chronicity of the disease, which often manifests itself only after months of infection. long-term studies in areas of toxicology, carcinogenesis, nutrition, and gerontology, in particular, have been affected. prior to the use of gnotobiotic techniques and barrier maintenance in rat production colonies, m. pulmonis was enzootic in nearly all commercial and institutional colo nies. today, vendors can be selected who offer mycoplasmafree rats. my coplasma pulmonis is highly contagious and in duces a disease that frequently results in debilitation or demise of the host after a long period of time. the clinical signs associated with mrm range from negligi ble upper respiratory tract signs to systemic signs associated with pneumonia. the earliest and most common signs include snuffling and serous or mucopurulent oculonasal discharge. extension of m. pulmonis infection from the nasopharynx via the eustachian tubes to the middle ears is common. however, torticollis and circling due to involvement of the inner ear are infrequently observed, even though one or both middle ear bullae may be impacted with exudate. the onset of upper res piratory signs is variable, but often occurs within several weeks postinfection. signs of penumonia include dyspnea, rales, and systemic effects such as weight loss, starry hair coat, and hunched posture. characteristically, signs of pneumonia occur - months postinfection, but this is quite variable and is a function of environmental influences, such as intracage ammonia levels and the immune competence of the host. in a small percentage of cases, the disease will be nearly subclinical even in the presence of extensive pulmonary lesions. mycoplasma pulmonis is transmitted both horizontally and vertically from dams to their litters. in most instances, trans mission from the female occurs postpartum by direct contact, but if the genital tract of the dam is infected, antenatal infec tion can occur. horizontal transmission between postweanling rats of any age readily occurs, and there appears to be no sig nificant age-related resistance to either infection or disease. although little is known about differences in resistance among rat stocks and strains, the lew rat has been shown to be more susceptible to mrm than the f rat. there is little evidence available to indicate that transmission occurs through fomites such as caging equipment and garments worn by personnel. since aerosol droplet and direct contact appear to be the prima ry modes by which m. pulmonis infections are spread, the rapidity with which the organism is transmitted is dependent upon environmental factors, such as ventilation rates, degree of recirculation of air, and animal density within rooms. the basis for the pathogenicity of m. pulmonis is not well understood. mycoplasma pulmonis adsorbs to the cell mem brane of the ciliated, columnar or cuboidal epithelia in the res piratory tract (fig. ) . it has been suggested that adsorption is a means by which mycoplasmas damage host cells by uptake of essential cellular metabolites; release of cytotoxic products, such as h ; or cross reaction of antibody with cell mem brane components that are antigenically similar to or altered by mycoplasmas. infection severely distorts or ablates ciliary structures (fig. ) , interfering with mucociliary clearance mechanisms. the gross lesions in the upper respiratory tract include mucopurulent exudate in the nasal cavity, sinuses, and middle ear bullae. later, the exudate becomes caseous within the bul lae. lesions in the lower respiratory tract reflect those of a bronchopneumonia. the earliest lesion is a mucopurulent exu date within the trachea, bronchi, and bronchioles. this pre cedes grossly evident lesions of the lung parenchyma that ini tially consist of atelectasis due to bronchial occlusion. later, bronchiectatic lesions appear as numerous cream-colored nod ular abscesses on the surface of the lung. these lesions may be restricted to only a portion of a lobe or may involve nearly all of the parenchyma (fig. ) . microscopically, the inflammatory response is characterized by a lymphocyte and plasma cell infiltrate in the submucosa and neutrophilic leukocyte response within the lumina of the epithelium. nasal cavity, eustachian tubes, middle ears, and tracheobronchial tree. a consistent and prominent lesion in the lung is the peribronchial lymphoid hyperplasia that often be comes quite massive. within the lumina of the bronchi and bronchioles, mucin and neutrophil exudation increases during the course of the disease to the point of bronchiectasis. con comitant with the impaction of bronchi is a change in the epithelia from a ciliated, columnar type to a squamoid type. this change in epithelial architecture is likely associated with cytotoxic enzymes from autolyzed neutrophils, although a di rect cytotoxic effect from mycoplasmas could be involved. a tentative diagnosis of mrm can usually be made by obser vance of the clinical signs and gross lesions described above. clinical signs alone are not particularly helpful, since nasal exudates are present in bacterial infections such as s. pneumoniae. in addition, the reddish porphyrin deposition seen in the nares and periorbitally in sialodacryoadenitis virus infec tion and water deprivation may be confused with exudation. the gross lesions of otitis media and bronchiectasis are rather distinct. however, c. kutschen lung lesions may grossly mim ic those of mrm. histopathology and serological evidence will differentiate mrm from sendai virus infection, although the two infections are often superimposed. recently a filamen tous bacterium has been associated with bronchiectasis in wild and laboratory rats (mackenzie et al., ) . however, the causal relationship of this organism with lesions is undefined since the rats were also infected with m. pulmonis. this fila mentous bacterium has not been successfully grown on artifi cial media, and its presence is best verified by either histology, using the warthin-starry stain, or electron microscopy (fig. ) . although a definitive diagnosis of mrm is made by isola tion of m. pulmonis from involved tissues, it is evident that the existence of other agents must be evaluated to determine if copathogens are contributory to lesions. prevention of mrm in either breeding or experimental colo-nies is dependent upon barrier systems that preclude the entry of m. pulmonis into the facility. hysterectomy derivation is the only means of establishing an m. pulmonis-frtt breeding colo ny from a previously infected stock. due to the frequent local ization of this microorganism in the uterus, it is necessary to ensure that neonates taken by hysterectomy have not been in fected in utero. rats used in research animal facilities are ob tained from various commercial and institutional sources. ac cordingly, it is essential that the mycoplasma status of these sources is known and that the rats are housed by vendor or in groups with a similar microbial status. for assessment of whether a group of rats is m. pulmonisfree, the best sites for isolation in animals without gross lesions are the nasal cavity, middle ear, trachea, and uterus-oviduct. mycoplasma pulmonis is not particularly fastidious and grows well in several types of mycoplasma media (cassell et al., ; lentsch et al., ) . most formulations have a ph indi cator that is useful since m. pulmonis ferments glucose. in broth media, moderate to heavy growth is reflected by ph and color of the broth. in broth cultures in which the titer is low, a perceptable ph change may not occur. tissue and washing samples should be placed in broth rather than agar media, since recovery of the organism is more likely in those samples con taining few mycoplasmas. samples from broth cultures are transferred to agar media when a ph change is readily evident or at - days if no ph change occurs. mycoplasma colonies are evident in - days by observation with x stereoscopic microscopy. although culturing and histopathology have been the usual means to survey rat colonies, elisa testing has recently been shown to be a very sensitive serological assay and one that can be performed quickly in most clinical laboratories (cassell et al., a) . in vitro sensitivity tests show m. pulmonis to be susceptible to tetracycline and tylosin. tetracyline, given at mg/ml drinking water, may be useful in some situations (lindsey et al., ) . however, treatment with antibiotics seldom influences the disease course of mrm in a colony situation. b. murine genital mycoplasmosis. mycoplasma pulmonis recently has become recognized as an important pathogen in the female genital tract of rats, and thus is being treated here as a distinct disease rather than as a sequella to mrm. infection of the genital tract is usually inapparent. however, reduced fertility and fetal deaths can occur. infection of the oviduct and uterus occurs frequently in rats who have respiratory my coplasmosis. it is unknown whether localization in the genital tract occurs due to a hematogenous spread or to an ascending infection of the genital tract. it has been shown that subsequent to intravenous inoculation, m. pulmonis almost invariably lo calizes in the female oviduct-uterus. gross lesions, when present, consist of a purulent oophoritis, salpingitis (fig. ) , and pyometra. the lew strain is particu-Ä^sfcvvv ( Ä ß f/g. . electron micrograph of filamentous bacterium (large arrow) and m. pulmonis (small arrow) attached to epithelium of respiratory mucosa. the morphology of size of the filamentous bacterium are similar to that of the cilia. (courtesy of dr. w. f. mackenzie.) larly prone to develop gross lesions. mycoplasmapulmonis ad sorbs to the epithelial cells in the genital tract in a manner similar to that seen in the respiratory tract. salpingitis occurs most frequently and is characterized by exudation of neutrophils into the lumen, hyperplasia of oviductal epithelium, and a lymphoid response in the submucosa. the lesions in the ovarian bursa include edema and inflammation. uterine le sions can vary from a mild inflammatory change to pyometra (casselle/fl/., b). genital mycoplasmosis in the male rat has not been well doc umented. however, it is known that experimental inoculation can include an inflammatory response in the ductus efferens and epididymis. moreover, it is known that m. pulmonis is capable of adherence to spermatozoa in an in vitro system. since pasteur ella pneumotropica can also induce similar le sions in the female rat, a diagnosis of mycoplasmosis is depen dent upon isolation of m. pulmonis from the lesions. methods for culturing and identification are similar to those used for respiratory mycoplasmosis. because the rat is widely used in various types of reproduc tive biology research, m. pulmonis colonization, even without gross lesions, would probably impact on the validity of data. the grossly evident caseous lesions in the ovary and oviduct can be mistaken for neoplasia if microscopy is not done. c. mycoplasmal arthritis. the etiological agent of this disease is mycoplasma arthritidis. this mycoplasma species colonizes the pharynx, middle ears, and lungs of rats, although few studies have been done to document the relative frequency of this mycoplasma in rat sources. within the respiratory tract, m. arthritidis colonization is thought to induce negligible le sions, and it has been shown to coexist with m. pulmonis. although it is often considered to be the principal agent in volved in arthritis in rats, the disease has been rarely reported. nearly all reports of its involvement in clinically apparent ar thritis have been made prior to . it has been suggested that poor cage sanitation and abrasions of the extremities are in volved in entry of the organism to the joints by hematogenous spread or extension from surrounding tissues (ward and cole, ) . since the organism appears to be of low virulence, the immunocompetence of the host may be a major factor in the outcome of infection. arthritic animals limp and move with difficulty due to pain associated with the polyarthritis. any of the joints in the limbs and vertebrae can be affected, but the tibiotarsal and radiocarpal joints are most often involved. affected joints are hyperemic and swollen. incised joints reveal a purulent exudate in both articular and periarticular tissues. microscopically, there is exudation of neutrophils into the synovial spaces, and a lym phocyte and plasma cell infiltration in the synovial mem branes. destruction of the articular cartilage occurs subsequent to the inflammatory response. since polyarthritis can occur subsequent to septicemias asso ciated with other bacteria, particularly c. kutschen, a diag nosis of m. arthritidis-'mduced arthritis is contingent upon the demonstration of m. arthritidis by isolation or immunofluorescence techniques. this mycoplasma species grows well in me dia used to isolate m. pulmonis if arginine is added to the for mulation (cassell et al., ) . tetracyclines have been used to prevent the onset of arthritis when the organism has been inoculated intravenously, but there are no reports of its efficacy in spontaneous cases. mycoplasma arthritidis, like m. pulmonis, may contaminate transmissible tumors and caution should be exercised to ensure transplanted tissues are not contaminated. hemobartonellosis. the causative agent of this rickettsial disease is hemobartonella muris. this organism is an extra cellular parasite of erythrocytes and induces inapparent infec tions that may persist for long periods. the ability of the host to restrict the infection to a subclinical mode rests with the integrity of the reticuloendothelial system. evidence of infec tion is usually limited to splenomegaly and laboratory findings of mild parasitemia and reticulocytosis. transmission of h. muris involves the blood-sucking louse, polyplax spinulosa. transmission can occur during a blood meal or when rats crush infected lice and are inoculated via pruritis-induced abrasions. the organism can also be transmit ted inadvertantly with transplantable tumors and other biolog ical products. diagnosis of hemobartonellosis is dependent upon identifica tion of the organism in the peripheral blood of infected ani mals. the usual method of detection is by splenectomizing rats suspected of harboring the organism. in these rats, severe para sitemia and hemolytic anemia occur within weeks after sur gery. hemobartonella muris can be visualized on the surface of erythrocytes in romanowsky-stained blood smears as coc- coid bodies arranged singly, in clusters, or chains (cassell et al., ) . the rarity of reported cases would indicate h. mûris is no longer a significant problem in barrier-maintained colonies. however, conventionally maintained colonies may be exposed to infected wild rats and p. spinulosa and, accordingly, the disease still is of importance in the laboratory rat. the disease has had a negative impact on investigations of various types, but principally with those in which the host's immune compe tence has been impaired. a. parvoviral syndromes. parvoviruses that can infect rats include rat virus (rv), toolan h-l (h-l) virus, and minute virus of mice (mvm). parvoviruses are small nonenveloped viruses that resist extremes in temperature, ph, and drying. rat virus, or kilham rat virus (krv), has several antigenically related strains (rv, h- , x- , l , hb, sprv, her, hhp, kirk), all of which have been isolated as inadvertant contami nants of rat tissue or rat-passaged biological material. toolan h-l related serotypes (h-l and h-t) are antigenically distinct from rv serotypes. both rv and h-l are experimentally pathogenic, producing similar lesions, but only rv has been associated with natural disease. neonatal rats can be experi mentally infected with mvm, but the virus does not seem to cause natural infection. minute virus of mice antibody reac tivity can be present in rat serum, but this is probably non specific, since it can be found in germfree rat serum and is reduced or eliminated by receptor destroying enzyme. rat virus infection is usually subclinical or latent, but a num ber of clinical syndromes have been associated with it. infec tion of pregnant females can cause fetal résorption and birth of small litters. pups are runted, atactic, or jaundiced. neonates develop similar signs following postpartem exposure. rats in troduced to an infected colony can develop ruffled fur, de hydration, and sudden high mortality. a similar syndrome oc curs in latently infected adults subjected to immunosuppressive regimens. the rat is the only natural host for rv and h- , although experimental infection can be established in a number of other species. seroconversion to both rv and h-l virus is common, with a high prevalence of infection within an enzootically in fected colony. horizontal transmission is achieved by the oral and probably respiratory routes, with virus excretion primarily in the feces. some strains of rv can be excreted in the milk or in utero. clinical signs are manifest transiently upon introduc tion of rv into a previously uninfected population, but, there after, the virus spreads rapidly to produce subclinical or inapparent enzootic infection. rat virus can persist as a true latent infection in the presence of high circulating antibody, but dis ease can be activated by immunosuppression. it must, there- fore, be assumed that seropositive rats are persistently infected and can serve as a source of infection to other rats. pups infected in utero or as neonates develop intranuclear inclusions and necrosis in the outer germinal cell layer of the cerebellum. the recovered animal has severe depletion of the internal granular layer and disorganized purkinje cells. intra nuclear inclusions are also in hepatocytes, kupffer cells, endothelial cells, and biliary epithelial cells, resulting in necrotizing hepatitis and the sequellae thereof (bile retention, jaundice, peleosis, bile ductal hyperplasia, parenchymal collapse, nodu lar hyperplasia). in adults, infection is usually inapparent, but when acute disease is precipitated, rv injures vascular walls and hematopoietic elements, causing coagulative disorders, thrombosis, hemorrhage, and infarction within the central ner vous system (hemorrhagic encephalomyelopathy). hemorrhagic and necrotic lesions have also been noted in the per itoneum, testis, and epididymis. rat virus has broad tissue tropism and lesions or clinical signs may potentially be varied, depending on virus and host factors (coleman et al., ; jacoby et al, ) . infertility and unthrifty pups caused by rv must be differenti ated from environmental and husbandry factors or infectious agents such as mycoplasma or sendai virus. adult disease must be differentiated from toxicity, nutritional deficiency, and trau ma. diagnosis is made by the typical lesions, if present, virus isolation, and serology. seroconversion to each virus (rv or h-l) can be detected by serum neutralization, hemagglutination inhibition, complement fixation, and immunofluorescence. hemagglutination inhibition is currently the most commonly used means of antibody determination (jacoby et ai, ) . since rv infection is usually silent and persistent and can be transmitted either vertically or horizontally, effective control is best achieved by destroying the entire population, decon taminating, and repopulating with clean stock. virus-free rats can be obtained from selected commercial vendors or by caesarean rederivation. rederived progeny must be tested for vertically transmitted strains of virus. colonies can be kept virus-free by limiting entry to seronegative, virus-free rats (as well as transplantable rat neoplasms or tissues), periodic serological testing, and adequate physical containment. although parvovirus infection of rats is usually inapparent, there can be adverse effects on the research usefulness of in fected rats. immunosuppression may exacerbate illness and mortality in latent carriers. the viruses often contaminate transplantable tumors and cell lines, can modify immune re sponsiveness or cause teratological effects. a decision to work with infected animals should be made carefully. b. other dna virus infections. rats are susceptible to rat cytomegalovirus, which has a predilection for the salivary and lacrimai glands. infection is widespread among wild, but not laboratory rats (jacoby et al., ) . rats also seroconvert to mouse adenovirus, but it is not known if infection is due to a mouse or rat strain of virus. adenovirus-like inclusions have been reported in the intestine of rats treated with cancer chemotherapeutic agents (ward and young, ) . c. siaiodacryoadenitis virus and related coronaviral infections. two strains of coronavirus have been identified as pathogens of laboratory rats: siaiodacryoadenitis virus (sdav) and rat coronavirus (rcv). furthermore, rats are experimen tally susceptible to the coronavirus of mice, mouse hepatitis virus (mhv). coronaviruses are large, pleomorphic enveloped rna viruses with surface peplomers or spikes that confer a corona-like appearance to the virion. viruses of this group have complex antigenic interrelationships and cross-react ex tensively. common antigens are shared by sdav, rcv, and mhv, particularly by complement fixation, but antibody reac tivity is highest with homologous virus. siaiodacryoadenitis virus and rcv represent different strains of the same virus, but whether different strains of the same virus or separate viruses, they are both important natural pathogens in rats. the signifi cance of mhv for rats is not known, but the virus can replicate in the respiratory tract of intranasally inoculated rats (taguchi et al., ) . natural antibodies to mhv can occur in rats, but this is probably due to the closely related antigenicity of mhv to sdav and rcv rather than natural mhv infection of rats (barthold, ) . clinical signs of sdav infection vary widely in severity, but include blepharospasm, sneezing, porphyrin-pigmented nasal and ocular discharge, and cervical edema (fig. ) . some rats develop keratoconjunctivitis and other ocular lesions. signs persist approximately one week, but ocular sequellae can be permanent. acutely infected rats become anorectic, and estrus can cease temporarily. infection is subclinical in weanling or older rats, but intranasally inoculated neonates die and suck lings develop lower respiratory disease. siaiodacryoadenitis virus is highly contagious and spreads rapidly among susceptible rats by contact, aerosol, or fomite. susceptible rats of any age can be infected. when enzootic within a colony, clinical disease occurs only in sucklings, since adults are immune. infection is acute, lasting only about week, at which time rats seroconvert with no carrier state. maintenance of sdav in a colony requires continuous intro duction of susceptible stock as weanlings or newly introduced rats. the epizootiology of rcv is presumed to be similar to sdav. within days of intranasal inoculation, sdav causes rhi nitis followed by necrosis of the ductular and acinar epithelium of salivary and lacrimai glands, accompanied by intense in flammation and edema. tracheitis and peribronchial lymphoid hyperplasia can also be found. salivary glands appear swollen, pale, with interlobular and periglandular edema. harderian glands are flecked with yellow-gray foci. one, some, or all of the salivary or lacrimai glands can be affected, with the excep tion of the sublingual glands, which are spared. cervical lymph nodes become enlarged. glandular repair ensues within week, with squamous metaplasia of ductular epithelium and hyperplasia of acinar epithelium. the repair phase subsides within days with minimal residual lesions. interstitial pneu monia can occur in suckling, but not adult rats. conjunctivitis, keratitis, corneal ulcers, synechia, hypopyon, and hyphema can arise due to lacrimai dysfunction. eye lesions usually re solve, but can proceed to chronic keratitis, megaloglobus (fig. ) , and retinal degeneration. rat coronavirus infection causes rhinotracheitis and focal interstitial pneumonia. salivary but not lacrimai gland infection is rare, but when present, resem bles wild sdav lesions. infection with rcv also lasts approx imately week (barthold, ; jacoby et al., ) . nasal and ocular signs must be differentiated from those caused by mycoplasma, sendai virus, pathogenic bacteria, ex cess ammonia, or hypovitaminosis a. cervical swelling may fig. . epiphora and swelling of the ventral neck in a rat naturally infected with sdav. (from barthold, ; courtesy of hemisphere publishing corp.) fig. . megaloglobus and hyphema in a young rat naturally infected with sdav. (from barthoid, ; courtesy of hemisphere publishing corp.) also occur in immunosuppressed rats infected with p. aeruginosa. microscopic sdav lesions are characteristic. mild lower respiratory tract lesions associated with rcv must be differentiated from those of sendai virus or pneumonea virus of mice (pvm). seroconversion or rising complement fixing antibody titers following acute disease is confirmatory. how ever, antibody may be low or undetectable with this method. serum neutralization is another test that can be used, but the most sensitive antibody tests are immunofluorescence or elisa. either mouse or rat coronaviruses are used as antigen in these latter tests (smith, ) . rats can be kept free of sdav and rcv if they are isolated and if newly introduced rats are immune or unexposed. intro duction of a single subclinically infected rat can precipitate epizootic disease among naive rats. if an outbreak occurs, the infection will run its course and die out within - weeks if new rats are not introduced into the room and if breeding is temporarily ceased. routine disinfection of rooms and equip ment is sufficient to destroy environmental sources of virus. sialodacryoadenitis virus lesions can be confused with or contribute to changes induced by test compounds or nutritional deficiencies, particularly vitamin a. sialodoacyoadenitis virus disease can predispose to anesthetic death due to airway hypersecretion. eye lesions resulting from sdav infection can in terfere with eye research. both sdav and rcv can potentiate other respiratory infections. d. sendai viral infection. sendai virus commonly infects laboratory rats, but its clinical significance is less than in mice. sendai virus is a parainfluenza virus of the paramyxovirus family. paramyxoviruses are pleomorphic, enveloped, labile rna viruses. sendai virus infection in rats is usually subclinical, but can be manifested as ruffled fur, dyspnea, or anorexia. a decrease in average litter size and runted pups is common during outbreaks in breeding colonies. sendai virus is highly contagious and disseminates rapidly. outbreaks subside following development of an immune popu lation, with the potential of recurrence several months later as the susceptible population enlarges. sendai virus induces an acute respiratory infection with no natural carrier state. excre tion and transmission of virus occurs via the respiratory tract (jacoby <?r Ö/., ) . sendai viral lesions in the rat are similar to those in genet ically resistant strains of mice. following an initial necrotizing rhinitis, tracheobronchitis ensues within week of infection. lungs become consolidated and plum-colored in a patchy, pri marily anteroventral, distribution. necrotizing bronchiolitis, focal nonsuppurative interstitial pneumonia, and perivascular and peribronchial lymphoplasmacytic infiltrates are the hall marks of the lower respiratory component. in the rat, acute lower respiratory lesions last only a few days and coincide with clinical disease. repair of bronchial mucosa is effected through hyperplasia and squamous metaplasia. residual le sions can last for months in the mouse, but probably not in rats. during the acute stage of sendai virus infection, mucosal necrosis and inflammation can extend up the eustachian tubes to the middle ear. bacterial otitis media is often preceded by sendai viral infection in rodents (brownstein et al., ; d. g. brownstein, unpublished observations, ) . mild nonsuppurative pulmonary lesions can be seen with sendai, pvm, rcv and to a lesser extent, sdav. sendai virus can be superimposed upon and contribute to respiratory mycoplasmosis. squamous metaplasia of respiratory mucosa can mimic hypovitaminosis a and also occurs in my coplasmosis. virus isolation and serocon version or rising titers to sendai virus confirm a diagnosis based upon characteristic lesions and clinical signs. sendai virus antibody can be accu rately detected by complement fixation and hemagglutination inhibition, but newly developed immunofluorescence and elisa are replacing these methods (smith, ) . rats can be kept free of sendai virus in a similar manner as with sdav and rcv. recovered sendai virus antibody-posi tive stock can be utilized as breeders to reestablish a virus-free population. pups born of these parents will be virus-free and antibody negative, following decay of their maternal antibody. mice and hamsters can serve as sources of infection with paramyxoviruses. sendai virus infection, although usually subclinical, can cause significant pulmonary changes and act as a copathogen with mycoplasma or bacteria. squamous metaplasia and hy perplasia induced by these agents influence respiratory carcinogenesis and interpretation. sendai virus can cause immunosuppression for prolonged periods in rats (garlinghouse and vanhoosier, ) . e. pneumonia virus of mice infection. pneumonia virus of mice is a pneumovirus of the paramyxovirus family and thus shares many features in common with sendai virus. it has not been associated with clinical disease in the rat or mouse under natural conditions. several rodent species, including hamsters, guinea pigs, and gerbils, seroconvert to pvm under natural conditions. the virus seems to spread rapidly among suscepti ble rodents, based on serocon version. it is presumed to be transmitted in a manner analogous to sendai virus. the pathogenesis of pvm has been investigated in mice, but has not been thoroughly examined in rats, even though it pro duces more significant pulmonary lesions in rats than mice un der natural conditions. in the mouse, pvm replicates in the upper respiratory mucosa without obvious cytolysis following low-dose exposure (d. g. brownstein, unpublished observa tions, ) . viral antigen and cytolytic lesions are seen in the bronchial and bronchiolar epithelium as well as alveolar walls following apparently higher doses of virus in a pattern reminis cent of sendai virus. the ensuring lesion, like that of sendai virus, is necrotizing bronchiolitis and interstitial pneumonia (carthew and sparrow, ) . the active infection lasts about week, after which time lymphocytic infiltration of peribronchial and perivascular tissues and focal interstitial pneumonia can persist for several weeks. rat lungs develop prominent nonsup purative perivascular and mild interstitial inflammation in re sponse to both experimental and natural pvm infections (d. g. brownstein, unpublished observations, ; vogtsberger et al., ) . infection is usually diagnosed retrospectively in rats, where pulmonary lesions are observed following seroconversion to pvm in the absence of other respiratory pathogens. pulmonary lesions mimic those of sendai virus or possibly coronaviruses. antibody to pvm has usually been detected by hemagglutination inhibition or serum neutralization, but these tests are being replaced by immunofluorescence and elisa. hemagglutination inhibition is least expensive and highly accurate, however (smith, ) . pulmonary lesions induced by pvm can inter fere with the interpretation of experimental studies, or possibly pulmonary function, although this has not been established. hemorrhagic fever with renal syndrome, korean hemorrhagic fever, or muroid virus nephropathy is a human disease com plex caused by one or more serologically related bunyaviruses, including hantaan virus. this syndrome is mentioned here be cause of its emerging eminence as a major zoonotic disease in which several species of the suprafamily muroidae, including laboratory rats, can serve as carriers. the virus is transmitted to man by excretions and aerosols from the lungs, saliva, and urine of healthy rodent carriers. pathology, if any, in rats has not been described. the disease in humans includes proteinuria, azotemia, and, less often, petechiae, hemoconcentration, hypotension, and renal failure. human mortality can oc cur, but recovery with immunity to reinfection is usual. hemorrhagic fever with renal syndrome viruses cause human disease throughout much of eurasia, but on the basis of serological evidence, hfrs viruses exist world-wide in feral rodents and humans, including the united states. transmis sion of hfrs to laboratory personnel by silently infected al bino laboratory rats (rattus norvegicus) has been documented in japan and belgium. virus-infected lung and kidney cell lines provide excellent sources of antigen for sensitive immu nofluorescence tests, but serological monitoring of laboratory rats for hfrs virus antibody is not currently a common prac tice (gajdusek et al, ; anonymous, ) . g. other virus infections. rats harbor endogenous retroviruses or genomic retrovirus sequences that (in contrast to the mouse) are of minimal significance in terms of natural disease. a number of endogenous rat sequences have been artificially / / / recombined with mouse leukemia virus sequences or naturally recombined with other rat retrovirus sequences to form defec tive rat sarcoma viruses, for example kirsten and harvey rat sarcoma viruses, which are experimentally oncogenic to rats, mice, and hamsters (shih and scolnick, ) . natural seroconversion to reovirus- and mouse encephalomyelitis virus have been noted with no apparent disease. an enterovirus with neurotropic properties has been isolated from naturally infected, asymptomatic rats. although rats can be experimen tally infected with ectromelia and lymphocytic choriomeningitis virus, these agents are not of practical significance as natu ral infections of rats. testing for antibody to these agents is therefore a vacuous exercise. a number of other viruses or viruslike agents have been isolated or incriminated as causes of disease in rats, including rat submaxillary gland virus, novy virus, enzootic bronchiectasis agent, gray lung virus, and wild rat pneumonia agent. these agents, if extant at all, are of du bious importance in contemporary laboratory rat populations (jacoby et ai, ) . recently, clinically silent infection with an unidentified pox virus has been found in soviet rats. micro scopic necrotizing and inflammatory changes were restricted to the nose (kraft et ai, ) . the agent is believed to be cow pox virus. laboratory rats are host to far fewer parasites than wild rats, since husbandry practices interrupt complex life cycles and caesarean rederivation has simply eliminated many agents al together. control of these agents in infected colonies is also best achieved by these means. this section will mention only agents that have been reported in laboratory rats, although most are unlikely or rare. table ix outlines selected treatment regimens for control of common metazoan parasites, but the reader must beware that these drugs may render the treated rat useless for experimental work. decisions to treat rather than eliminate infected rodents must be made with care, in concert with the needs of the scientific investigator. a. protozoal infections. several flagellates can parasitize, rats but few are truly pathogenic. hemoflagellates (tripanosoma cruzi, t. lewisi) occur in wild rats, but these agents require anthropod vectors. nevertheless, t. lewisi can be en countered if a colony is infested with rat fleas. trypansoma lewisi is only mildly pathogenic even in heavy infections, and infection is short term. rats harboring this organism become ill when stressed or immunosuppressed. enteric flagellates (tritrichomonas sp., tetratrichomonas sp., pentatrichomas sp., trichomitis sp., hexamastix sp., enteromonas sp., retortamonas sp., chilomastix sp., monocercomonoides sp., and octomitus sp.) are very common but nonpathogenic in labora tory rats. giardia muris and spironucleus (hexamita) muris application - gm/liter drinking water for days, stop days, re peat days . gm/liter drinking water for days mg/liter drinking water or . mg/kg feed for weeks . mg/gm feed for day mg/g sc, single dose mg/kg po, single dose . - . % dust with or without pyrethrin hr/week on cage top for several weeks - gm of pellets in bedding for days, change bedding and cage, repeat at days, change and repeat at month . % dip - % dust, up to . % spray or up to % dip, repeat at days up to . % dip % spray "information kindly supplied by dr. j. w. streett, yale university. ; 'a number of these compounds have adverse effects on the experimental usefulness of treated rats and therefore should be used with discretion and full knowledge of the investigator. are also common and considered potentially pathogenic, caus ing unthriftiness, diarrhea, and intestinal lesions in severely affected animals. however, these are generalizations made from other species, since natural clinical disease has not been reported among rats. sporozoan parasites, common in wild rats, are encountered only on occasion in laboratory rats. hepatozoon muris infec tion is usually subclinical, but severely affected rats can have leucocytosis, splenomegaly, anemia, hepatic degeneration, emaciation, or death. infection requires ingestion of the vec tor, laelaps echidninus. toxoplasmosis (toxoplasma gondii) is subclinical in rats, requiring the ingestion of cat feces or contaminated tissues containing asexual stages, but can also be transmitted transplacentally. intracellular t. gondii organisms occur in several tissues, particularly lung and reticuloendothelial organs, and cysts are found in the central nervous system. sarcocystis muris, once common but now rare in labo ratory rats, has a life cycle similar to Ã. gondii. cysts are found in the muscle. frenkelia sp. has been described in a colony of laboratory rats, causing large thin-walled cysts and inflamma tion in the central nervous system, but no clinical signs. encephalitozoon cuniculi is apparently now uncommon in labora tory rats and seldom induces signs or lesions. its prevalence and significance are far greater in the laboratory rabbit. single or clusters of small spores form within cysts in a number of tissues. nonsuppurative inflammation can be found in the brain and kidneys. unlike t. gondii, e. cuniculi organisms are gram-positive. encephalitozoon cuniculi can be transmitted via contaminated transplantable tumors, tissue, or tissue fluids from infected rats. intestinal sporozoans include eimeria nieschulzi and e. miyairii, which infect the small intestine, and e. separata, which infect the large intestine of wild rats. they are exceedingly rare or nonexistent in laboratory rats. pneumocystis carinii, once considered to be a yeast but now considered a sporozoan, is an ubiquitous organism that infects the lung of a variety of species, including laboratory rats. prev alence of infection is high within infected colonies, but preva lence among various geographic areas in unknown. it seems to be very common among both conventional as well as barrierreared rats. normally p. carinii is not pathogenic, but infected rats can develop disease after several weeks of repeated steroid injections and starvation, particularly in younger rats. remark ably, rats from many commercial sources can be so treated and shown to be infected, even if free of other pathogens. under these circumstances, the lungs are expanded and rubbery. al veolar walls are thickened and hypercellular, and alveoli are distended with proteinaceous, eosinophilic foamy material containing macrophages and organisms in different stages of development. tissues can be stained with special stains, such as methanamine silver or periodic acid-schiff, to visualize the organisms, which are not visible with hematoxylin and eosin. other protozoa, including entamoeba muris and balantidium coli, are found in the large bowel of rats, but are not pathogenic. the reader is referred to more exhaustive coverage of protozoa for detailed means of diagnosis (flynn, ; hsu, ) . b. nematodiasis. syphacia muris is a common oxyurid (pinworm) of the cecum and colon. embryonated eggs are passed in the feces or deposited on the anus. rats are infected by ingestion of eggs or by larval migration into the colon from the anus. syphacia muris closely resembles s. obvelata, the mouse pinworm. both species of syphacia can patently infect rats or mice, but preference seems to be shown for the homolo gous host. another common oxyurid of rats and mice is aspicularis tetraptera, which is also found in the cecum and colon. aspicularis eggs are nonembryonated, and are not de posited near the anus. depending on parasite species, the pré paient period is - days. diagnosis is achieved by exam ination of large bowel contents (particularly the cecum and proximal colon) for nematodes, feces for ova, and, in the case of syphacia, cellophane tape impressions of the anus for ova. speciation is easily achieved by the differential morphology of ova. aspicularis ova are symmetrically ellipsoidal, while syphacia ova are flattened on one side. since eggs are ex tremely resistant to environmental factors and disinfectants, and autoinfection can occur with s. mûris, infection is difficult to eradicate. anthelminthic treatment ameliorates infection (table ix) , but all rats must be treated simultaneously and the room must be thoroughly cleaned. despite these actions, rein fection frequently occurs. caesarean rederivation and subse quent strict sanitation are the most effective means of control. pinworms are generally considered nonpathogenic, but heavy parasite loads can be deleterious to the host. concern has been raised regarding the effects of these agents on research but no effects have been documented in the rat. trichosomoides crassicauda is common in some rat colonies but is not generally encountered in commercially raised ani mals. it inhabits the transitional epithelium and lumen of the urinary tract. the small male resides within the reproductive tract of the female, whose anterior end embeds within the mucosa. embryonated ova with bipolar opercula are shed in the urine. following ingestion, eggs hatch in the stomach, lar vae penetrate the gastic wall, then migrate via the lungs, kidney, and ureter to the urinary bladder. migrating larvae can incite eosinophilia and formation of granulomata, particularly in the lung. drug treatment (table ix) is effective, as is cesarean rederivation. parasites do not incite an inflammatory response in the bladder, but can serve as a nidus for calculi or enhance natural and experimental bladder carcinogenesis. other nematodes are common in wild but rare in laboratory rats. trichinella spiralis adults occur in the intestine, and lar vae migrate extensively, encysting in muscle. transmission is effected by ingestion of contaminated meat. capii i aria hepatica eggs are ingested, hatch, and migrate through the cecum to the liver through the portal veins. adults mature and lay characteristic bipolar operculate ova in the liver, where they lie dormant until ingested by a carnivore. gongylonema neoplastium burrows in the squamous epithelium of the tongue and anterior stomach, where it incites a proliferative response or ulcération. the life cycle involves an intermediate insect host. angiostrongylus cantonesis, which requires the ingestion of a mollusk intermediate host, infects the brain and lung. the intestinal nematodes heterakis spumosa, nippostrongylus brasiliensis, strongyloides ratti, and trichuris muris lack in termediate hosts, but rarely occur naturally in laboratory rats. none is known to be very pathogenic. further details on nematode diagnosis and ova morphology are available (flynn, ; hsu, ; oldstone, ) . taenia taeniaformis, the cat tapeworm. following ingestion of ova, oncospheres migrate to the liver and form strobilocerci (cysticercus fasiolaris). host connective tissue capsules can give rise to sarcomas. this parasite has been used as a model of parasite-induced oncogenesis in the rat. cysticercus fasciolaris is on occasion encountered in laboratory rodents with contaminated food or bedding. hymenolepis nana and diminuta infect the small intestine of several species, including the rat. hymenolepis nana, the dwarf tapeworm, has a -mm wide segmented body ranging from to mm in length. the life cycle may be either direct or indirect. following ingestion of ova, oncospheres penetrate the intestinal mucosa, form cysticercoid larva, then emerge into the lumen and mature into adults that shed eggs in the feces. nonimmune hosts can be autoinfected; eggs are pro duced, hatched, and complete their life cycle within the intes tine of a single host. the indirect cycle involves intermediate hosts, such as grain beetles or fleas, in which cysticercoid lar vae form and await ingestion by the definitive host. hymenolepis diminuta is - mm wide and - mm long. the eggs are passed in the feces but must be ingested by an intermediate host (flour beetle, flea, moth), which in turn must be ingested by the definitive host to complete the life cycle. enteritis can occur in heavy infestations, which are most likely to be encountered with h. nana. both can infect primates, in cluding humans. hymenolepis nana, which does not need an intermediate host and can cause autoinfection, is a particular public health hazard. anthelmintic treatment is effective (table ix) . diagnosis of hymenolepis is made by finding ova containing hexacanth embryos in the feces or adults in the lumen of the bowel. differential features are detailed else where (hsu, ) . d. insect infestation. two anopluran (sucking) lice infest wild rats, polyplax spinulosa (the spined rat louse) and hoplopleura pacifica (the tropical rat louse). the latter has not been reported among laboratory rats. polyplax spinulosa is now rare in most laboratory rat populations. it completes its entire life cycle within the fur of the host and is transmitted by direct contact. infested rats are unthrifty, irritable, pruritic, restless, and anemic. polyplax spinulosa can transmit a num ber of infectious agents, which include hemobartonella muris and t. lewisi. diagnosis is made by identifying organisms in the fur. rat fleas (xenopsylla sp., leptopsylla sp., and nosopsyllus sp.) are rare in laboratory rats, since their life cycles are usu ally interrupted by proper sanitation. fleas can transmit other agents and can serve as the intermediate host of h. nana and diminuta (flynn, ; hsu, ; oldstone, ) . see sec tion iii,a, ,e for methods of control. e. arachnid infestation. mesostigmate mites can be en countered on occasion in laboratory rats. they are rapid blood suckers and have a nonselective host range. they are on the host only during feeding, spending much of their life cycle secreted in the environment. diagnosis must be attained by finding engorged mites on bedding, cages, and crevices. other hosts, including humans, fall prey to their painful and irritating bites. the most important mesostigmate is ornithonyssus bacon (tropical rat mite). heavily infested colonies contain de bilitated, anemic rats with reduced reproductive efficiency and occasional deaths. liponyssoides sanguineus has not been re ported in laboratory rats, but may be unrecognized because of its similarity to ornithonyssus sp. laelaps echidninus (spiny rat mite) does not adapt well to the laboratory rat environment unless husbandry is poor. it is a vector of hepatozoon. prostigmate mites have a more selective host range and spend their life cycle in the fur (or follicles) of the host. radfor dia ensifera, the rat fur mite, can be common in some rat colonies. this mite produces few ill effects, but heavy infesta tions can induce self-inflicted trauma (flynn, ; hsu, ; oldstone, ) . demodex sp. is also encountered, but its prevalence is unknown, since it lives deep within hair follicles and sebaceous glands where it produces minimal lesions (walbtxgetal., ) . astigmate mites include the mange mites, which have a se lective host range and complete their life cycle on the host. notoedres muris, the ear mange mite, infests the skin of the ear and to a lesser extent, the nonglabrous regions of the body. this mite burrows in the cornified epithelium, and elicit a pruritic dermatitis (flynn, ; hsu, ) . it is seldom seen in contemporary rat colonies. control of ectoparasites is gained by preventing the introduc tion of infected animals (including wild rodents), sanitation, treatment of rats, and, in the case of mesostigmates and fleas, treatment of the environment (table ix) . treatment seldom completely eliminates infestation and can have an adverse ef fect on the usefulness of the treated rats for research. repopulation or caesarean rederivation and subsequent preven tion by proper management is the best approach. deep mycoses are generally not considered to be significant natural diseases in laboratory rats. pulmonary aspergillosis is occasionally observed in rats and mice, particularly when immunocompromised. aspergillus sp. can be readily isolated or observed in affected lung tissue with selected histochemical techniques. pulmonary lesions consist of miliary granulomata containing langhans giant cells in areas with characteristic uniformly branched, septate hyphae. phycomycotic encepha litis has been reported as a natural disease in -to -week-old rats. phycomycotic fungi have thick, irregularly branched, nonseptate hyphae in tissue. dermatomycosis is probably more likely to be encountered than deep mycosis, but is also rare in laboratory rats. ring worm in rats is restricted largely to infection by trichophyton mentagrophytes. infected rats can be lesionless carriers or dis play patchy alopecia and erythema with scale, papule, or pus tule formation. fungal elements are visible within the stratum corneum and hair follicles. ecothrix spore formation in hair shafts can be present. the disease can be diagnosed by exam ination of skin scrapings treated with % potassium hydrox ide, histological sections of skin prepared with fungal stains, or isolation (weisbroth, ) . a. genetic anomalies. genetic anomalies in coat color and character, organ development, immune responsiveness, obesity, hypertension, metabolism, and others have been iden tified and in many cases developed as specific characteristics of various stocks and strains. the reader is referred tö several more reviews for further information (altman and katz, ; hansen et al., ; robinson, robinson, , . some strains nat urally develop disease syndromes, such as brattleboro rats with diabetes insipidus, while other strains have less obvious characteristics but react uniquely to different research vari ables. the researcher must be aware of these variations and judiciously select the appropriate stock or strain to accomplish the goals of the experiment. each stock, strain, substrain, and even group of rats that has drifted from its parental stock can develop its own unique dis eases, manifest its own incidence and rate of development of various spontaneous lesions, and react in different ways to in fectious and research variables. expression of genetic traits is further influenced by extraneous factors such as husbandry and diet. b. nutritional deficiencies. more is known about the nu tritional requirements and deficiencies of rats than perhaps any other species. natural deficiencies of single dietary compo nents are rare and seldom recognized. this is because rats ef fectively store fat-soluble vitamins (and b ), manufacture vi tamin c, and fulfill much of their requirement for b vitamins by coprophagy. furthermore, some deficiencies are influenced or compensated for by other dietary elements. the most com mon signs of nutritional deficiency are vague, such as poor hair coat, reduced weight gain or growth, diminished fertility, and susceptibility to infectious disease. commercially avail able rodent diets effectively supply balanced diets to rodents, but diets should not be utilized beyond their expiration date and ideally should be kept in cold storage. several components deteriorate with prolonged storage, heating, or sterilization, in cluding lysine, vitamins a and e, and, to a lesser extent, riboflavin and thiamin. nutritional requirements vary with the genetic strain, stage of life, and microbiological association. axenic or specific pathogen-free (spf) animals can lack gut microflora necessary for supplying certain vitamins, particu larly vitamin k. they must receive supplemented diets both because they need more and because autoclaving or pasteuriza tion reduce the levels of certain essential nutrients. antibiotic treatment, which affects intestinal microflora, can reduce the bacterial source of vitamins by coprophagy. nutritional ade quacy goes far beyond the needs of the rat, since the composi tion of nutritionally adequate diets is known to profoundly in fluence the biological responsiveness to research manipula tion, infectious disease, and expression of spontaneous lesions (national research council, ; rogers, ) . the signs of nutritional deficiency are often vague and must be differentiated from other syndromes. hemorrhage due to hypovitaminosis k can be confused with rv disease (which in itself can be precipitated by nutritional deficiency). infertility or poor production can be caused by rv, sdav, sendai virus, m. pulmonis, or nuturitional deficiency. squamous metaplasia in the salivary and lacrimai glands of rats recovering from sdav or in the respiratory tract of rats infected with sendai virus or m. pulmonis must be differentiated from hypo vitaminosis a, which in turn predisposes to respiratory infec tions. environmental factors, such as excess heat or low hu midity, contribute to infertility or skin disease which mimic hypovitaminosis e or other deficiencies. ordinarily mild infec tious diseases can become severe, such as pseudotuberculosis, due to nutritional deficiency. the diagnosis of many rat dis eases therefore requires review of nutritional practices. practices other than nutrition can also influence or cause dis ease in rats. sanitation is of obvious import. cage design, pop ulation density, and temperature influences have been men tioned in sections ii,d and e. outbreaks of opportunistic in fectious disease, such as pseudotuberculosis, can be precipi tated by sudden fluctuations in temperature and humidity. pseudomonas aeruginosa can be introduced and spread within a colony through inappropriate sanitation, particularly of water bottles. refilling water bottles at a faucet in the animal room is not advised for this reason. bottles and sipper tubes should be sanitized and water should be acidified or chlorinated. exces sive ammonia from dirty bedding, overcrowded cages, or poor ventilation predisposes to respiratory infections, particularly mycoplasmosis. a number of management-related syndromes can occur that are not related to sanitation or infectious disease. rats adapt well to automatic watering systems, but animals unfamiliar with these devices can become dehydrated. similarly, mal function of these systems and blockage of water bottle sippe tubes with metal filings or other detritus can have a similar effect. rats maintained for long periods on wire-bottom cages develop foot sores, which can result in severe lesions, discom fort, hemorrhage, and anemia. aging rats must be examined periodically for overgrowth of their incisors, a very common problem in rats held long term. teeth must be carefully clip ped, avoiding splitting or shattering. dusty bedding and food can result in foreign body pneumonia. plant, mineral, and even bone fragments can be found in sections of lung obtained from rats under these circumstances. softwood shavings used as bedding can cause intestinal impaction, particularly in young rats. high ambient light, or even light levels within the recom mended range, can cause retinal degeneration and cataracts in albino rats. rats maintained near ceiling light fixtures develop retinal degeneration, while those near the floor are spared. low relative humidity (< %) in concert with high tempera ture and other factors can cause one or more annular constric tions of the tail skin (ringtail) or toes. the segments distal to the constrictions swell or undergo dry gangrene (fig. ) . this is most apt to occur in suckling or preweanling rats and in rats kept in wire-bottom cages. increasing humidity, reducing air turnover rate, placing rats in solid-bottom cages, and providing nesting material for nursing dams and their litters are all ame liorative for this condition. axenic rats normally have enlarged ceca, which become ap proximately five times normal size. there is net efflux of fluid into the lumen in response to increased osmotic pressure of luminal content, reduced availability of ions needed for mucosal solute-coupled water résorption and vasoactive com pounds which exert their effect on smooth muscle tone. cecal enlargement can interfere with reproduction. dietary manip ulation can reduce the enlargement substantially (foster, ) . the effects on smooth muscle tone become pronounced in aging axenic rats, which can develop progressive cecal en largement due to atony. volvulus and torsion can result. the reader must be cognizant of the much larger list of en- vironmental variables that significantly alter physiological re sponsiveness of test animals (baker et al., a; gralla, ) . unlike the mouse, traumatic skin disease in the rat is more likely to be due to self-inflicted trauma than fight wounds. rats infested with ectoparasites or dermatophytes can develop excoriative dermatitis due to self-inflicted trauma. roughly sym metrical ulcerative dermatitis over the dorsal and lateral neck and shoulders has been reported (fig. ) . the etiology re mains to be determined, but coagulase-positive staphylococcus aureus is consistently isolated from the skin lesions, and colonies of gram-positive cocci are microscopically visible on the surface of the lesion. attempts to induce the disease in other rats with this agent have yielded inconsistent results (fox et al, ) . the rat is often considered "resistant" to infection and ac cordingly submitted to a variety of experimental procedures without regard to aseptic techniques. peritonitis and wound in fections are often found under such conditions. inappropriate handling of large rats by the tips of their tails will cause the skin to tear and slip off when they struggle. adynamic ileus occurs in rats given intraperitoneal injections of anesthetic preparations containing chloral hydrate or related compounds (fig. ) . for variable periods up to weeks after treatment, rats become lethargic, anorectic, and constipated with distended abdomens that may lead to death. gross nec ropsy findings reveal segmental atony and distension of the bowel, usually jejunum, ileum, and cecum. focal serosal in flammatory changes and fibrosis are seen microscopically (fleischman et al., ) . this syndrome mimics megaloileitis (tyzzer's disease), but can be differentiated on the basis of history, lack of characteristic bacteria in tissues, and lack of liver and heart lesions. in.addition, it should not be confused with the bowel dilatation observed in axenic rats. space exists in this chapter to only summarize the more fre quently reported tumors in the rat, and the topic will not in clude experimentally induced tumors. the reader is encouraged to refer to the articles and monographs referenced in the text for more complete information. there have been numerous studies done to assess the inci dence of neoplasia in various stocks and strains of rats (burek, ; coleman et al., ; international agency for research on cancer, mackenzie and garner, ; ringler and dabich, ; sher, ) . some of these have led to the development of animal models and provided baseline data for experimental carcinogenesis work for which the rat is regularly used. there is a wide range of values reported in regard to the prevalence and type of tumors found in a particular rat stock or strain. these differences are a function of genetic and environ mental influences and differences in sample selection. nutri tion is well known as a factor that influences the development of neoplastic disease. for instance, it has been shown that a high fat diet enhances tumorigenesis, that the protein-calorie ratio influences the occurrence of some tumors, and that re stricted food intake of post weanlings for or more weeks de creases tumor risk (ross and bras, ) . most of the surveys on tumor occurrence in rats either state nothing in regard to the presence of enzootic infectious disease or acknowledge that infectious diseases were present in the population. in many instances, infectious diseases can signifi cantly influence data on tumor risk because of their effect on longevity, preneoplastic changes, and masking of small tumors. with the exception of mammary fibroadenomas in many stocks and testicular tumors in f rats, the prevalence of tumors is quite low in rats under months of age. according ly, the age at which rats are surveyed is very important in de fining the incidence of neoplasia in a particular stock or strain. the f rat is used in the bioassay program of the national cancer institute and is widely used in toxicology studies. it has been selected for these types of studies because of its small size, longevity, and low incidence of most tumors. however, this strain has a moderate to high incidence for some tumors, such as those of the testis, mammary gland, uterus, and hematopoietic and endocrine systems (sher, ) . a sprague-dawley-derived stock that is commonly used in carcinogenesis studies is the crl:cd(sd)br rat. this stock has a high inci dence of mammary tumors and pituitary adenomas. papillomas and squamous cell carcinomas occur infre quently, but have been reported in many stocks of rats. squam ous cell carcinomas are usually located on the face and head, and tend to be highly invasive but not metastatic. most of these arise as sebaceosquamous tumors from the glands of zymbal in the ear. similar tumors occur on the prepuce and clitoris. in females from most stocks and strains, the mammary gland is the most frequent site of neoplasia, with incidences of - % being common. these tumors occur in males but much less frequently. benign fibroadenomas are the most common type, but adenocarcinomas may also occur. fibroadenomas have ductal epithelium and periductular connective tissue compo nents and tend to be less vascular than carcinomas. adenocar cinomas can metastasize to regional lymph nodes and the.lung. tumors of the mammary gland, which may arise at any site from the neck to the inguinal region, often attain a very large size (fig. ) . the prevalence of tumors in the alimentary tract is extremely low, with most cases involving the colon. although hepatic tumors are readily induced by chemical carcinogens, very few spontaneously occurring malignant neoplasms have been re ported. more commonly occurring are neoplastic nodular le sions. the term ò 'neoplastic nodule" is used to describe cir cumscribed nodules of proliferating hepatocytes that compress the parenchyma (altman and goodman, ) . there is some fig. . mammary fibroadenoma in an aged rat. controversy as to whether these nodules are in all cases neo plastic. however, there is evidence that some develop into carcinomas. leukemia is rare in many stocks and strains of rats. howev er, mononuclear cell leukemia is very frequently seen in f and wf rats. in one survey of male f rats, an incidence of % proved leukemia second in occurrence only to the testic ular interstitial cell tumors. the leukocyte count in leukemic rats ranged from , to , cells/ìÀ with an average of , (coleman et al., ) . other studies indicate a simi lar incidence for the wf rat. in the bn/bi rat, myelomonocytic leukemia has been reported to be the most common lymphoreticular tumor, with female and male incidences of and %, respectively (burek, ) . both types of leukemia oc cur in rats over months of age. neoplasms of the pituitary occur frequently with a higher proportion in females. surveys have shown an incidence of - % in f rats, % in om (osborne-mendel) rats, and - % in sprague-dawley rats. the most common pituitary tumor is the chromophobe adenoma. these are benign tumors that frequently become quite large, and, due to compression of the brain by the tumor, hydrocephalus and neurological signs may occur. they appear dark due to hemorrhagic areas arising from a vascular sinusoid component of the tumor. clinical manifestations may include neurological signs or cachexia, but these are often absent. pancreatic islet cell tumors are common in some stocks and strains. surveys have reported a % incidence in f rats, . % in holtzman rats, and a - . % in sprague-dawley stocks. tumors of the thyroid are usually parafollicular cell ade nomas. these benign tumors occur in many stocks with an incidence varying between and %. follicular cell car cinomas, which occur infrequently, can metastasize to the lung. adenomas of the adrenal cortex are very common in several strains. in at least three strains, buf/n, m /n, and om/n, over % of the females and % of the males have cortical tumors. pheochromocytomas are the most common medullary tumors. they are particularly common in the buf/n, f /n, m /m, and wn/n strains, and, unlike cortical tu mors, they appear more often in males. tumors of the brain have been found to occur rarely in those studies that included examination of the cns. less is known about the prevalence of tumors in the cns than in other sys tems because many studies have not included brain and spinal cord examination. the astrocytoma is the most commonly re ported tumor of the cns. less often reported tumors include oligodendroglioma, meningioma, ependymoma, and granular cell tumors. primary tumors of the lung are rare in the rat. the most fre quently reported types are bronchogenic carcinomas, car cinomas, sarcomas, and adenomas. tumors of the bladder are rare in most stocks and strains of rats. however, the bn/bi strain has an unusually high inci dence of carcinomas of the bladder and ureter. males of this strain have been reported to have a % incidence of bladder carcinoma, while % of females have carcinoma of the ureter (burek, ) . nephroblastomas, renal tubular adenomas, and adenocarcinomas have been reported in the rat kidney. tumors of the prostate are common in the axc rat. in one report, adenocarcinoma of the ventral prostate was histologically detected in % of -to -month-old axc rats. prostatic tumors are reportedly uncommon in most stocks and strains of rats. reportedly, however, some of these surveys did not include adequate sectioning to locate small adenomas and carcinomas. interstitial cell tumors are the most common testicular tumor type. nearly all aged f rats and the majority of aci/n rats develop these tumors, but the tumor is rare in most other strains. tumors of the vagina and cervix are rare, except in aged bn/bi rats. tumors of the uterus and ovary are common in several strains of rats. one om strain was reported to have a % incidence of granulosa cell tumors, and a high incidence of endometrial tumors has been reported in the f and m strains. congenital disorders in the rat are infrequently reported. this is probably a reflection of two factors; the first being that evidence indicates the incidence is extremely low in outbred stocks, and the second being that commercial/institutional sup pliers select against breeders who have produced abnormal young. the genitourinary system and the eye have been most associated with congenital disorders. one substrain of the axc rat has a greater than % inci dence of unilateral renal agenesis and hydronephrosis (fujikura, ) . a high incidence of unilateral and bilateral hy dronephrosis has been found in bn/bi rats (cohen et ai, ) . pseudohermaphroditism has been reported in rats that are phenotypically female but who have an xy karyotype. testicles are present either in the abdomen or inguinal canal. this condition is due to a sex-linked recessive gene that is expressed by an insensitivity of tissues to androgens (bardin et al., ) . a number of ocular disorders have been reported in the rat. retinal dystrophy is inherited as a single autosomal recessive gene (rdy). homozygotes have a progressive loss of the retinal photoreceptor cells and an overproduction of rhodopsin early in the disease process (lavail et al., ) . a retinal degener ation reported in the wag/rij rat appears to be an expression of an autosomal dominant gene. end-stage lesions include disap pearance of photoreceptor cells, migration of the pigment epi thelium, and disorganization of remaining retinal layers. this retinal disease appears to mimic the pathogenesis of retinitis pigmentosa in humans (lai et al., ) . other reported devel opmental disorders of the eye include colobomas and cataracts. reports of congenital heart disease are rare. among the few reports is one involving an inbred strain of long-evans rats (fox, ) . in this strain, approximately % of the neonates have interventricular septal defects. this malformation is be lieved to be of polygenic origin. unlike the mouse, there are few reported mutants that have disorders of the central nervous system. recently, a partially inbred strain has been developed in which one subline has nearly a % incidence of hydrocephalus (kohn et al., ) . the mode of inheritance appears to be polygenic. the hydrocephalus is classified as communicating and the pathogenesis may be due to poorly developed veins in the periosteal and durai layers, and to underdeveloped pia-arachnoid cells. neoplastic and nonneoplastic lesions vary in type, onset, and prevalence due to the hereditary and environmental factors as sociated with a specific colony of laboratory rat. the rat is widely used in gerontological research. those who use the rat must be clearly aware of the factors that can influence lesions and death in aged rats (anver and cohen, ; burek, ) . neoplastic disease, a cause of death in many aged rats, has been discussed previously, and this section will summarize the more commonly seen nonneoplastic lesions in aging rats. a. chronic progressive nephropathy (cpn). chronic pro gressive nephropathy is among the most commonly encoun tered lesions in the rat, particularly in the aged animal where it is a major life-limiting disease. because of its complex nature, cpn has a large number of synonyms, often with inaccurate descriptive titles. sequential development of lesions and fac tors that modify the course of cpn have been thoroughly stud ied, but the actual pathogenesis remains undetermined. chron ic progressive nephropathy occurs in most rats, but its rate of development is significantly influenced by numerous factors, including sex, genetics, age, diet, association with microflora, hormone treatment (particularly testosterone), and others. male rats have an earlier onset and more rapid progression of cpn than females, and albino strains and stocks seem to be more predisposed. diet is an extremely important predisposing factor, particularly the quality and quantity of dietary protein. significant differences in severity of cpn can be observed among rats of the same sex, strain, age, and source when fed different diets for their lifetime. axenic rats do not seem to develop significant cpn. rats with cpn develop progressively severe proteinuria, first with the selective excretion of only small molecules such as albumin. in late cpn there is nonselective excretion, and the electrophoretic pattern of urinary protein resembles that of serum. proteinuria related to cpn should not be confused with the normal urinary excretion of a-globulin in male rats, proba bly of tubular origin. the histopathology of cpn is charac terized as progressive basement membrane thickening of the glomerulus, bowman's capsule, and proximal tubule. base ment membranes split and wrinkle as tubules become atrophie and collapse. glomeruli enlarge, with mesangial deposition of protein and lipid, adhesion to bowman's capsule, and segmen tai sclerosis. tubular epithelium may contain granules of resorbed protein, which later is mixed with lipofuscin and hemosiderin. as glomerular protein leakage becomes more severe, tubules dilate and accumulate hyaline eosinophilic castes of protein within their lumina, resembling colloid. tubular epi thelium may undergo atrophy with collapse or dilatation. in terstitial fibrosis and leukocytic infiltration is frequently ob served. grossly, the kidneys become enlarged, pale with irregular surfaces and pigmented (fig. ) . cystic tubules can be observed. hypoproteinemia, parathyroid hyperplasia with serum chemical changes indicative of hyperparathyroidism, and nephrotic syndrome are observed in rats with severe cpn (barthold, ) . b. nephrocalcinosis. this disease, which occurs more fre quently in females, has been reported in numerous strains of rats. in most cases, affected rats have been fed purified diets (e.g., . renal lesions usually involve the corticomedullary junction and consist of mineral deposition in the lumina of the proximal tubules (nguyen and woodward, ) . e. polyarteritis nodosa. this disease is of unknown etiol ogy and affects muscular arteries, most commonly the mesenteric, pancreatic, and spermatic. the lesions may be either acute or chronic. in the acute stage, there is intimai and medial fibrinoid necrosis, focal thrombosis, disruption of the elastic lamellae, and an infiltration of polymorphonuclear leukocytes and mononuclear cells. in the chronic stage, the arteries are nodular, tortuous, and thick-walled. affected vessels fre quently have aneurysms and thrombi. both acute and chronic phases may be observed in the same artery in an animal. d. myocardial degeneration. degeneration is a commonly observed lesion that occurs in most stocks and strains, with an onset at - months of age. it occurs more frequently in males. the lesions are usually microscopic but, in advanced stages, can appear grossly as grayish foci. the papillary mus cles and their attachment sites in the wall of the left ventricle are the most frequent sites of the lesion (anver and cohen, ) . e. radiculoneuropathy. spinal nerve root degeneration has been reported in a number of rat stocks and strains. lesion distribution may vary among strains; however, the cauda equi na and ventral spinal nerve roots are commonly involved. pos terior paresis and paralysis have been associated with these lesions, but some suggest that these signs are associated with a separate degenerative process of the skeletal muscle (anver and cohen, ) . an enzyme linked immunosorbent assay for detection of antibodies to corynehacterium kutschen in experimentally infected rats copulatory behavior can inhibit preg nancy in female rats neoplastic diseases inbred and genetically defined strains of laboratory animals. part . mouse and rat rat-bite fever in animal research laboratory personnel muroid virus nephropathies lesions associated with aging reproduction and breeding housing to contro! research variables selected norma tive data. appendix i pseudohermaphroditic rat: end organ insensitivity to testoster one chronic progressive nephropathy in aging rats research complications and state of knowledge of rodent coronaviruses reproduction and breeding techniques for laboratory animals morphophysiology sendai virus infection in genetically resistant and susceptible mice observa tions on enzootic paratyphoid infection in a rat colony age-associated pathology a comparison in germ-free mice of the pathogenesis of sendai virus and pneumonia virus infection my coplasmal and rickettsial diseases detection of natural mycoplasma pulmonis infection in rats and mice by an enzyme-linked immunosorbent assay (elisa) adherence and colonization of mycoplasma pulmonis to genital epi thelium and spermatozoa in rats tissue weights of the rat. i. normal values determined by dissection and chemical methods immunopathology. in "cell pathology heritable hydronephrosis in a mutant strain of brown norway rats pathological changes during aging in barrier-reared fischer male rats naturally occurring lethal parvovirus infection of juvenile and young-adult rats breeding of the rat adynamic ileus in the rat induced by chloral hydrate parasites of laboratory animals gnotobiology ulcera tive dermatitis in the rat corynebacterium kutschen pneumonia in rats on long term tobacco inhalation studies evidence suggesting a multigenic origin of membranous septal defect in rats kidney malformations in fetuses of axc line rats bibli ography of hemorrhagic fever with renal syndrome (muroid virus nephropathies) effect of heat and selected chemical disinfectants upon infectivity of spores of bacillus piliformis (tyzzers disease) studies on adjuvantinduced arthritis, tumor transplantability and serologie response to bovine serum albumin in sendai virus-infected rats scientific considerations in monitoring and evaluating toxicological research nih rodents catalogue anatomy of the laboratory rat parasitic diseases viral diseases. /// "the laboratory rat tyzzer's disease in the rat pathogenicity of mycoplasma pulmonis in laboratory rats a new model of congenital hydrocephalus in the rat morphological evi dence for natural poxvirus infection in rats animal model: hereditary retinal degeneration in wag/rij rats the ufaw handbook on the care and management of laboratory animals photoreceptor pig ment epithelial cell relationships in rats with inherited retinal degenera tion comparison of techniques for primary isolation of respiratory mycoplasma pulmonis from rats historical foundations murine chronic respiratory disease. significance as a research complication and experimental production with mycoplasma pulmonis comparison of neoplasms in six sources of rats a filamentous bac terium associated with respiratory disease in wild rats salmonella enterocolitis in the rat clinical biochemical and hematological reference values in normal experimental animals intranephronic calculosis in rats helminths, ectoparasites and protozoa in rats and mice hematology and clinical biochemistry genetics of the norway rat nutrition lasting influence of early caloric re striction on prevalence of neoplasms in the rat tumors in control hamsters, rats, and mice: literature tabulation molecular biology of mammalian sarcoma viruses state of the art detection methods for rodent viruses the microscopic anatomy of the white rat asymptomatic infection of mouse hepatitis in the rat histological and serological response of b c f, mice and f rats to experimental pneumonia virus of mice infection demodicidosis in laboratory rats (rattus norvegicus) latent adenoviral infection of rats: intranuclear inclusions induced by treatment with a cancer chemotherapeutic agent the role of mycoplasmas and l forms of bacteria in disease bacterial and mycotic diseases respiratory disease in a colony of rats. ii. isolation of mycoplasma pulmonis from the natural disease, and the experimental disease induced with a cloned culture of this organism craigie's neuroanatomy of the rat key: cord- - zvqvumc authors: hamm, ronda l; kaufman, phillip e; reasor, colleen a; rutz, donald a; scott, jeffrey g title: resistance to cyfluthrin and tetrachlorvinphos in the lesser mealworm, alphitobius diaperinus, collected from the eastern united states date: - - journal: pest manag sci doi: . /ps. sha: doc_id: cord_uid: zvqvumc the lesser mealworm, alphitobius diaperinus (panzer), is an important pest in poultry facilities. the toxicity of cyfluthrin and tetrachlorvinphos to five strains of the lesser mealworm was compared with the toxicity to a susceptible laboratory strain. bioassays were carried out with both larvae and adults. for the susceptible strain, cyfluthrin and tetrachlorvinphos had similar toxicity to adults, but cyfluthrin was times more toxic to larvae when compared with tetrachlorvinphos. high levels of resistance to tetrachlorvinphos in two beetle strains were detected in both larvae and adults, although these strains were heterogeneous and still contained susceptible individuals. resistance to cyfluthrin ranged from . ‐ to . ‐fold for adults and from . ‐ to ‐fold for larvae at the lc( ). overall, the patterns of resistance did not mirror the insecticide use patterns reported at these facilities. the implications of these results to management of the lesser mealworms are discussed. copyright © society of chemical industry the lesser mealworm, alphitobius diaperinus (panzer), is the primary structural pest of the poultry industry. the lesser mealworm is a known reservoir of many avian pathogens and parasites, including salmonella typhimurium cast & chalm, escherichia coli (mig) cast & chalm, tapeworms, avian leucosis virus, turkey coronavirus and turkey enterovirus. - chicks feeding excessively on larvae have poor weight gain, and mortality may result. high beetle populations consume significant feed. under dry conditions in the broiler house, beetles will bite the skin of birds resting at night. to prevent these bites, agitated birds will rest for only short periods of time, leading to reduced weight gain and feed conversion. in addition, mature beetle larvae climb building walls and posts and chew into building support structures and insulation, seeking pupation sites. , the cost of lesser mealworm control in georgia alone was over $ , with estimated damage costs of $ . two insecticides registered for the control of lesser mealworm in poultry facilities in the usa are cyfluthrin and tetrachlorvinphos, which are typically applied following manure removal in cagedlayer systems. in spite of the widespread use of these compounds for control of lesser mealworm (and houseflies), minimal information about resistance to these materials in this species in the usa is available. vaughan and turner examined several formulated insecticides for their toxicity to a fieldcollected (virginia, usa) strain of lesser mealworm, and found that tetrachlorvinphos was highly effective. poultry facilities are typically treated with pesticides for lesser mealworm infestations following manure removal, when beetles in the pupal and adult stage are hiding in building structural and insulation harborages. savage states that, if litter is stockpiled near the poultry house, half the beetles will move back in when resources are no longer available or the temperature falls below optimum. he also describes how lesser mealworm adults can fly mile in one night, although the methods used to determine this are not described. therefore, it is reasonable to assume that, even with manure removal from the poultry house, many of the displaced beetles are capable of reinfesting poultry houses. in the present study, the susceptibility of five strains of lesser mealworms from georgia, maine and new york was compared with that of a susceptible laboratory strain using a residual contact method of insecticide exposure. bioassays were carried out with both larvae and adults against the two currently registered insecticides used for control, cyfluthrin and tetrachlorvinphos. an insecticide-susceptible strain (denmark-s) of a. diaperinus was obtained from saturnia (bjerringbrovej rødovre, denmark). lesser mealworms were also collected in from four cagedlayer poultry farms, three in new york (cayuga county, onondaga county and wayne county) and one in kennebec county, maine. an additional strain was obtained from an infestation occurring in a commercial cricket colony in waycross, georgia. alphitobius diaperinus is likely affected by insecticides used for housefly (and possibly ectoparasite) control in poultry facilities, as well as insecticides used specifically for lesser mealworm control. therefore, information was collected on all insecticide use at each facility. insecticide use for fly control at the cayuga county facility included premise applications of permethrin, tetrachlorvinphos and carbaryl, and feed mixtures containing cyromazine and methomyl fly bait. in this facility, walls and support beams were sprayed with either cyfluthrin or tetrachlorvinphos twice following each manure removal (approximately every - months). the birds were occasionally treated with carbaryl for northern fowl mites, ornithonyssus sylviarum (canestrini and fanzago). insecticide use (based on interviews with the facility managers) for fly control for at least the previous years at the wayne county and onondaga county facilities was limited to pyrethrin space sprays and methomyl baits. treatments for fly control at the maine facility included nearly weekly applications of pyrethrins from to , one application of dichlorvos, two applications of dimethoate and weeks per year of cyromazine feed-through use, as well as methomyl and nithiazine baits. all facilities remove manure every - months. no pesticide use history is available for either the denmark-s or waycross county strains. however, because both of these strains were obtained from insect cultures, it is assumed that these strains received no pesticide exposure for at least the last years. insect colonies were maintained at • c, - % rh and provided a diet of + cracked corn + wheat bran (agway, ithaca, ny) ad libitum. cyfluthrin ( % mix of isomers) and tetrachlorvinphos ( . %) were from chem services (west chester, pa) and were evaluated against six strains of beetles at two life stages. a residual contact bioassay method was used for all the insecticides tested. glass jars ( ml volume, treated surface area cm , total internal surface area cm ) were treated with . ml of insecticide in acetone, or acetone only for control treatments. jars were put on an orbital shaker to ensure complete coverage of the bottom and about one-quarter of the wall (beetles could not climb up the jars). the acetone was allowed to evaporate for at least h before the introduction of beetles. a minimum of three doses giving > % and < % mortality after insecticide treatment was used for each experiment. each experiment was replicated at least times. the specimens used in the assays were - day post-eclosion adult beetles or mature larvae that passed through a no. us standard sieve but were retained by a no. sieve. some - insects were placed inside a glass jar that had been treated with either insecticide or acetone only. mortality was assessed after h. typically, a total of - adults and - larvae were treated per concentration per strain. all bioassays were kept at . • c with a : h light:dark photoperiod. adults and larvae were considered dead if they were unable to move out of a cm circle within min. bioassay data were pooled and analyzed on the basis of standard probit analysis as adapted to personal computer use, using abbott's correction for control mortality. strains were considered 'resistant' if their lc value was significantly greater than that of the susceptible (denmark-s) strain. cyfluthrin and tetrachlorvinphos had similar toxicities to adults of the susceptible strain (denmark-s). however, cyfluthrin was times more toxic to susceptible larvae than tetrachlorvinphos (see tables to ). significant levels of cyfluthrin resistance (resistance ratios varied from . -to . -fold at the lc and from . -to . -fold at the lc ) were detected in adults from four of the five strains ( table ). the highest levels of resistance were fig. ). * significantly different from . based on non-overlap of % ci. found with the kennebec strain, which is somewhat surprising given the reported lack of cyfluthrin use at this facility. however, this population was from a facility that had been repeatedly treated with pyrethrins, which could result in cross-resistance to pyrethroids such as cyfluthrin. four of the five strains had less steep cyfluthrin log concentration-probit (lcp) lines relative to the susceptible strain, resulting in higher resistance ratios at the lc and indicating that these strains are more heterogeneous than the susceptible strain. this suggests that resistance alleles are likely present in these populations. adults from the wayne strain were not significantly resistant to cyfluthrin at the lc . larvae from the wayne strain were significantly more susceptible to cyfluthrin at the lc , but not at the lc ( table ), suggesting that there is some small amount of variation in cyfluthrin toxicity between different susceptible strains. larvae from the kennebec strain proved most resistant, having resistance ratios of and at the lc and lc , respectively. larvae from the waycross cricket colony strain were significantly resistant ( . -fold), but only when lc values were compared. generally, the patterns of resistance to cyfluthrin in larvae were similar to those in adults, with the exception of the onondaga and cayuga strains in which resistance was detected only in adults. adults in two of the strains tested (kennebec and waycross) contained individuals that were highly resistant to tetrachlorvinphos, as indicated by the plateau in the lcp line for these strains (fig. ) . both strains contained individuals that survived a concentration that was approximately -fold greater than the susceptible strain lc . since the waycross strain (from a cricket colony) had not been exposed to insecticides for many years, and given that highly resistant individuals can still be found in this strain, the present data indicate that the gene(s) responsible for the resistance have a negligible fitness disadvantage in the absence of insecticide use. adults from the wayne and onondaga strains from caged-layer poultry farms were also resistant to tetrachlorvinphos, but these strains did not have plateaus in their lcp lines. adults from the cayuga strain were susceptible to tetrachlorvinphos (table ) , even though this poultry farm reported using this insecticide for premise treatments. the kennebec and waycross strains also contained individual larvae that were highly resistant to tetrachlorvinphos, as seen from the plateau in the lcp line for these strains (fig. ) . although similar to the results observed for adults of these strains, the plateau occurred at a slightly lower percentage mortality value. larvae from the other three strains were not significantly different from the susceptible strain in their response to tetrachlorvinphos ( table ). the similar lcp lines obtained for adult and larval beetles in the kennebec and waycross strains suggest that a similar mechanism may be causing resistance in both life stages. identification of this mechanism will require further study. the present results indicate that resistance is very likely to be having a negative impact on the successful management of the lesser mealworm in caged-layer poultry facilities, especially in the case of tetrachlorvinphos at the kennebec facility. a goal for any resistance monitoring study is to identify the level of resistance detected in the assay, and relate it to the level of control seen in the field. to achieve this goal it will be necessary to conduct additional studies with these strains to examine the mortality response of adults and larvae to formulated materials applied to plywood (as would occur in a commercial setting) or to monitor the effectiveness of these insecticides directly at the poultry facilities. clearly, the gene(s) involved in resistance to tetrachlorvinphos (in adults and larvae) do not exert a strong fitness cost as they are maintained in the waycross strain even after several years of being reared in the lab without insecticide exposure. similarly, lambkin also reported retention of fenitrothion resistance for approximately years in a. diaperinus. since tetrachlorvinphos and fenitrothion are both organophosphates, it is possible that a similar mechanism of resistance exists for these two organophosphates in the usa and australia. if so, this could explain why tetrachlorvinphos and fenitrothion resistance alleles are preserved in lesser mealworm populations in the usa. identification of the gene(s) involved in the resistance will require further study. poultry integrated pest management: status and future. integ pest manag rev manure breeding insects responsible for cestodiasis in caged layer hens ecology and management of arthropod pests of poultry transmission of enteric pathogens of turkeys by darkling beetle larvae (alphitobius diaperinus) transmission of eimeria, viruses, and bacteria to chicks: darkling beetles (alphitobius diaperinus) as vectors of pathogens reservoir competence of alphitobius diaperinus (coleoptera: tenebrionidae) for escherichia coli (enterobacteriales: enterobacteriaciae) limited transmission of turkey coronavirus (tcv) in young turkeys by adult lesser mealworms feeding behavior and growth of broiler chicks fed larvae of the darkling beetle, alphitobius diaperinus reducing darkling beetles residual and topical toxicity of various insecticides to the lesser mealworm (coleoptera: tenebrionidae) construction profiles of high rise caged layer houses in association with insulation damage caused by the lesser mealworm, alphitobius diaperinus (panzer) in virginia summary of losses from insect damage and costs of control in georgia in pesticide management education program susceptibility of lesser mealworm (coleoptera: tenebrionidae) to beauveria bassiana (moniliales: moniliaceae): effects of host stage, substrate, formulation, and host presentation d'un programme basic d'analyse logprobit pour micro-ordinateur a method of computing the effectiveness of an insecticide changes in cross-resistance patterns of houseflies selected with natural pyrethrins or resmethrin ( -benzyl- -furylmethyl (+,−)-cis-trans-chrysanthemate) baseline responses of adult alphitobius diaperinus (coleoptera: tenebrionidae) to fenitrothion and susceptibility status of populations in queensland and new south wales, australia the authors thank k murray, c sheppard, j deacutis, g howser, e kingsley, m lunoe, e lastro and a meisner for their assistance, and m hardstone for help with the figures. they also extend their appreciation to the new york and maine poultry producers who most generously provided labor and assistance with this study. this research was supported by the northeast regional ipm competitive grants program project no. nyc- received from the cooperative state research, education, and extension service, us department of agriculture, and by the multi-state hatch project s- and the daljit s and elaine sarkaria professorship. any opinions, findings, conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the us department of agriculture. key: cord- -txk qic authors: li, zi; he, wenqi; lan, yungang; zhao, kui; lv, xiaoling; lu, huijun; ding, ning; zhang, jing; shi, junchao; shan, changjian; gao, feng title: the evidence of porcine hemagglutinating encephalomyelitis virus induced nonsuppurative encephalitis as the cause of death in piglets date: - - journal: peerj doi: . /peerj. sha: doc_id: cord_uid: txk qic an acute outbreak of porcine hemagglutinating encephalomyelitis virus (phev) infection in piglets, characterized with neurological symptoms, vomiting, diarrhea, and wasting, occurred in china. coronavirus-like particles were observed in the homogenized tissue suspensions of the brain of dead piglets by electron microscopy, and a wild phev strain was isolated, characterized, and designated as phev-cc . histopathologic examinations of the dead piglets showed characteristics of non-suppurative encephalitis, and some neurons in the cerebral cortex were degenerated and necrotic, and neuronophagia. similarly, mice inoculated with phev-cc were found to have central nervous system (cns) dysfunction, with symptoms of depression, arched waists, standing and vellicating front claws. furthmore, phev-positive labeling of neurons in cortices of dead piglets and infected mice supported the viral infections of the nervous system. then, the major structural genes of phev-cc were sequenced and phylogenetically analyzed, and the strain shared %– . % nt identity with the other phev strains available in genbank. phylogenetic analysis clearly proved that the wild strain clustered into a subclass with a hev-jt strain. these findings suggested that the virus had a strong tropism for cns, in this way, inducing nonsuppurative encephalitis as the cause of death in piglets. simultaneously, the predicted risk of widespread transmission showed a certain variation among the phev strains currently circulating around the world. above all, the information presented in this study can not only provide good reference for the experimental diagnosis of phev infection for pig breeding, but also promote its new effective vaccine development. for the upper respiratory tract and is propagated through the neural route (andries & pensaert, ) . the disease caused by phev was first reported in canada in (roe & alexander, ) , and the pathogen was first isolated from the brains of suckling piglets with encephalomyelitis in (greig et al., ) . since then, the infection has been reported in the united states, japan, argentina, belgium, south korea, china and other pig-raising countries (gao et al., ; hirano & ono, ; pensaert & callebaut, ; quiroga et al., ; rho et al., ; sasseville et al., ) . today, many serological surveys have revealed that phev is widespread, and there are frequent subclinical infections (li et al., ) . in china, phev infection first occurred in beijing in , and the outbreaks caused enormous economic losses for the pig industry (chen et al., ; dong et al., ; gao et al., ) . here we report that there is a suspected outbreak of phev infection on a farm in changchun of jilin province, in , resulting in serious economic losses. many infected piglets characterized with vomiting and nerve symptoms, and some cases were accompanied by screaming or diarrhea; all of the piglets with clinical symptoms died finally. in this paper, the diagnosis was made on the basis of pathologic features, immunohistochemistry, microbiological detection, and rt-pcr. a phev field strain was isolated from the brain tissue of infected piglets, and the major structural proteins of the strain were sequenced to identify genetic relationships with other coronaviruses of the genus betacoronavirus. on march , there was an acute outbreak of suspected porcine hemagglutinating encephalomyelitis in suckling pigs on a farm with a total of sows in changchun, jilin province, china. at the time of the outbreak, these pigs had not been immunized with any phev vaccines; the total proportion of deaths in piglets that had not been weaned was . % ( dead piglets). the collected samples were tested for phev using real-time reverse transcription-polymerase chain reaction (rt-pcr) targeting the he gene, as well as for other viruses that cause similar clinical symptoms among swine, including porcine epidemic diarrhea virus (pedv), porcine transmissible gastroenteritis virus (tgev), porcine deltacoronavirus (pdcov), and pseudorabies virus (prv). all experiments on piglets research were performed in accordance with animal welfare ethical committee of jilin university guidelines and regulations (permission number -cvm- ). the involved rt-pcr primers were designed based on the most conserved segment of their genomes (table ) , and subsequently validated by blast (http://www.ncbi.nlm.nih.gov/blast) with sequences from genbank. the original samples were diluted -fold with phosphatebuffered saline (pbs) and were centrifuged at , × g at • c for min. the supernatant was filtered through a . -µm syringe filter, and was used as inoculums for balb/c mice or for virus isolation in neuro- a cell culture. postmortem examinations were performed and samples submitted for histopathologic examination, including tissues from the brain, heart, spleen, liver, kidneys, and lungs. paraffin-embedded sections of brain that had characteristic microscopic lesions were thirty -week-old male balb/c mice were randomly and equally divided into three groups. the mice in group were inoculated with the original filtered brain tissue by the intranasal route, the mice in group were inoculated with hev n (genbank: ay ) in the same manner, and the mice in the third group formed a negative control group. the permission to work with laboratory animals was obtained from the animal welfare ethical committee of the college of veterinary medicine, jilin university, china (permission number -cvm- ). all of the mice experiments were carried out at bio-safety level (bsl- ) facilities at the key laboratory of zoonosis, ministry of education, college of veterinary medicine, jilin university. clinical signs were monitored, and immunofluorescence assay (ifa) was performed. the phev monoclonal antibody (diluted : ) was used as the primary antibody, and a : dilution of affinity purified fluorescein-labeled goat anti-mouse igg was used as the second antibody. cell staining was examined using a fluorescence microscope. the neuro- a cell line was used to isolate phev from the original field and from mousepassaged phev samples. cultured cells were propagated in dulbecco's modified eagle medium (dmem, gibco, usa) supplemented with % heat-inactivated fetal bovine serum (hyclone, logan, ut, usa) and % antibiotic-antimycotic (gibco, grand island, ny, usa). briefly, a monolayer of cells was washed twice with % dmem, and then was inoculated with the filtered samples. after adsorption for h at • c in % co , the cells were washed times, and % dmem was added. the cell cultures were examined daily for cytopathic effect (cpe). when more than % cpe was evident in the inoculated cell monolayers, the cells and supernatants were harvested together and used as seed stocks for the next passage. after serial passage, the cell cultures were clarified by centrifugation at , × g for min at • c and then were further ultracentrifuged at , × g for h at • c using an ultracentrifuge. the pellet was resuspended and submitted for virological investigation using electron microscopy (em). the virus isolate was designated phev-cc . the % confluent neuro- a cells in -well plates were used for plaque assays of phev-cc propagation and purification. briefly, the wells were inoculated with -fold serially diluted virus ( . ml/well), followed by adsorption for h at • c in % co ; then, the wells were washed times, and ml of the agarose/mem mixture ( : ) were added. after the plaques were counted and confirmed, uniform and clear plaques were chosen to inoculate -well plates directly. when cpe was observed, the positive clones were harvested, and the viral titers were determined. when the neuro- a cells were confluent in -well plates, ml of -fold dilutions of the purified virus were absorbed for h. viral cpe was monitored for to days, and virus titers were determined by % tissue culture infectious dose (tcid ). all five of the main structural protein genes, hemagglutinin-esterase (he), spike (s), small membrane (e), membrane (m) and nucleocapsid (n), of phev in the original specimen, in the balb/c mice infected with passaged phev-cc , and in cell culture were amplified, cloned and sequenced. all of the primers were designed according the sequence of the hev n genome. viral rna was extracted from the brain tissue suspensions of symptomatic piglets using a commercial kit (qiagen, hilden, germany), and the rna was quantified using a spectrophotometer (bio-rad, usa). the rna was converted to cdna by an oligo (dt)-priming strategy, and the genes were amplified using primestar max dna polymerase (takara, kyoto, japan). the purified pcr products were cloned into the pmd -t vector (takara, kyoto, japan) and were introduced into e. coli dh a by transformation. the recombinant plasmids were extracted and verified by pcr and then were sequenced at shanghai sangon biological engineering technology and services co., ltd. (china). the sequence data were assembled and analyzed using dnastar and ncbi blast (http://blast.ncbi.nlm.nih.gov/blast.cgi). the percentage similarities of the nucleotides and amino acids were analyzed using dnaman and dnastar software. the structural gene sequences and other coronavirus strains sequence were subjected to phylogenetic analysis using the neighbor-joining method in mega software, version . . statistical analysis was performed with either student's t -test or one-way anova with a bonferroni post hoc test with software provided by graphpad prism version . data were presented as means ± s.e.m. p values of < . were considered statistically significant. clinical signs of these suspected infected suckling piglets were consisted of vomiting, diarrhea, wasting, dullness, screaming, anorexia, trembling, and ataxia (fig. a) . pathological examination showed that the main changes in the piglets were congestion, edema and hemorrhage in brain tissue (fig. b) . none significant histopathological changes were found in other substantive organs. a total of homogenized tissue suspensions of the brain, spinal cord, lungs, kidneys, spleen and intestinal contents from nine suspected piglets were tested for phev by rt-pcr. of these tested samples, eight of nine brain samples from young nursing pigs on the farms were phev positive, as well as eight of nine spinal cord samples and four of nine intestinal content samples (table ) . of the phev-positive samples, all were negative for pedv, tgev, pdcov, and prv. postmortem examinations were performed on seven infected piglets for pathologic evaluation. samples submitted for histopathologic examination included brains from phev-infected piglets and antigen-negative piglets. microscopic examination of brain samples showed characteristics of non-suppurative encephalitis. a large number of glial cells were aggregated to glial nodules in the infected brains ( figs. a and b) . neurons in the cerebral cortex were degenerated and necrotic, and neuronophagia was widespread (figs. c and d). selected paraffin sections of brain samples that had characteristic microscopic lesions were examined for phev antigen by ihc tests with an anti-phev monoclonal antibody. in the brains, antigen-positivity in the cytoplasm of nerve cells was distributed widely in the cortical neurons (fig. e ). brain samples from the healthy pig were normal (fig. f ). mice in two infected groups were inoculated with hev n and phev-cc , respectively, and were monitored daily for clinical signs of disease. mice in the hev n-infected group showed typical neurological damage, with symptoms of depression, arched waists, standing and vellicating front claws at three days post-inoculation (dpi). the same symptoms occurred in the phev-cc -infected group (figs. a and b), but the emergence time was slightly delayed (fig. c , p < . ). all of the infected mice died within a week, and the mice in the control group survived normally. paraffin-embedded sections of the infected mouse brain samples were positive for phev in the cytoplasm of nerve cells by ifa using a mouse anti-phev monoclonal antibody. in the brain, antigen-positive neurons were distributed widely in the cerebral cortex and hippocampus (fig. ) . in the cerebellum, viral-specific antigen was detected in the purkinje cells ( fig. ) but in only a few granular cells. the neuro- a cell monolayer was inoculated with original field and mouse-passaged phev-positive samples. at dpi, the inoculated cell monolayer showed visible cpe, in the form of gathering pyknosis and rounded cells that rapidly detached from the monolayer on dpi (fig. a) , the mock-inoculated neuro- a cells showing normal cells (fig. b) . the virus was further serially passed in neuro- a cells for a total of passages. virus growth was confirmed by ifa using the antiserum phev, and the antigens were mostly located in the cytoplasm (fig. c ). to confirm phev replication, viral rna was extracted from the culture supernatants and was tested by rt-pcr. the presence of phev particles in the infected cells was also examined by em. the em results showed multiple virus particles approximately to nm in diameter with typical coronavirus morphology (fig. d) . thus, the phev strain was successfully isolated and was designated as phev-cc . plaque assay was used to plaque isolates and to purify phev on neuro- a cells, and large clear plaques were evident under an agar overlay medium on the cells. the cloned virus phev-cc was tested by rt-pcr and was further serially passaged to passages on neuro- a cells ( . log pfu/ml). during the serial passages, significant increases in viral rna titers were observed following each cell passage. the infectious titers of phev-cc were determined by tcid and were calculated according to the reed-muench method. as shown in fig. , there were no significant differences in replication or proliferation between the phev-cc and hev- n strains in the neuro- a cells, but the rna virus titers in the phev-cc -infected cells ( . tcid /ml) were slightly higher than those in the hev n-infected cells ( . tcid /ml) after h post-inoculation. to examine whether genetic changes occurred in the phev-cc strain (genbank: ku ) compared with other phevs available in genbank, the major structure genes were amplified by specific primers (table ) and sequenced. a total of , nucleotides were determined for strain phev-cc , covering five complete structure genes-he, s, e, m and n-and the locations of the organization of the targeted genes were sketched in a conceptual map (fig. a) . therewith, the corresponding nucleotides and deduced amino acid sequences of the phev-cc strain were compared with the homologous sequences of phevs. the results showed that the phev-cc strain shared %- . % nt identities with the other phev strains available in genbank. the structural genes of the phev-cc strain had the greatest nucleotide sequence similarity ( . %) to the hev-jt strain (genbank: ed . ), and it shared % with hev n (genbank: ay . ). compared with the hev n strain, there were four nucleotide sense mutations at positions and in the he gene, in the s gene, in the m gene. these nucleotide changes all induced corresponding amino acid (aa) changes (s g and t i in the he protein; r h in the s protein; a t in the m protein). however, residues in the e and n genes of phev-cc strains were highly conserved in identity with other phev reference strains in the genbank database. a phylogenetic tree was constructed using the five genes (he, s, e, m, and n) of phev-cc with some other phev strains obtained from genbank database, as well figure the growth curves of phev strains. neuro- a cells were, respectively, inoculated with phev-cc and hev n. the tcid was measured at different time points, and the growth curves were plotted. there was no significant difference in replication or proliferation between the phev-cc strain and hev- n strain (p > . ). as several members of the coronaviruses (fig. b ). phylogenetic analysis of the five genes clearly showed that the phev-cc strain clustered into a subclass with a hev-jt strain from china isolated in , and a similar finding showed that the phev stains in china were highly homologous with a north american strain (ay ). additionally, the homology of the deduced amino acid sequences between the phev-cc strain and hcov-oc (genbank: kf ) was as high as %. in march , there was a suspected outbreak of phev infection on a farm in changchun in the jilin province of china. the clinical signs consisted of vomiting, twitching, wasting, and diarrhea were observed in suckling piglets. the histologic autopsy showed that there were pinpoint petechiae in the kidneys, thinning of the intestinal wall, and hemorrhage in the brain. due to the great similarity between phev and pseudorabies virus (prv) infection in piglets, it was difficult to distinguish them only by clinical and autopsy symptoms. therefore, prv and phev were first detected by pcr and rt-pcr, respectively, and the test results showed that prv was negative, and phev was positive, thus excluding prv infection. at the same time, no sow abortions or stillbirth phenomena were found in the pigs, also supporting the above results. immunohistochemical staining results further confirmed phev infection in the brains of the dead piglets. vomiting and neurological symptoms are common in piglets infected with phev, but the symptom of diarrhea is relatively rare. because the outbreak was characterized by vomiting and diarrhea, some other viral infections with similar clinical symptoms have been reported in pigs, including pedv, tgev and dpcov (ma et al., ; song, moon & kang, ; tanaka et al., ) . in this study, these viruses were further detected to exclude misdiagnosis or mixed infection. thus, the case was identified as simple phev infection, and we successfully isolated a phev field strain as phev-cc . phev has a typical neural tropism, and it invades the central nervous system via the peripheral nervous system (hirano et al., ; lafaille et al., ; lee et al., ; zhang et al., ) . previous studies have shown that the virus successfully killed -to -weekold mice readily by different routes, and viral antigen was detected in both peripheral nerves and the cns (hirano et al., ; hirano et al., ; hirano et al., ) . in this paper, three-week-old balb/c mice were chosen for inoculation with phev-cc by the intranasal route, and the results of the experiment confirmed that phev-cc had strong pathogenicity to mice. viral titers of phev-cc or hev n were determined by tcid , and the growth curve showed that there was no significant difference in replication or proliferation between them. to characterize the virus isolates, the complete structural genes were sequenced and analyzed, and the phylogenetic relationships among the coronavirus strains were determined. phylogenetic trees showed that the wild type and hev-jt shared the highest homology, and that identified with hev n was %. these findings suggested that phev strains currently circulating in china are closely related. notably, the phev strain jt isolated from jilin province, china, in was most closely related to the emerging phev-cc strains, suggesting that they could be derived from a similar ancestral strain. furthermore, we performed sequence alignment and homology analysis between phev-cc and other coronaviruses, which including mhv, bcov, hcov-oc and bat cov, and we found it shared up to % homology with hcov-oc (genbank: kf ) (gonzalez et al., ; li, ; snijder, horzinek & spaan, ) . this finding suggested that, although phev infection in humans has not been reported currently, there is a definite potential threat to human health. according to the deduced amino acids of the phev-cc strains, genetic evolution and variation analyses were performed. it was found that the broad variation occurred in the encoded structural proteins and functional region. there were five major structural proteins of phev, he, s, e, m and n proteins, encoded from utr to utr (vijgen et al., ; weiss & navas-martin, ) . sequence analyses of the five structural proteins of the phev-cc field isolate suggested that phev has remained more genetically stable in the e, m and n proteins (schultze et al., ; vieler et al., ) . the he proteins of some coronaviruses are involved in the release of virions from the host cell, and it has been shown to have acetylesterase activity and to function as a receptor-destroying enzyme, which might be related to the early adsorption of coronavirus (schultze et al., ) . compared with hev n, there were two amino acids (s g, t i) in the he protein of phev-cc that were meaningful mutations, while six amino acid variations were observed (s g, s g, k n, t i, v a, l f) when blasted with the iaf- strain. we hypothesize that the amino acid variation of the he protein might have a certain effect on replication and virulence, but it was difficult to explain the differences in virulence among the pigs, based on the amino acid changes. in addition, there were some variations of the amino acids in the s protein, which plays vital roles in viral entry, cell-to-cell spread, and the determination of tissue tropism (dong et al., ; lu, wang & gao, ) . therefore, the differences in virulence of phev strains might be caused by multiple factors, and the variation of the whole genome has resulted in changes in their antigenic differences. briefly, the outbreak on the pig farm in northern china was caused by phev, and the virus was isolated, systematically characterized and designated phev-cc . this work will enrich the data on the genome and molecular epidemiology of phev and will provide material for further study of the virulence of phev, which should have a certain theoretical and practical significance. immunofluorescence studies on the pathogenesis of hemagglutinating encephalomyelitis virus infection in pigs after oronasal inoculation 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receptor-binding activity the coronaviruslike superfamily porcine epidemic diarrhea: a review of current epidemiology and available vaccines molecular epidemiological study of feline coronavirus strains in japan using rt-pcr targeting nsp gene genomic relationship of porcine hemagglutinating encephalomyelitis virus to bovine coronavirus and human coronavirus oc as studied by the use of bovine coronavirus s gene-specific probes evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus evidence for borna disease virus infection in neuropsychiatric patients in three western china provinces this study was supported by the national natural science foundation of china (nos: , , ), the national key r&d program of china (nos: yfd , yfd ), and the youth scientific research foundation of jilin province (no. jh). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the following grant information was disclosed by the authors: the authors declare there are no competing interests. • zi li and wenqi he conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables.• yungang lan, kui zhao and xiaoling lv conceived and designed the experiments, performed the experiments, analyzed the data.• huijun lu, ning ding, jing zhang, junchao shi and changjian shan performed the experiments, contributed reagents/materials/analysis tools.• feng gao analyzed the data, reviewed drafts of the paper. the following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):the permission to work with laboratory animals was obtained from the animal welfare ethical committee of the college of veterinary medicine, jilin university, china (permission number -cvm- ). the following information was supplied regarding the deposition of dna sequences:genbank: ku ; http://www.ncbi.nlm.nih.gov/nuccore/ . the following information was supplied regarding data availability: genbank: ku . key: cord- - oqzsd authors: domanska-blicharz, katarzyna; lisowska, anna; sajewicz-krukowska, joanna title: molecular epidemiology of infectious bronchitis virus in poland from to date: - - journal: infect genet evol doi: . /j.meegid. . sha: doc_id: cord_uid: oqzsd the presence of infectious bronchitis virus (ibv) was identified for the first time in the poultry population in poland at the end of the s. from this time a few waves of epidemics caused by different ibv variants spread across the country. in order to gain more insight into the molecular epidemiology of ibv in poland, in the present study the s coding region of ibv isolates and nearly whole genome of strains collected over a period of years was characterized. phylogenetic analysis showed that these strains belonged to five recently established ibv lineages: gi- , gi- , gi- , gi- and gi- . additionally, two strains from and formed a separate branch of the phylogenetic tree categorized as unique early polish variants, and one strain was revealed to be the recombinant of these and gi- lineage viruses. irrespective of year of isolation and s -dependent genotype, the genome sequences of polish ibv strains showed the presence of six genes and orfs: ′utr- a- b-s- a- b-e-m- b- c- a- b-n- b- ′utr, however their individual genes and putative proteins had different lengths. the phylogenetic analyses performed on the genome of ten polish ibv strains revealed that they cluster into different groups. the polish gi- , gi- and gi- strains cluster with other similar viruses of these lineages, with the exception of the two strains from and which are different. it seems that in poland in the s and s ibv strains with a unique genome backbone circulated in the field, which were then replaced by other strains belonging to other ibv lineages with a genome backbone specific to these lineages. the recombination analysis showed that some polish strains resulted from a recombination event involving different ibv lineages, most frequently gi- and gi- . infectious bronchitis virus (ibv) is the etiological agent of a highly contagious disease of chickens known as infectious bronchitis, but the virus can replicate in epithelial cells of different organs, also affecting the urogenital or digestive tracts beside the respiratory tract (cavanagh, (cavanagh, , . together with genetically similar viruses isolated from other domesticated galliformes, ibv belongs to the igacovirus subgenus within the gammacoronavirus genus (nidovirales order, cornidovirinae suborder, coronaviridae family, orthocoronavirinae subfamily). the nonavian sw gammacoronavirus isolated from beluga whales was recently assigned to the separate cegacovirus subgenus (dong et al., ; king et al., ) . the virus genome is an approximately kb long single-stranded, positive-sense rna consisting of several open reading frames (orfs). two thirds of the genome in the ′ end are occupied by two overlapping orfs encoding viral rna-dependent rna polymerase. the a and b orfs encode non-structural polypeptides (nsp - ) which are associated with rna replication and transcription. in the ′ end are genes that among other products encode the four major structural proteins: spike (s), envelope (e), matrix (m), and nucleocapsid (n). the s glycoprotein is post-translationally cleaved into s and s subunits of about and amino acids during viral maturation. the s subunit anchors the spike into the virus membrane whereas s forms the extracellular part of the spike and plays a major role in tissue tropism and induction of protective immunity (cavanagh and gelb, ) . ibv undergoes many genetic changes generated both by recombinations and mutations such as substitutions, deletions and insertions, which could lead to the emergence of new variants. among factors that create favorable conditions for such events are characteristic features of coronaviruses in the genome structure (large singlestranded rna) and virus biology (minimal proofreading activity of viral polymerase) and modern poultry-rearing habits and immunological pressure caused by the worldwide use of vaccines (ovchinnikova et al., ; woo et al., ) . mutations within the s gene particularly result in new geno-or serotypes, and currently there are many such types around the world (de wit et al., ) . their number, diversity and naming and the plurality of methods used for their determination for years have caused much confusion. to avoid it, new classification rules based on the whole s gene phylogeny (about nt) and new nomenclature have been proposed. this system distinguished and named lineages, aggregating into genotypes (gi to gvi) (valastro et al., ) . however, in the last three years, two more lineages (gi- and ) and even one more genotype (gvii) have been described in china jiang et al., ; ma et al., ) . in poland, the first suspicion of ib was based on clinical observations, as respiratory symptoms incurable with antibiotics in some flocks and/or misshapen eggs from commercial flocks came to notice. laboratory confirmation of ibv infection was obtained at the end of the s. between and , sera from two hundred ten chicken flocks at the age of - months were examined in an agar gel precipitation test and only % of them were positive although % of flocks contained birds with positive serum (karczewski and cakala, ) . outbreaks of ib with respiratory signs and a drop in egg production and egg quality in non-vaccinated breeding and particularly laying chicken flocks were recorded in the mid- s (bugajak et al., ) . since the mid- s, outbreaks of ib-nephritis have been reported in broiler flocks (minta et al., ) . a multiplex-pcr testing strains isolated between and revealed that the most of them belonged to the b type (capua et al., ) . the emergence of qx ibv was detected in (domanska-blicharz et al., ; domanska-blicharz et al., ) . more recently, the next variant of ibv called var which had been circulating only in the middle-east region for the previous years was also detected in poland . in this study we attempted to molecularly characterize the field ibv strains detected in poland during the period between and . strain determination was accomplished by phylogenetic analysis of the full s coding region sequences against reference strains representing all genotypes and lineages recently described (valastro et al., ) . additionally, we also analyzed the complete genome sequences of ten polish ib viruses. recombination analysis was also performed using the obtained sequences of these strains. thirty four field ibv strains isolated between and in poland were included in the study. these strains originated from poultry experiencing clinical forms of the disease as respiratory or enteric symptoms, nephritis, or problems with egg production. epidemiological information of the studied isolates is summarised in table . the samples were named to fulfill the previously described criteria, but to make it easier to follow the results of the analysis, in subsequent parts of the text they were shortened to the individual symbol given in the laboratory and the year of identification (ducatez, ) . the earliest virus materials from the s were available in the form of a lyophylizate of allantoic fluids from commercial chicken eggs. after propagation in spf embryos, materials from the s were in the form of allantoic fluids stored deep frozen. field materials delivered to the department of poultry diseases for diagnostic purposes between and were isolated in specific pathogen-free (spf) chicken eggs as described previously (gelb and jackwood, ) . virus genome presence confirmation and genotype determination preceded the spf egg isolation. materials from to as referred to above were also refreshed using the virus isolation method on spf embryonating eggs. harvested allantoic fluids were processed using an rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's recommended procedure for rna extraction, and isolated rna was stored at − °c until analysis. the rt-pcrs were conducted on the one-step model using the one step rt-pcr kit (qiagen, hilden, germany) according to the manufacturer's instructions. various combinations of primer pairs described recently as well as additional primers specifically constructed for some strains (appendix a supplementary material) were applied for amplification and sequencing of the whole s coding region (binns et al., ; boursnell et al., ; dolz et al., dolz et al., , lisowska et al., ; worthington et al., ) . the reactions were run according to the recommended protocol for the kit with different annealing temperatures depending on the melting temperature of the primer pair used. amplified pcr products were visualized by electrophoresis on a % agarose gel stained with ethidium bromide and then purified using a qiaquick gel extraction kit (qiagen, hilden, germany). typically, for the s coding region of polish ibv strains, - pcr products were sequenced in both directions using sanger sequencing technology by genomed (warsaw, poland). the complete genomes of these ibv strains were generated using illumina miseq technology (illumina, san diego, usa) in several laboratories. the five ibv strains / , / , / , g / and g / were processed in the department of microbiology of the swedish national veterinary institute (sva, uppsala, sweden), four subsequent virus strains / , / , / and g / were analyzed in the department of omics analysis in our institute, and one ibv strain g / was sequenced by genomed (warsaw, poland). analyses in these organizations were made according to the standard procedure. briefly, rna extracted directly from the allantoid fluid was retrotranscribed into dna using a superscript iv first-strand cdna synthesis kit (invitrogen, waltham, usa) and the second strand was synthesized with the addition of klenow polymerase (new england biolabs, ipswich, usa). a bp-long paired-end dna library was prepared using a nextera xt sample preparation kit (illumina, san diego, usa) and sequencing was performed using a miseq reagent kit v (illumina, san diego, usa). sequences of s coding region fragments obtained by sanger sequencing were trimmed based on quality and assembled into consensus sequences using geneious v . . (biomatters, auckland, new zealand). sequences of polish viruses were searched with blast (basic local alignment search tool) to find these ones with the highest similarity and include them in the phylogenetic analyses. then the full s sequences were aligned with sequences representing lineages in their ibv genotype groupings and unique variants as valastro et al. recommended (valastro et al., ) using clustal w. additionally, eight sequences representing the two newly identified gi- and gi- lineages and one gvii- genotype were included in the analysis. sequencing data from miseq technology obtained from the sva (uppsala, sweden) and genomed were processed with the clc genomics workbench (qiagen, hilden, germany). the reads obtained in the department of omics analysis of our institute were assembled into contigs with the spades assembler using the website at http://spades.bioinf. spbau.ru (bankevich et al., ) . for phylogeny of the complete genomes of the polish ibv strains, a preliminary analysis was carried out using all gammacoronaviruses available in the niaid virus pathogen database and analysis resource (vipr) through the website at http://www.viprbrc.org/ (pickett et al., ) . next, strains were selected for further analysis, taking into account their clustering in the vipr analysis. alignments of nucleotide sequences were performed using the multiple alignment using fast fourier transform (mafft) method in geneious software, v . . (biomatters, auckland, new zealand). the alignments were then exported to the mega program, v . . (tamura et al., ) . maximum likelihood (ml) phylogenetic analyses of the s coding region and of the complete genome were then conducted using the best-fitting nucleotide substitution models (the lowest bayesian information criterion (bic) scores in each analysis were for the general time reversible (gtr) model and a discrete gamma distribution (+g) with five rate categories, assuming that a certain fraction of sites are evolutionarily invariable (g + i)). bootstrap analyses of the resultant trees were performed using replicates. to detect any recombination events in the analyzed sequences, rdp software v . was used (martin et al., ) . the full s coding region sequences of ibv strains were screened to check if unusual clusters formed by polish ibv strains are viruses representing real new ibv lineage or recombinants. ten full genomes of polish ibv strains were also analyzed for recombination events using the complete genomes of representative viruses selected for analysis as described above. the rdp analysis was accomplished using different available methods with their default parameters, however recombination events were only considered proven if detected by at least seven programs (rdp, geneconv, bootscan, maxchi, chimaera, siscan and seq) and the p-value was calculated at below . × e− . full s sequences of the analyzed polish ibv isolates as well as complete genomes of ten of them were submitted to the genbank database and accession numbers were assigned as given in table . the nearly full genome sequences of ten ibv strains were obtained with the ′ and ′utr fragments incomplete. in all genomes the analysis predicted genes consisting of open reading frames (orfs) with a typical order for ibv of ′utr- a- b-s- a- b-e-m- b- c- a- b-n- b- ′utr, but with their individual genes and putative proteins having different lengths ( table ). the orfs/proteins with a constant conservative amount of nt and amino acids (aa) were a ( nt/ aa), b ( nt/ aa), and n ( nt/ aa). the next orfs/proteins of conservative length were b, c and a counting nt/ aa, nt/ aa and nt/ aa respectively in strains, whereas in two ibvs each of these structures was different (longer or shorter by one nt codon/aa). the difference in orf/protein length of accessory b protein was also slight as it fell within - nt/ - aa. the most diverse in terms of length was the orf coding e protein, ranging from to nt ( - aa) in polish gi- and gi- ibv strains to - nt ( - aa) in polish gi- ibvs. a similar relationship was also observed in the case of the orf encoding m protein, which was the shortest ( - nt/ - aa) in ibv strains of gi- and g- lineages and the longest ( nt/ aa) in ibvs of gi- lineage. the orf of the s protein was of varying lengths from nt/ aa to nt/ aa and did not show any dependence for length on identified ibv lineages. the molecular relatedness extents of the compete genome and the individual orfs between polish and selected ibv strains were - % (appendix b -b supplementary material). genotyping based on phylogenetic analysis of full s coding region sequences of polish ibv strains from the years - grouped (caption on next page) k. domanska-blicharz, et al. infection, genetics and evolution ( ) them into six groups: five distinct, previously known lineages and an additional new one (fig. ) . five isolates comprised of four early ones from the s and one identified in were assigned to the gi- lineage. one strain identified in affiliated to the gi- lineage. the group of gi- lineage contained eight ibv strains: two from the late s isolated between and , two identified between and , and four strains isolated after . the gi- lineage comprised ten strains detected between and and the group of gi- lineage held eight isolates detected between and . two isolates, / and / , were in the separate cluster designated early polish on the phylogenetic tree. sequence analysis revealed that five polish gi- strains shared nucleotide identities of . - % and formed two clades. in one clade were the dutch h , north american, south african, indian and four polish isolates but the strain / formed a distinct branch in this gi- subtree with low nucleotide identity of . - . % to the rest of this group. the analysis of the s coding region sequences of eight polish isolates of gi- lineage showed that four strains formed a common branch, sharing . - . % nt identity with the / strain from the united kingdom, whereas two earlier strains from and which were similar to each other with . % identity form a sister group with the more recent viruses and had . - . % nt similarity with the / strain. the earliest polish gi- strains from and were visibly different and had nt identity of . - . % to the rest of the g- ibv strains. one of them, strain / , occupied positions close to the israeli variant strain from with identity of . %, and its similarity to the moroccan gi- lineage prototype g strain from was . %. the similarity of the other, strain / , was . % and % to strains from israel and morocco, respectively. the gi- lineage contained only one polish strain, g / , which shared . % nt identity with the dutch d virus. eight polish strains were in the gi- lineage and the similarity of their s coding region sequences was between . and . %. their identity with the pathogenic israeli is/ / strain from showed as from . to . %. the subtree of gi- lineage contained polish qx strains in two branches, of which one contained nine strains with nt identity of . - . % to the european qx prototype dutch l- k/ ibv. one strain, g / , with similarity to the previous ones of . - . %, constituted the offshoot branch. the two polish strains / and / , isolated at an interval of years from each other, formed a separate branch in the phylogenetic tree and they shared . % nt identity. the phylogenetic analysis of the analyzed full ibv genomes showed that ten polish ibv strains grouped into four phylogenetic groups (fig. ) . two early strains, / and / , clustered together with massachusetts-like strains (mass , peafowl/gd/kq / and ses ab- ) showing the highest nt identity of . % with the sequence of the prototype gi- lineage beaudette strain. the two most recent strains, g / and g / , clustered with ibvs of gi- lineage. the sequence identities of g / and g / to the previously described polish gammacov/ck/poland/g / strain were . and . %, respectively. the three polish strains / , g / and g / were in the same cluster as other qx strains from europe and africa and were distantly related to chinese qx ibvs analyzed in this study (sdzb and p ). they had nucleotide similarity to each other of . - . % and were located in two subclades. a polish strain from clustered together with swe/ / , the first described full-genome ibv strain of qx type in europe, with similarity of . %. the other two polish qx strains were . % similar to each other and formed a common branch on the phylogenetic tree. three early polish strains / , / and / were in a separate branch on the phylogenetic tree and showed nucleotide similarity with each other in the range of . - . %. recombination analysis of all aligned full s sequences was performed to assess the existence of possible recombinants among the analyzed polish ibv strains, especially those with less obvious membership to the lineage, i.e. / , / and / . our analysis identified only one s coding region which resulted from recombination events and it belongs to the / strain; this event having taken place was supported by seven different methods with a very good global ka p-value of . e− . we confirmed this recombination breakpoint with phylogenetic trees. the region from to nt of the / ibv strain clustered together with / and / isolates, the viruses which formed the separate early polish cluster on the full s coding region phylogenetic tree (fig. a) . in turn, the region from to nt clustered with viruses belonging to gi- lineages (strains / , / , ibv india and h ) (fig. b) . the relevant s coding region fragments of the other ibv strains analyzed in this study grouped in the same way as they did in the phylogenetic analysis of the entire, intact s coding region. to check if any of the analyzed genomes of polish ibv strains result from recombination events, their sequences were thoroughly examined using the rdp program. our analysis revealed many such events. however, we selected five of them identified in six strains and they were supported with seven different methods (rdp, geneconv, bootscan, maxchi, chimaera, siscan and seq) and a very good global ka p-value below . x e− (table ) the retrospective phylogenetic analysis of ibv included field strains collected over a period of years, between and . we investigated the s coding region of ibv strains and the whole genome of ten strains. polish ibv strains showed different molecular features of the s coding region allowing their genotype or lineage to be determined, and their appearance in time reflects the history of ibv epidemics in europe (de wit et al., ) . the plot showing the timeline of various ibv lineages's detection and introduction of different vaccines to poultry population in poland is given in fig. . the first identified ibv isolates in europe belonged to the mass type. in the netherlands they were diagnosed in the middle of the s and one of them was even attenuated for vaccine development purposes (bijlenga et al., ) . the first ibv material available in our laboratory originates from and its s sequence displayed the features of gi- lineage, although no information was provided about the disease symptoms observed in the chicken flock where it was identified. later on, especially in the middle of the s, numerous cases of a drop in egg production were recorded. the problem was so serious that polish veterinary authorities decided to allow the first ib vaccine introduction, but only for immunization of commercial layer flocks. the health problems in layers were significantly mitigated, but at the end of the s respiratory fig. . phylogenetic tree of the s gene of reference and polish ibv strains (bold underlined letters). the tree was constructed using mega using the maximum likelihood method based on the gtr + g + i model and bootstrap replicates (bootstrap values shown on the tree). to make the tree clearer visually, branches with ibv lineages only distantly correlated with studied polish strains are collapsed. problems and mortality manifested in broiler chicken flocks, and so the vaccination of chickens of this production type was also started (minta et al., ) . we thoroughly examined four virus strains from that time, / / , / / , / / and / / . two of them, strains / / and / , have the s structure typical of the gi- lineage. similarly, their entire genomes revealed the highest identity to masslike strains such as h or beaudette. these two viruses came from broiler chickens on farms in the silesia region separated by only a few kilometers. the two other viruses, / and / , were identified in broilers delivered to the laboratory near the same period (in june ) but from farms about km from the previous ones. however, our investigation revealed a distinction between them. phylogenetic analysis of the full s coding region showed that strain / forms a separate branch on the tree designated as early polish and was classified as a unique variant of ibv within the gi genotype. deep analysis of the / strain strongly suggests that its s coding region was created as the result of a recombination event between the mass-like strains and the unique early polish variants circulating in the field at that time. it should be emphasized that the identified recombination breakpoint ( nt) was in the intermediate region between highly variable regions (hvrs) and and hvr previously described as the most frequent locations of variations between ib viruses, and moreover, it exactly matches the breakpoint ( and nt) of recombinants between viruses of gi- and gi- lineages (valastro et al., ) . the introduction of vaccines based on the mass-like strains for chicken immunization significantly reduced the economic losses caused by ib. this state of ib control lasted for about years until , when ib disease inducing kidney damage appeared, caused by b-like ibv strains. the first case of nephritis was in -week-old broilers in the south of poland. the birds showed signs of severe enteritis and the observed gross lesions were congested tracheas and lungs and swollen and pale kidneys with the presence of urine. in subsequent months, further broilers with nephritis were provided for diagnostic purposes and the diseased flocks from which they came were located in all regions of poland; most of them had not been vaccinated against ib, but some had been immunized with mass-like vaccine in the first days of life. the strains identified at that time, / and / , have an s sequence similar to ibv strains of gi- lineage. surprisingly, one of the first isolates known to cause nephritis, strain / together with strain / inflicting respiratory disorders, were located in the early polish branch of the phylogenetic tree with well supported uniformity to others (bootstrap value of ) (hillis and bull, ) . the next polish gi- strains identified between and revealed the highest nt similarity to the s sequence of the / strain contained in the most commonly used vaccine in poland at that time (adzhar et al., ) . the next epidemic wave of ib in poland was caused by qx strains. the first report of disease induced by this virus type was published in , but our studies showed the presence of the virus in poland in (domanska-blicharz et al., ) . it was from this year that qx strains were first identified in holland, germany, belgium and france, and next year they became dominant in some of these countries phylogenetic tree of the s gene fragment between potential recombination breakpoints and (a) and and (b) among ibvs included in the analysis. sequences of polish ibv strains are marked with bold underlined letters and recombinants with black dots. the tree was constructed using mega using the maximum likelihood method based on the gtr + g + i model and bootstrap replicates (bootstrap values shown on the tree). to make the tree clearer visually, branches with ibv lineages only distantly correlated with studied polish strains are collapsed. (worthington et al., ) . the analysis of the migration history of gi- strains suggests that most european ones came from a single introduction from china, which then spread in european countries, evolving in them separately, since they tend to cluster by country. however, the genetic variability of gi- ibvs sometimes identified within countries suggests subsequent introduction of the virus in epidemic waves (franzo et al., ) . the division of polish gi- strains into four clusters could reflect a separate introduction or epidemic wave of this virus variant into the country. the last large epidemic wave of ib was caused by gi- (var ) strains. the first strain of this lineage was identified in december and in the following months it was the most common virus type detected in field samples delivered to our laboratory for diagnostic purposes apart from strains of b . the s coding region of most polish gi- strains is in the same phylogenetic cluster, except for strain g / , which constitutes a separate one and could result from a separate virus introduction or from its intensive evolution. the single polish g / virus strain of gi- lineage analyzed in our study was identified in a -week-old broiler flock vaccinated with poulvac ib primer so it is highly probable that the identified strain originated from vaccine virus. although ball et al. (ball et al., ) showed that after vaccination of -day-old broilers with this vaccine only rna of the mass strain was detected in tissues and swabs and explained it through the higher replication potential of the mass virus. it cannot be ruled out that, as some other ibv strains are, the d virus is deposited in the body of chickens (possibly in the cecal tonsils) and after some time it is shed with cloaca (alexander and gough, ; naqi et al., ) . it should be noted that the ibv strains discussed here in detail are those that caused the greatest losses in polish poultry farming. during this period, strains of other genotypes and lineages also circulated in the field but their detection or type determination was not possible using available methods. the comprehensive studies of polish ibv isolates from to using serological and molecular tests conducted in cooperation with italian researchers showed that the b type was a major component of the ibv population in poland during this period, but serologically the presence of /i isolates was also identified and one isolate even showed no serological cross-reaction in an hi test nor amplification in rt-pcr (capua et al., ) . recently, the /i and q types were determined to affiliate to the gi- lineage, which has been present in europe (italy) since and persists until now (franzo et al., ) . in the period - numerous cases of d ibv (gii- lineage) were detected, however, in subsequent years ( ) ( ) , the number of d -positive samples dropped to . %, and currently we do not detect these viruses at all (domanska-blicharz et al., ; domanska-blicharz et al., ) . the complete genome sequences of all polish field ibv strains showed the presence of six genes and orfs in the order previously reported, irrespective of the year of first isolation (abolnik, ; gomaa et al., ; hewson et al., ) . most accessory proteins are conservative in their lengths, in contrast to the structural ones which differ by even as much as aa (m protein of gi- / and gi- ). an interesting observation is clustering of polish ibvs based on the complete genome sequences. the earliest strains / and / belonging to the gi- lineage cluster with other representatives of this lineage such as the beaudette and dutch h strains, which could indicate the common origin of mass-like viruses. the viruses from the next epidemic wave are / and / , and they were on the separate branch of the phylogenetic tree together with the strain / . the / virus was located on the s coding region phylogenetic tree with the / strain in the separate ibv branch of early polish ibv. on the other hand, the third virus of this separate group, / , found its place in the gi- lineage on the phylogenic tree based on the s coding region. thorough analysis using rdp software revealed that the s gene of this virus was acquired from ibv k. domanska-blicharz, et al. infection, genetics and evolution ( ) strains of gi- lineage during a recombination event. the results suggest that in the late s and s two ibv variants circulated in the polish poultry population: gi- and unique early polish ones. these viruses differ not only in the s coding region, which is the basis for the differentiation of lineages, but also in the remaining part of the genome. the viruses with such a genome backbone recombined with other viruses that were donors of the s coding region. grouping on a phylogenetic tree based on the complete genome of the other five polish strains was as expected. three g- strains, / , g / and g / , took positions among other qx-like viruses from europe (sweden and italy) and africa (south africa and sudan) (abolnik, ; abro et al., ; ducatez et al., ; naguib et al., ) . in turn, two strains of gi- lineage, the viruses g / and g / , were in the branch with the previously characterized polish g / and iranian is- strains of gi- lineage isolated in . it seems that in poland in the s and s ibv strains with a unique genome backbone circulated in the field, which were then replaced by strains belonging to other ibv lineages with a genome backbone specific to these lineages. in addition to the aforementioned recombination, five such events were also identified in polish ibv strains. three strains of the gi- lineage had orf a and orf b which revealed a high frequency of recombination events with / and sdzb -like strains (qx type strains from china from ). two strains of the gi- lineage exhibited recombination with the italy/ / -type ibv, and a similar recombination pattern was also previously indicated . in conclusion, phylogenetic analysis performed on the s coding region of polish ibv strains collected during a -year period showed that these strains belonged to five recently established ibv lineages: gi- , gi- , gi- , gi- and gi- . additionally, two strains formed a separate branch of the phylogenetic tree described as unique early polish variants and one strain revealed itself to be the recombinant of gi- lineage viruses and these unique early polish variants. the phylogenetic analyses performed on the complete genome of ten polish ibv strains showed that they cluster into different groups. polish gi- , gi- and gi- strains cluster with other similar viruses of these lineages, with the exception of the strains from to which are different. the recombination analysis showed that polish strains are a mosaic of different parental viruses most likely resulting from recombination events involving different ibv lineages, most frequently gi- and gi- . it should be also stressed that the major epidemics of ib in poland appeared every - years: gi- in , gi- in , gi- in and gi- in . these subsequent ibv lineages could have reached chickens in poland in various ways: carried by wild birds, or as a result of international trade, including uncontrolled movement of animals across borders (domanska-blicharz et al., ; hussein et al., ; kahya et al., ) . despite the apparent regularity in the appearance of subsequent ib epidemics, it is absolutely impossible to predict when the next one will appear. the most important impediment to prediction are the visible climate changes forcing changes in bird behavior, but another is the extraordinary intensification of the poultry industry in poland. taken as a whole, the molecular characteristics of polish ibvs presented here could help to understand the origin, spread and evolution of ib viruses in europe and the rest of the world. none. genomic and single nucleotide polymorphism analysis of infectious bronchitis coronavirus emergence of novel strains of avian infectious bronchitis virus in sweden molecular analysis of the /b serotype of infectious bronchitis virus in great britain a long-term study of the pathogenesis of infection of fowls with three strains of avian infectious bronchitis virus infectious bronchitis vaccine virus detection and part-s genetic variation following single or dual inoculation in broiler chicks spades: a new genome assembly algorithm and its applications to 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: molecular evolutionary genetics analysis version . s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification infectious bronchitis virus variants: a review of the history, current situation and control measures coronavirus diversity, phylogeny and interspecies jumping a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from the authors wish to acknowledge dr. siamak zohari and karin ullman (department of microbiology, national veterinary institute -sva, uppsala, sweden) and dr. ewelina iwan and arkadiusz bomba (department of omics analysis, national veterinary research institute, puławy, poland) for their support while conducting ngs. we also acknowledge justyna opolska for her help in molecular diagnostic tests.an ethical statement is not required as samples from animals were delivered to our laboratory by the owners or veterinarians for diagnostic purposes. chickens on the farms were under the supervision of appropriate persons, who took different samples as part of their routine work.this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. supplementary data to this article can be found online at https:// doi.org/ . /j.meegid. . . key: cord- -p bl authors: gao, mengying; wang, qiuling; zhao, wenjun; chen, yuqiu; zhang, tingting; han, zongxi; xu, qianqian; kong, xiangang; liu, shengwang title: serotype, antigenicity, and pathogenicity of a naturally recombinant tw i genotype infectious bronchitis coronavirus in china date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: p bl since , strains of the naturally recombinant tw i genotype of infectious bronchitis virus (ibv) have caused considerable damage to the chinese poultry industry. to better understand the antigenicity and pathogenesis of this genotype, the characteristics of the ck/ch/ldl/ strain were compared to those of four commercial ib vaccine strains that are used commonly in china, as well as four attenuated viruses that represent two types of ibv strains, which are believed to have originated in china and are the predominant ibv types circulating in chicken flocks in china and many other parts of the world. the results showed that all eight strains were genetically and serotypically different from the strain ck/ch/ldl/ . furthermore, neither the vaccine strains nor the attenuated viruses could provide complete respiratory protection of chickens against a challenge with the ck/ch/ldl/ strain, indicating that it is necessary to develop new live vaccines or to evaluate the use of established vaccines in combination to control naturally recombinant tw i-type ibv strains in the future. our results showed that strain ck/ch/ldl/ is very pathogenic, and that it is able to cause cystic oviducts in a high percentage of birds, as well as mortality due to nephritis and respiratory distress with complete tracheal ciliostasis, especially in chickens infected at day of age. being a coronavirus and, therefore, a single-stranded rna virus, infectious bronchitis virus (ibv) has an enormous capacity to change by spontaneous mutation and genetic recombination. it is believed that spontaneous mutations and genetic recombination can occur randomly in the ibv genome; however, if these events occur in the spike (s) gene, especially in its hypervariable regions, these events are most likely to result in the emergence of many different antigenic or genotypic types, which are commonly referred to as variants. therefore, ibv is ubiquitous in most parts of the world where poultry are reared, and it is able to spread very rapidly in non-protected chicken flocks, leading to heavy economic losses in poultry industries (cavanagh and gelb, ) . in general, while many new variants are unable to replicate or survive for a long time, a few variants that are of economic importance have emerged worldwide or in restricted geographic areas. therefore, effective surveillance, which is primarily based on the isolation and identification of the virus type causing disease, is of great importance. classification or typing of ibv strains is very important for implementing control measures, research purposes, and understanding the epidemiology and evolution of ibvs. thus far, two major groups of classification systems, including functional tests that examine the biological functions of a virus (immunotypes, protectotypes, and serotypes) and non-functional tests that assess the viral genome (genotypes), are commonly used for ibv typing (de wit, ) . for a specific ibv strain, although evidence from some studies suggests that there is a high correlation between the genotype and serotype, other studies have presented conflicting data (de wit, ; zhang et al., ; chen et al., ) , which may lead to contradictory results. the disadvantages of each system are that they only analyze one or several characteristics of a virus strain. hence, data from only one system has to be interpreted with caution, while a more objective and accurate conclusion can be drawn by completely analyzing the results from different systems, although it is suggested that the preferred typing system usually depends on the goal (e.g., selection of vaccination programs or epidemiological studies), available techniques, experience, and costs (de wit, ) . a large number of ibv genotypes and variants have been isolated in china in recent years (han et al., ) , among which two genotypes, the lx and ck/ch/ldl/ i types (also known as qx-and q -like, respectively), were first isolated in china and subsequently have become widespread worldwide (valastro et al., ) . among these ibv genotypes, the tw type was first isolated in in taiwan, and it was considered to have a different genotype than all of the other ibvs (wang and tsai, ) . the majority of tw ibvs isolated from taiwan are nephropathogenic, and they have mortality rates ranging from to % in -day-old specific-pathogen-free (spf) chickens (wang and tsai, ) . however, the naturally recombinant tw i (nrtw i) genotype was first isolated in in china, and it was thought to have originated from a natural recombination between lx and tw viruses (xu et al., ) . despite a previous study that characterized the genetic characteristics of an nrtw i type virus and its nephropathogenicity in -day-old spf chickens (xu et al., ) , there is no further information on this important ibv variant. to better understand the nrtw i type, a series of experiments was performed to investigate its antigenicity and pathogenicity in the oviducts of spf layers, and to evaluate the protection provided by commercial vaccines and attenuated viruses. nine ibv strains, including four vaccine strains (h , ldt -a, / , and connecticut (conn)), four attenuated strains (ck/ch/ldl/ [ldl/ ], ck/ch/lsd/ [lsd/ ], ck/ch/ lgx/ [lgx/ ], and ck/ch/ldl/ i [ldl/ i]) and the ck/ch/ldl/ (nrtw i) strain (xu et al., ) , were used in this study. the ibv strains ldl/ , lsd/ , and lgx/ (chen et al., ) were isolated in china and belong to the lx type. these three strains were attenuated by serially passaging them (p ), (p ), and (p ) times, respectively, in -day-old spf chicken eggs. the ldl/ p , lsd/ p , and lgx/ p strains were shown to be fully attenuated in spf chickens (liu et al., a) . furthermore, vaccination with attenuated viruses provided complete protection against their virulent parental viruses (data not shown). the ldl/ i strain was also passaged times (p ) in spf chicken eggs (liu et al., b) . each of the virus stocks was prepared in -dayold embryonated spf chicken eggs by the allantoic route of inoculation, and the infectious allantoic fluid was collected h post-inoculation as previously described (liu et al., a) . the titers of the viruses were determined as previously described (chen et al., ) , and the median embryo infectious dose (eid ) was calculated using the method of reed and muench ( ) . white leghorn spf layer chickens and fertile spf chicken eggs were obtained from the harbin veterinary research institute. the birds were maintained in isolators with negative pressure, and food and water were provided ad libitum. all experimental procedures were approved by the ethical and animal welfare committee of heilongjiang province, china. the s genes of strains lsd/ p and lgx/ p were amplified and sequenced as previously described . the sequences have been submitted to genbank, and they have been assigned accession numbers kt and kt , respectively. the s gene sequences of the remaining eight viruses, including the h , ldt -a, / , conn vaccine, ldl/ p , and ldl/ i p , nrtw i and the tw i-type tw / strains, were selected from the genbank database and used for s gene comparison. both the sequences of amino acid and nucleotide were assembled and the similarities were calculated using the clustal w method available in the bioedit software package (version . . . ., available at: http://www.mbio.ncsu.edu/bioedit/bioedit). in addition, other reference strains in which the s subunit sequences were available in genbank (www.ncbi.nlm.nih.gov/genbank/) were also selected for a phylogenetic analysis. the characteristics of the reference viruses are listed in table . a phylogenetic tree based on the s gene was constructed from aligned amino acid sequences by the neighbor-joining method with bootstraps using the mega program (tamura et al., ) . four ibv vaccine strains (ldt -a, / , h and conn), the attenuated ldl/ i p and nrtw i were used for virus crossneutralization test in this study. because strains ldl/ p , lsd/ p and lgx/ p belong to the same genotype (lx -type or qx-like), hence, only the strains ldl/ p and lsd/ p were used. in addition, the ibv strain ck/ch/lsc/ i, which represents another important genotype in china (han et al., ) , was also used for virus cross-neutralization test in this study. sera against these ibv strains were prepared as previously described (guo et al., ) . briefly, -day-old spf chickens were inoculated by a combined intraocular and intranasal route using a total dose of eid of each virus per bird, respectively. after weeks, a booster dose of each virus with eid was administered by intravenous inoculation to the bird. birds were exsanguinated week after the last inoculation and the separated sera from chickens inoculated with the same virus were pooled. all sera were inactivated at c for min and stored in . ml aliquots at À c until required. for virus neutralization, sera were serially diluted two-fold with sterile phosphate-buffered saline (pbs) and mixed with eid of the ibv strains. after incubation for h at c, virus-serum mixtures were inoculated into the allantoic cavity of spf chicken embryos, which were observed for d. the end-point titer of each serum sample was calculated using the method of reed and muench ( ) . fifty-five -day-old spf layer chickens were separated into four groups, and they were provided with food and water ad libitum. groups - included birds, and they were challenged with the nrtw i strain when they were , , and days old, respectively. the challenge strain ( eid in . ml of diluent per bird) was applied by the intranasal and ocular routes. group , which served as a negative control, included birds that were not challenged. the chicks were examined daily for clinical signs of infection, such as tracheal rales, nasal discharge, watery eyes, and wheezing. the clinical signs from all of the birds in each group were counted by three people over a -min period. morbidity and mortality were recorded daily. gross lesions were also carefully examined from the dead chickens, and the kidneys of the dead chickens were subjected to immunohistochemistry (ihc) using monoclonal antibody d , which is directed against the nucleoprotein, as previously described (de wit et al., ; xu et al., ) . blood samples were collected on , , , , , and d post-challenge (dpc) from all birds, and they were examined for the presence of antibodies against ibv using a commercial enzyme-linked immunosorbent assay (elisa) kit (idexx, portland, me, usa) according to the manufacturer's instructions. at months of age, the birds were killed humanely using carbon dioxide/oxygen, followed by exsanguination. a post-mortem examination was performed, and special attention was paid to abnormalities in the oviducts and kidneys. meanwhile, samples from the kidney were collected to detect the presence of ibv by ihc. ami no acid substit uti ons (x . phylogenetic trees constructed from the nucleotide sequences of the s subunit gene of the four vaccine strains, four attenuated viruses, and reference ibv strains. the trees were computed using the neighbor-joining method. the significance of the tree topology was assessed by bootstrapping calculations. genbank accession numbers are indicated in table . one hundred -day-old spf chickens were separated into experimental groups, each containing birds. birds in groups - were vaccinated with eid of the h , ldt -a, / , and conn vaccines strains, and the ldl/ p , lsd/ p , lgx/ p , and ldl/ i p attenuated strains, respectively, in a total of . ml of pbs per bird, by the intranasal and ocular routes. birds in groups and received ml of sterile pbs via the same routes. blood samples were collected at , , , , and d post-vaccination (dpv) . at dpv, birds in groups - were challenged with eid of the virulent nrtw i strain, in a total of ml of pbs per bird, by the intranasal and ocular routes. birds in group were not exposed to virus and served as a negative control. nasopharyngeal and blood samples were collected from all of the birds in each group at , , , , and dpc. nasopharyngeal swabs were used to recover viruses from day-old spf chicken eggs, and viruses were subsequently detected by reverse transcription polymerase chain reaction (rt-pcr) as previously described (liu et al., a) . the serum was stored at À c for detecting antibodies against ibv using the aforementioned elisa kit as previously described (liu et al., a) . as illustrated in fig. , the four commercial vaccine strains (h , ldt -a, / , and conn), which represent different serotypes (massachusetts (mass), tl/ch/ldt / , /b, and connecticut, respectively) of ibv, were genetically different from the nrtw i strain, as indicated by an analysis of their s genes. of the four attenuated strains, the ldl/ p , lsd/ p , and lgx/ p strains belonged to the lx genotype, while the ldl/ i p strain belonged to the ck/ch/ldl/ genotype. these two genotypes were also genetically different from those of the nrtw i strain and the four vaccine strains. in accordance with these results, strain nrtw i did not share more than % nucleotide and amino acid similarities with the s genes and s proteins of the vaccine and attenuated virus strains in this study (table ). in addition, similar to previous results (xu et al., ) , strain nrtw i shared the highest nucleotide ( . %) and amino acid ( . %) similarities with the tw i prototype strain tw / . the results of the two-way cross-neutralization tests using the ibv strain nrtw i and the antisera against the four vaccine strains, which represented the mass, tl/ch/ldt / , /b ( / ), and conn serotypes, showed that the strains belonged to different serotypes ( table ). the results using the nrtw i strain and the antisera against the deduced parental virus types, the ldl/ p , lsd/ p , lgx/ p , and ldl/ i p strains, also showed non-detectable levels of cross-neutralization. none of the chickens that were vaccinated with the ldt -a vaccine strain or the attenuated ldl/ and ldl/ i viruses showed clinical signs or mortality when challenged with the ibv strain nrtw i, thereby demonstrating that vaccination with these three viruses provided a high degree of clinical protection. in contrast, respiratory signs (sneezing, nasal discharge, and tracheal rales) developed at - dpc and lasted until dpc in some of the chickens in the groups that were vaccinated with the h , / , and conn vaccines, and the attenuated lsd/ and lgx/ viruses when challenged with the nrtw i ibv strain at days of age. in addition, some of the chickens died in these groups, indicating that vaccination with these viruses could not provide complete protection against the nrtw i strain. no birds died during the experiment and no clinical signs were observed in the negative control group. as listed in table , the nrtw i challenge virus was re-isolated from the tracheas of nearly all of the birds in the eight vaccinated groups at dpc, as well as from non-vaccinated birds that were challenged. as expected, none of the chickens in the negative control group tested positive for virus isolation. the serological responses induced by the ibv vaccines and the challenge virus are presented in table . all of the birds in each of the vaccinated groups seroconverted at dpv, as well as at dpc with the nrtw i strain. none of the birds in the negative control group seroconverted during the experiment. table nucleotide and amino acid similarities of the s gene and protein among strain nrtw i and the eight vaccine/attenuated strains. a amino acid identity (%) -, not tested. a reciprocal titer. all of the chickens in the -and -day-old groups that were challenged with the nrtw i strain exhibited the aforementioned respiratory signs at - dpc, and nine and two chickens, respectively, died at - dpc. similar to the negative control group, no birds died and no clinical signs were observed in the day-old group (table ) . obvious hyperemia of the tracheal mucosa with catarrhal exudation was observed in all of the dead chickens. the most remarkable lesions were detected in the kidneys of the dead chickens. the affected kidneys were enlarged and pale, and urate deposition was observed in the tubules and ureters ( fig. a) . similar to our previous results (xu et al., ) , viral antigen was detected by ihc in the kidneys of all of the dead birds. antigen was found in the cytoplasm of the tubular epithelial cells and in the mucous membrane of the ureters and collecting ducts (fig. b) . interestingly, neither gross lesions nor ihc-positive cells were observed in the kidneys of the surviving chickens at the end of the experiment. in addition, characteristic dilatation of the oviduct developed in most ( / ) of the birds in the -day-old group. in contrast, one and two of the birds in the -and -day-old groups, respectively, exhibited cystic oviducts ( fig. ; table ). surprisingly, the dilatation and serous fluid accumulation in the oviducts were observed not only in the chickens in the -and -day-old groups, but also in chickens in the -day-old group, although only / of the birds in the -day-old group showed these lesions. as expected, no such lesions were observed in the birds of the negative control group. all of the birds in the -and -day-old groups seroconverted at and dpc, respectively, with strain nrtw i, which was earlier than that in the -day-old group, in which all of the birds seroconverted at days of age (table ) . we did not observe seroconversion in any chickens of the negative control group. in this study, most ib vaccines, including two types of government-approved vaccines (h and ldt -a) and two types of non-government-approved vaccines ( / and conn) that are used commonly in china, as well as the nrtw i type strains, were selected for genotyping, an s gene comparison, serotyping, and vaccination-challenge tests. in addition, we also selected four attenuated strains representing two types (the lx type strains ldl/ , lsd/ , and lgx/ , and the ck/ch/ldl/ i type strain ldl/ i) of ibv strains that are believed to have originated in china and are the predominant ibv types circulating in chicken flocks in china and many other parts of the world (valastro et al., ) . a phylogenetic analysis and an s gene comparison showed that the aforementioned six types of viruses are genetically different from each other and from strain nrtw i. this is in accordance with the results of the cross-neutralization tests that showed that the serotype of strain nrtw i differs from those of the selected six serotypes in this study and likely represents a novel serotype. the cross-neutralization tests did not include the tw i prototype strain tw / from taiwan, china, which was unavailable for the comparison. table results of the vaccination-challenge tests (groups of chickens were vaccinated with ibv vaccine or attenuated strains, and challenged with the nrtw i strain). / / / / / / / / / / / / / / / / / ldt -a / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / conn / / / / / / / / / / / / / / / / -ldl/ p / / / / / / / / / / / / / / / / -lsd/ p / / / / / / / / / / / / / / / / -lgx/ p / / / / / / / / / / / / / / / / -ldl/ i p / / / / / / / / / / / / / / / / / nrtw i d / / / / / / / / / / / / / / / / -negative control / / / / / / / / / / / / / / / / / a number of chickens that seroconverted/number inoculated. b two procedures, including virus isolation and subsequent rt-pcr, were used for virus recovery after challenge as described previously (liu et al., ) . the results from the two procedures were identical. number of chickens that were positive for virus recovery/number challenged. c days after challenge. d the chickens in this group were not vaccinated with the vaccine or attenuated viruses, but were challenged at days of age with the ibv nrtw i strain. the nrtw i strain was isolated from chickens that had been vaccinated against ibv with the commercial live attenuated h vaccine at day of age and then boosted at days of age (xu et al., ) , possibly indicating that the vaccine does not provide sufficient protection against a challenge with strain nrtw i. thus, further examination of the protection conferred by commercially available ibv vaccines and attenuated viruses against challenge with the novel serotype nrtw i strain was important for evaluating the extent of protection against distinct antigenic types. comparatively, the vaccine strain ldt -a and two attenuated viruses, ldl/ p and ldl/ i p , provided good clinical protection against challenge with the nrtw i strain, although all the strains analyzed in this study have actually percentages of similarity with nrtw i that are of almost the same value (they ranged from . to % of similarity at a nucleotide level, and from . to . % of similarity at an amino acid level). some antigenic epitopes that have roles in the protection have also been identified on the s and n proteins of ibv (ignjatovic and sapats, ) and may attribute to the difference of the cross-protection. further studies are needed to better understand the relationship between antigenic epitopes on s (both s and s ) and n proteins and the level of protection provided in challenge studies. as illustrated in table , the currently available vaccines and attenuated viruses did not provide sufficient respiratory protection against nrtw i challenge, which is in agreement with the genotyping and serotyping results. the results showed that the challenge virus was shed by some chickens in all of the vaccinated groups for at least dpc. additionally, the high rates of challenge virus isolation (less than % at dpc) from chickens that were vaccinated with the heterologous vaccines indicated that none of these vaccines and attenuated viruses provided sufficient protection against the ibv nrtw i strain, especially if a reduction of ibv transmission is considered to be important. these results revealed that it is necessary to develop new live vaccines or evaluate the use of established vaccines in combination to control nrtw i-type ibv strains in the future. over the past years, nephropathogenic ibv strains have emerged as the most predominant ibv strains in the poultry industry, and they are responsible for many outbreaks of kidney disease on chicken farms (de wit et al., ) . generally, the nephropathogenic ibv strains have a tropism for the epithelial cells of the respiratory tract, which leads to clinical signs such as excessive water consumption and wet droppings, followed by a severe renal infection that leads to increased mortality, as shown in the case for the nrtw i strain in our previous study (xu et al., ) , which was corroborated in the present study. our results also confirmed that the morbidity and mortality caused by nrtw i infection was age-dependent (cavanagh and gelb, ) . it was shown that many of the infections in young chickens caused by ibv strains result in permanent damage to the oviduct (broadfoot et al., (broadfoot et al., , , which leads to a significant reduction in egg production and quality upon sexual maturity. the effect of ibv on the oviduct of chickens is extremely variable. there are at least four major factors that influence the severity of oviduct lesions following ibv infection, as well as the outcome regarding the induction of false layers: the ibv strain that is involved, the age at infection, the presence of strain-specific, virus-neutralizing, maternally-derived antibodies at the time of infection, and the early protection induced by vaccinating young chickens (crinion and hofstad, ; de wit et al., ) . infections with different strains of lx type (qx-like) viruses can induce cystic oviducts with water-like fluid accumulation in some chickens that survive the infection, and cystic oviducts were observed at different ages after challenge (benyeda et al., ; de wit et al., ) , whereas no lesions could be observed in the oviducts after infection with the pathogenic / strain (benyeda et al., ; de wit et al., ) . for the mass serotype viruses, it appears that the induction of cystic lesions/false layers differs among different strains (crinion, ; jones and jordan, ; benyeda et al., ). these differences are not related to virulence because it was shown that a live attenuated strain, h , was capable of producing similar pathological changes (duff et al., ) . a recent study showed that cystic oviducts were also found in some spf female chicks that were infected with an is/ / -like virus (awad et al., ) , which was first detected in israel (meir et al., ) and later found in other parts of the world (awad et al., ) . in this study, we found that the nrtw i strain could induce cystic oviducts in spf chickens of different ages within month post-challenge. the nrtw i strain emerged recently in china, and it originated from recombination events between tw i-and lx -like (qx) viruses (xu et al., ) . one of the parental viruses, the tw ibv strain, was first isolated in in taiwan, and it is a nephropathogenic ibv strain; however, it was unknown whether it could induce cystic oviducts (wang and tsai, ) . in contrast, it was clearly shown that another parental virus (the lx type or qx-like) could induce both nephritis and cystic oviducts in chickens that survived infection (benyeda et al., ; de wit et al., ) . the nrtw i strain acquired the sequences of the nsp and s genes from a tw i-like virus, and the rest of its genome from an lx -like virus, although the tissue tropism and pathogenicity determinants of ibv remain unknown (xu et al., ) . in addition, our results also confirmed that as they age, chickens become more resistant to oviduct damage caused by nrtw i infections (cavanagh and gelb, ) . similar to other reports (benyeda et al., ) , our results showed that viral antigens in the kidneys and oviducts of the surviving chickens became undetectable immunohistochemically by the end of the experiment; this is in agreement with our field experience, which showed that ibvs could not be isolated (in most cases) from chickens exhibiting cystic oviducts. in conclusion, these experiments confirmed previous observations that showed that the nrtw i strain has a novel genotype, and that it is a pathogenic ibv strain that can result in high mortality rates by causing nephritis in susceptible birds. our results also showed that nrtw i infection induced age-dependent cystic oviducts. in addition, a cross-neutralization test showed that the nrtw i strain of the nrtw i genotype has a novel serotype that differs from those of the other strains used in this study. a vaccination-challenge test using different heterologous live vaccines and attenuated viruses in -day-old chickens did not induce high levels of protection against challenge at days of age, which suggests that it is necessary to develop new live vaccines or evaluate the use of established vaccines in combination to control nrtw i type ibv strains in future. experimental infection of is/ / -like infectious bronchitis virus in specific pathogen free and commercial broiler chicks comparison of the pathogenicity of qx-like, m and /b infectious bronchitis strains from different pathological conditions effects of infectious bronchitis on egg production effects of infectious bronchitis in baby chicks infectious bronchitis molecular and antigenic characteristics of massachusetts genotype infectious bronchitis coronavirus in china pathogenicity of four serotypes of avian infectious bronchitis virus for the oviduct of young chickens of varying ages egg quality and production following infectious bronchitis virus exposure at one day old induction of cystic oviducts and protection against early challenge with infectious bronchitis virus serotype d (genotype qx) by maternally derived antibodies and by early vaccination infection of day old chicks with infectious bronchitis (ib) virus and subsequent anatomical abnormalities a comparative study of pigeons and chickens experimentally infected with ppmv- to determine antigenic relationships between ppmv- and ndv strains a -year analysis of molecular epidemiology of avian infectious bronchitis coronavirus in china fine level epitope mapping and conservation analysis of two novel linear b-cell epitopes of the avian infectious bronchitis coronavirus nucleocapsid protein identification of previously unknown antigenic epitopes on the s and n proteins of avian infectious bronchitis virus persistence of virus in the tissues and development of the oviduct in the fowl following infection at day old with infectious bronchitis virus altered pathogenicity, immunogenicity, tissue tropism and - kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos evaluation of the protection conferred by commercial vaccines and attenuated heterologous isolates in china against the ck/ch/ldl/ i strain of infectious bronchitis coronavirus characterization of a recombinant coronavirus infectious bronchitis virus with distinct s subunits of spike and nucleocapsid genes and a untranslated region identification of a novel nephropathogenic infectious bronchitis virus in israel a simple method of estimating fifty percent endpoints mega : molecular evolutionary genetics analysis (mega) software version . s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification genetic grouping for the isolates of avian infectious bronchitis virus in taiwan emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks serotype shift of a /b genotype infectious bronchitis coronavirus by natural recombination the authors declare that they have no competing interests. key: cord- -hk qx k authors: rodrigues, juliana falcão; lourenço, rogério ferreira; maeda, denicar lina nascimento fabris; de jesus cintra, mariana; nakao, naomi; mathias-santos, camila; luiz, wilson barros; de souza ferreira, luís carlos title: strain-specific transcriptional and posttranscriptional regulation of heat-labile toxin expression by enterotoxigenic escherichia coli date: - - journal: braz j microbiol doi: . /s - - - sha: doc_id: cord_uid: hk qx k enterotoxigenic escherichia coli (etec) represents one of the most important etiological agents of diarrhea in developing countries and characteristically produces at least one of two enterotoxins: heat-labile toxin (lt) and heat-stable toxin (st). it has been previously shown that the production and release of lt by human-derived etec strains are variable. although the natural genetic polymorphisms of regulatory sequences of lt-encoding (eltab) genes may explain the variable production of lt, the knowledge of the transcriptional and posttranscriptional aspects affecting lt expression among etec strains is not clear. to further understand the factors affecting lt expression, we evaluated the impact of the natural polymorphism in noncoding regulatory sequences of eltab among clinically derived etec strains. sequence analyses of seven clinically derived strains and the reference strain h revealed polymorphic sites at both the promoter and upstream regions of the eltab operon. operon fusion assays with gfp revealed that specific nucleotide changes in the pribnow box reduce eltab transcription. nonetheless, the total amounts of lt produced by the tested etec strains did not strictly correspond to the detected lt-specific mrna levels. indeed, the stability of lt varied according to the tested strain, indicating the presence of posttranscriptional mechanisms affecting lt expression. taken together, our results indicate that the production of lt is a strain-specific process and involves transcriptional and posttranscriptional mechanisms that regulate the final amount of toxin produced and released by specific strains. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. diarrhea represents the third leading cause of child morbidity and mortality in the world, with serious impacts on public health policies and economies, particularly of lower-income countries [ ] . enterotoxigenic escherichia coli (etec) remains an important etiological agent of diarrheal illness in childhood and is the most common cause of traveler's diarrhea [ ] [ ] [ ] [ ] . the disease developed by etec strains requires the production of colonization factors (cfs), responsible for bacterial adhesion to enterocytes, as well as heat-stable toxins (st) and/or heat-labile toxins (lt) [ , ] . lt alone, or in combination with st, is expressed by more than half of the etec isolates [ , ] . once produced and released into the small intestine, lt binds to host cells via the pentameric b subunit, which leads to increased intracellular cyclic adenosine monophosphate (camp) levels due to indirect enzymatic activity of the a subunit, causing water/electrolyte losses and, thereby, watery diarrhea of different severities [ , ] . although clonal and stable lineages of etec are spread worldwide, epidemiological studies have revealed high genetic and phenotypic diversity among etec strains regarding serotypes, virulence features, mainly cfs and lt types, and genotypes disclosed by molecular approaches [ , [ ] [ ] [ ] [ ] [ ] [ ] . our group previously showed the occurrence of lt types among etec strains derived from asymptomatic and diarrheic brazilian children [ ] . these lt types were grouped into four phylogenetic clusters, two of which (a and d) comprised the majority of the variants closely related to the types lt and lt , respectively. more recently, these results were confirmed by further studies, and twelve additional lt variants were found among clinical strains isolated from diverse geographic areas [ ] . genetic polymorphisms in noncoding regulatory sequences and in lt-structural genes have also been ascribed to different lt types [ , , ] . however, considering the data available in the literature, thus far it has not been possible to correlate the ability to express lt and the natural genetic polymorphisms detected among different lt types. lt expression and secretion are also variable traits observed among etec strains [ , [ ] [ ] [ ] . the amount of toxin secreted by strains producing only lt correlates with the severity of diarrhea in animal models and depends on the capability of the bacteria to produce and release the toxin [ ] . lt expression is affected by different growth conditions, such as temperature, ph, osmolarity, and the presence of glucose, which indicates the presence of diverse regulation mechanisms [ , ] . the a and b subunits of lt are encoded by the elta and eltb genes, which are under the control of a single promoter and a transcriptional terminator [ ] [ ] [ ] . lt production is repressed by heat-stable nucleoid-structural (h-ns) proteins, which bind a region of dna near the transcriptional start site and a site at the end of the elta gene [ ] . under environmental changes, particularly at °c, the dna-h-ns complex is destabilized, allowing the binding of rna polymerase, leading to the synthesis of eltab polycistronic mrna [ , ] . in addition, it was reported that the camp receptor protein (crp) negatively regulates lt expression, but there are doubts about the direct binding of this protein to the eltab regulatory region [ , ] . posttranslational regulation of lt expression has also been reported, but the available evidence is weak [ , , ] . in the present study, we demonstrated that specific nucleotide changes at the promoter region of the eltab operon impact lt expression among clinically derived etec strains of different phylogenetic clusters. in addition, our results revealed that lt stability is affected by posttranscriptional mechanisms, which demonstrate that intrinsic transcriptional and posttranscriptional factors contribute to the strain-specific lt expression observed among etec strains. the lt-producing etec strains listed in table were previously characterized regarding genetic and phenotypic features [ , , ] . the h (lt + st + cfa-i + ) is the most studied etec strain, particularly in regard to sequencing data and regulation mechanism of the eltab operon, and, therefore, it was used as a reference strain. the h strain was kindly provided by ann-mari svennerholm (university of gothenburg, gothenburg, sweden). the bl competent e. coli were transformed with plasmids harboring the transcriptional fusions or with a control plasmid, generating the recombinant strains. the bacterial strains were cultivated in ca-ye medium ( % casamino acids, . % yeast extract, mm nacl, mm k hpo , and . % trace salt solution consisting of mm mgso , mm mncl , and mm fecl ; ph = . ) at °c and rpm [ ] . the dna sequences upstream of the translation start codon of the elta gene, comprising nucleotides, were amplified by pcr using genomic dna from etec strains and sequenced by the sanger method (abi sequencer, big dye version , perkinelmer applied biosystems) as previously described [ ] . the forward and reverse primers for pcr and sequencing were constructed based on the sequence of the eltab operon from plasmid p (genbank: fn . ), and their sequences are described in table . representative nucleotide sequences for each polymorphic group were deposited in genbank: mn ( i), mn ( iii), mn ( v). the phylogenetic relationship among the etec strains based on the sequenced regulatory nucleotides was represented by a rooted tree generated using the clustalw program and neighbor-joining methods. the eltab operon regions from up elements to the start codon of translation (position − to + according to the transcription start), harboring the eltab promoter observed in the tested etec strains, were genetically fused to the dna sequence encoding the green fluorescent protein (gfp) (genbank number: kf . ). three eltab promotergfp gene transcriptional fusions were drawn regarding the polymorphisms observed in the eltab promoters of the etec strains using the eltab operon from h as a reference ( table ). the pbsk(+) vectors harboring the transcriptional fusions were manufactured by the biomatik company (wilmington, delaware, usa). a plasmid containing the gfp gene under the control of the lepa promoter was used as a positive control for gfp production. the bl e. coli strains harboring the plasmids (recombinant strains) or not (mock) were grown in ca-ye medium for h at °c and rpm. culture aliquots were centrifuged, and the cell sediments were resuspended in phosphatebuffered saline (pbs) x, generating bacterial suspensions equivalent to an od nm of . the samples ( μl/well) were transferred to a microtiter plate in triplicate, and the green fluorescence was detected with excitation at nm and emission at - nm in a fluorometer (glomax-multi detection system, promega). the results were expressed as fluorescence arbitrary units. for rna isolation, etec strains were grown in ca-ye medium to an od nm of . . cells were harvested by centrifugation at , x g for min, and total rna was extracted by the trizol method (invitrogen). rna samples were treated with dnase i (fermentas) and tested for the absence of dna contamination by pcr. rna quality was evaluated by agarose/formaldehyde gel electrophoresis, and the yield was determined by nanodrop (thermo fisher scientific). reverse transcription was performed using . μg of rna, u of revertaid reverse transcriptase (fermentas), and ng of random hexamer primer (fermentas). quantitative pcr amplification of the resulting cdna was carried out with maxima sybr green/rox qpcr master mix (fermentas) and . μm oligonucleotides specific for each gene ( table ) . primers were designed using the primer express software (applied biosystems), and their sequences are described in d polymorphisms in the eltab promoter region at the positions − and − , according to the transcription start site, using h as reference etec table oligonucleotides used in the present study oligonucleotide sequence oligonucleotides labeled as rt were used for real-time pcr. fw and rv denote forward and reverse, respectively. the target transcripts for rt-pcr are indicated in the oligonucleotide names ** oligonucleotide used for sequencing of the region upstream of the etxa gene, corresponding to nucleotides. underlined nucleotides indicate the kpni restriction site table . the results obtained with the eltab primers were normalized using the rpoa gene as the endogenous control, which was shown to be constant in the samples analyzed. in addition, efficiency analysis of the primers was tested using different amounts of eltab-specific cdna into the real-time pcr reactions. the rpoa gene-specific primers resulted in efficient amplification according to ct-based standard curve analysis (equation = − . × + . ; r = . ). relative expression levels were calculated using the -ΔΔct method [ ] . measurement of the cell-associated and secreted lt levels among different etec strains was based on previously described protocols [ , ] . the culture samples of the reference strain h were adjusted to an od nm equal to . and allowed to grow in ca-ye medium for h until an od nm is equivalent to approximately . or overnight cultures ( h). the cell samples were adjusted to an od nm equal to or , with samples harvested after h, or to an od nm of , with samples cultivated overnight. after cell lysis, culture supernatants and debris-free extracts of the bacterial pellets were recovered and kept at °c before lt quantification (ng/ml) by elisa. alternatively, bacterial strains were cultivated for h, processed without normalizing the culture optical density, and the lt concentrations were adjusted according to the total protein content, which was determined using the bradford assay. the last protocol allowed us to normalize lt concentration both in the bacterial cell extracts and supernatants and generate lt values from cell extracts similar to those obtained with cellular density adjustments equivalent to (fig. s , supplementary material) . for routine analyses of lt expression in the tested etec strains, we applied the protocol based on the normalization of lt expression by total protein content. the total lt production for each bacterial strain was calculated according to the following formula: secreted lt + cell-associated lt (ng/ml)/total protein in the supernatant + cell-associated total protein (mg/ml), and the resulting values were expressed by ng of lt/mg of total protein. quantification of lt in the bacterial fractions was performed by capture elisa as previously described [ ] . briefly, the microtiter plates (nunc maxisorp-thermo fisher scientific, waltham, massachusetts, usa) were coated with rabbit anticholera toxin serum (titer equal to × ) diluted to : in pbs x. after overnight incubation, twofold serial dilutions of the samples were placed on the plates, and the captured lt was detected by serial exposure to mouse anti-lt serum (titer = × ) and horseradish peroxidase-conjugated goat anti-mouse igg antibodies (sigma-aldrich, st. louis, mo, usa) diluted to : and : , respectively. the reactions were read at nm in an epoch™ multi-volume spectrophotometer (biotek instruments, vermont, usa). the bradford assay was carried out to measure total protein in the bacterial samples according to the manufacturer's instructions (coomassie plus protein assay kit, thermo fisher scientific). standard curves (regression analysis with r > . ), generated with the absorbance values versus concentrations of purified lt or bovine serum albumin (pierce-thermo fisher scientific), were used to determine lt and total protein concentrations, respectively, in the samples. purified lt was obtained as previously reported [ ] . determination of the lt stability in cell cultures of different etec strains was based on a protein synthesis inhibition assay using chloramphenicol [ ] . etec strains were grown in ca-ye broth for h, and then the cultures were supplemented with chloramphenicol ( μg/ml). alternatively, the bacterial cultures were supplemented with chloramphenicol ( μg/ml) and a protease inhibitor cocktail according to the manufacturer's instructions (proteoblock™ protease inhibitor cocktail, fermentas, thermo fisher scientific). aliquots ( ml) were collected at different times ( , , , and min), and the bacterial cells were sonicated to release the lt toxin. the lt concentration was measured and normalized according to the total protein concentration. the lt stability was determined according to the following formula: (lt concentration in the cultures after - min × )/lt concentration at h. the resulting values were expressed as percentages. all data were represented as arithmetic means ± se and analyzed for two-way anova with tukey's post hoc test using graphpad prism v software (graphpad software, inc., la jolla, ca). genetic polymorphisms have been reported in the promoter and upstream regions of the lt and lt eltab operons in etec strains [ ] . we sequenced the region encompassing nucleotides upstream of the translational start codon of the elta gene of etec strains expressing different serotypes and different lt types ( , , , , and ), according to a previously reported classification based on the eltab gene sequences (table ) [ ] . according to the kyoto encyclopedia of genes and genomes (kegg) (pmid ), this bp region may also contain the promoter elements of the open reading frames (orfs) divergently transcribed to the eltab operon (fig. a) . the sequence of the etec reference strain h was identical to that derived from plasmid p and previously deposited in genbank (genbank: fn . ). altogether, seven polymorphic sites were found in the sequence upstream of the transcription start site: four were located at presumed nonregulatory dna sequences (t- c, a- g, t- c, a- g), two at the promoter region (spacer and pribnow box: g- t and c- t, respectively), and one at the up regulatory element (deletion at position − from start site of transcription) (fig. a) . two of these polymorphic sites were not previously reported (a- g and a deletion at position − ), and the a- g polymorphic site was characteristic of strains belonging to the o serogroup that express lt /lt types. in addition, no polymorphic sites were found at the . the values represent the means ± se from three independent experiments. ***p < . and **** p < . , significant differences with regard to the mock group and between values indicated by brackets (two-way anova with tukey's test) previously reported crp-binding sites [ ] . based on the determined eltab sequences, the tested strains were grouped into three clusters according to the serotypes and/or lt types ( fig. b and table ). as previously shown [ ] , our results confirmed the natural genetic polymorphism in the eltab transcriptional regulatory elements among lt -and lt producing etec strains and, for the first time, also among strains encoding lt , lt , and lt types. the impact of sequence polymorphism on the transcriptional activity of eltab genes the impact of the observed sequence polymorphisms on the transcriptional activity of the elt operon was determined after cloning a reporter gene downstream of the eltab promoter. the transcriptional fusions were constructed by combining the nucleotide changes at positions − and − (g- t and c- t): peltab(g- , c- ), from the reference strain h , peltab(h ), peltab(c- t), and peltab(g- t, c- t) (fig. ) . the recombinant e. coli strains harboring the transcriptional fusions showed similar growth patterns to the nontransformed e. coli (data not shown). the presence of peltab(h ) resulted in a two-to fourfold increase in gfp production compared to the expression observed with peltab(g- t, c- t) and peltab(c- t), respectively (fig. c) . these results indicate that the tested nucleotide changes affected the transcriptional activity of the eltab genes. to confirm these findings, we monitored the production of ltspecific mrna in different etec strains. in our experimental conditions, with the primers designed for rpoa, a housekeeping gene, and for eltb, as the target gene (fwrteltb and rvrteltb), we observed clear correlations among ct values and the amounts of cdna ( table , fig. a and b) . based on this set of primers, the levels of eltb-specific transcripts in the h strain were approximately -and -fold higher than those expressed by the tested o -or o -associated etec strains, which harbor peltab(g- t, c- t) and peltab(c- t), respectively ( figs. and c ). similar to the results generated with the operon fusion experiments, it was possible to demonstrate that the observed sequence polymorphisms in the promoter sequence affected the elt operon transcriptional regulation in all tested etec strains. additionally, etec strains sharing we also determined the action of posttranscriptional regulatory factors by measuring the amount and stability of lt produced by the different etec strains. for that purpose, we standardized the quantification using the total protein content present in both bacterial cell lysates and culture supernatants as a reference to measure the amount of cell-associated and secreted lt level (see materials and methods, fig. s , supplementary material). the total amount of lt produced by the tested etec strains ranged from to ng of toxin/ mg of total protein, which included both cell-associated and secreted toxin (fig. ) . similar to the results based on the measurement of lt-specific mrna levels, the h strain expressed the highest lt concentrations among all tested etec strains (figs. c and ) . nonetheless, the amount of lt produced by h [peltab(g- , c- )] did not correspond to the amount of transcript when compared with the o :h strains [peltab(c- t)] and o -h /h strains [peltab(g- t, c- t)]. this observation indicates that the amount of lt produced by etec strains is also regulated by posttranscriptional factors. to demonstrate the differential stability behavior of lt produced by different treatments, we measured the amount of cell-associated lt in bacterial cultures treated with a protein synthesis inhibitor. as observed in fig. , the stability of lt produced by h was significantly lower than those observed with the other tested etec strains (fig. a-c) . in addition, incubation of the cells in the presence of a protease inhibitor mixture recovered the stability of lt produced by the h strain (fig. d) . collectively, these results demonstrated that lt production by etec strains is affected both by transcriptional and posttranscriptional elements. etec strains have shown remarkable heterogeneity regarding genotypes, phenotypic and serological traits, as well as virulence factor profiles [ , , ] . etec may also be distinguished according to the expressed lt type and amount of toxin produced and secreted [ , , , ] . in the present study, we demonstrated that the natural polymorphism in the eltab promoter region may lead to different amounts of transcripts. additionally, we demonstrated that the final amount of lt produced by the etec strain may also be affected, as demonstrated with the reference strain h , by the presence of cellular proteases that reduced the stability of synthesized lt. altogether, our results demonstrate that the genetic diversity observed among the eltab operon of etec strains may impact, both at the transcriptional and posttranscriptional levels, the expression of lt. bacterial gene expression is controlled by rna polymerase and regulatory proteins that are dependent on the promoter-sigma factor interaction [ ] . we identified polymorphic sites at the eltab promoter region from different etec strains expressing the main lt phylogenetic groups. these mutations were previously shown by joffré and sjöling ( ), but it was not possible to correlate them with distinct levels of eltab gene-specific transcripts so far [ ] . here, we demonstrated differences in transcriptional activity according to the three clusters of mutations found in the promoters of the eltab operon from the etec strains using gfp as a reporter gene. the data obtained with the promoter-gfp gene fusions were correlated with the distinct patterns of specific mrna expression exhibited by the strains. the presence of cytosine in the pribnow box proved to be critical to better the performance of the promoter activity of the eltab operon since the transition of c- t diminished gfp and lt expression. supporting these results, lt producers harboring the promoter peltab (c- ) [ ] showed lt production profiles more similar to the h reference strain [peltab(c- )] fig. expression of lt by the different etec strains. total lt production was measured by quantitative capture elisa and normalized by the total protein content of each sample. the values represent the means ± se from three independent experiments. * p < . and **p < . , significant differences with regard to the h strain (two-way anova with tukey's test) than the lt -associated etec strains evaluated in the present study [peltab(c- t)]. in all allelic variants, the − region of the eltab operon remains a highly at-rich promoter, which may not interfere with promoter opening. however, cytosine instead of thymine at the − position may increase recognition by sigma factor and consequently may improve gene transcription. a similar behavior was observed when promoters that regulate genes belonging to the adaptive response to dna alkylating agents were mutated [ ] . together, these results demonstrate that genetic polymorphisms in the promoter impact the transcriptional activity of the eltab operon and contribute to the diversity of lt expression in etec. although the c- t mutation accounts for the decreased expression of eltab in the etec strain analyzed, we cannot state that this mutation reduced the affinity of the rnapolymerase holoenzyme for the − promoter element. we found two open reading frames (orfs) divergently transcribed to the eltab genes -orfs and . therefore, the promoter region of the and even could be located within the region encompassing − to + relative to the transcription start site (+ ) of eltab, which was used in the gfp gene constructions. by changing the occupancy of this region with transcriptional factors involved in expression of and , the c- t mutation could alter the accessibility of rna-polymerase to the − promoter element of the eltab operon instead of a direct effect of this mutation on the binding affinity of the enzyme for the promoter. single nucleotide polymorphisms (snps) were also observed in the up element (deletion at position − ) and region upstream from it among tested etec strains. considering that deletion of the up element did not interfere with eltab transcription according to yang and coworkers [ ] , the polymorphism in this region of the eltab operon may not be responsible for the variability in the lt-specific mrna levels among the etec strains tested in the present study. on the other hand, the four polymorphic sites upstream of the up element could contribute to the eltab-specific transcript levels. additional analyses are required to evaluate the impact of snps upstream of the promoter on the diversity of lt expression among the etec strains. reports in the literature have also demonstrated modulation of lt expression in etec by global regulators such as h-ns and crp proteins [ , , , ] . although nucleotide the values correspond to the analysis carried out at h after incubation with the inhibitors. the values represent the average ± se of the lt relative amounts (%) in comparison to those obtained before treatments for each strain. the assays were repeated three times. **p < . and *** p < . , significant differences with regard to the h strain (b-c) or as indicated by a bracket (d); ns values without statistically significant differences (twoway anova with tukey's test) substitutions at the h-ns-or crp-binding sites as well as genes encoding each regulator are rarely found in bacteria sharing close phylogenetic relationships, such mutations may affect the activities of these regulators [ ] [ ] [ ] . data obtained here and by other researchers did not detect polymorphic sites at the crp-binding sites previously described by bodero and munson ( ) [ , ] . however, we found variability in the amount of lt produced in the presence or absence of glucose among etec strains, indicating that eltab gene repression or activation by crp is a strain-specific phenomenon (data not shown). sahl and rasko ( ) also observed diversity in the effect of glucose and bile salts on the expression of lt-encoding genes upon comparing h and e a strains [ ] . together, these data demonstrate that etec strains may diverge regarding the elt operon regulation networking under different environmental stimulus. additionally, our results are in accordance with the previous observation that crp, in fact, does not bind to the eltab operon and indirectly controls lt expression. we did not observe a strict correlation between transcription levels of the eltab operon and total amount of lt produced by the tested etec strains, indicating that posttranscriptional events were also regulating lt production. one polymorphism was found in the rbs of the eltb gene from h and could contribute to compensation at the translation level of the strong transcriptional activity of the eltab operon in comparison with other tested strains. however, additional experiments are required to confirm this observation. on the other hand, we consistently detected instability of h -derived lt (lt ) under culture growth conditions, which at least partially explains why h [peltab(g- , c- )] expressed four to ten times more eltab transcripts but produced only approximately two times more lt than etec strains from serogroups o and o [peltab(c- t) and peltab(g- t, c- t), respectively]. the stability of this protein was recovered under treatment with protease inhibitors, indicating strong control of lt production by the cellular protein degradation machinery in h . this susceptibility to proteolysis does not seem to be due to lt type and may be ascribed to specificities of the proteolytic machinery of h . the proteolytic routes have been reported in the literature particularly the control of expression and activity of virulence and physiology regulators in bacteria [ , ] . however, proteolysis of lt as regulatory mechanism of expression has not been described previously. collectively, the results showed in the present study demonstrate that lt production is regulated by both transcriptional and posttranscriptional mechanisms of protein regulation, allowing the control of overexpression of lt by some etec strains. despite the natural diversity of etec, clonal relationships among some strains have been demonstrated by phenotypic and genotypic methods [ , , , ] . as such, etec strains sharing determined serotypes or serogroups have shown the same pattern of virulence factors; for example, the o :h strains are cfa-ii + lt + st + [ ] . here, we observed that strains sharing the same serogroup (o or o ) and expressing phylogenetically clustered lts (lt /lt or lt /lt , respectively) show similar patterns of lt production and the same allelic variant in the etxab promoter [peltab(c- t) or peltab(g- t, c- t), respectively] in comparison with reference etec h [peltab(g- , c- )]. in fact, such lineages sharing the same serogroup present a close genetic relationship defined by the mlee, pfge, and rapd profiles [ ] . additionally, our results together with those reported by [ ] reveal that lt -producing strains with different genetic backgrounds show distinct lt production profiles. in fact, previous reports at the global or local geographic scale have demonstrated the diversity of lt expression among etec strains, even among strains expressing the same lt type [ , , ] . collectively, these findings suggest that the determined allelic variants of the eltab genes encoding phylogenetically related lts may have been distributed by horizontal transfer to e. coli strains with different genetic backgrounds and that the production of the toxin would be impacted by specificities of the regulatory regions in the eltab operon and, particularly, by strain-specific characteristics of the bacterial cellular machinery. considering that the level of lt production and secretion may contribute to the severity of diarrhea, further studies focused on the diversity of lt expression, and the virulence of the strain shall contribute to the design of more rational preventive and therapeutic approaches to control diarrhea associated with etec infection. global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for causes of death, - : a systematic analysis for the global burden of disease study prevention and self-treatment of traveler's diarrhea aetiologyspecific estimates of the global and regional incidence and mortality of diarrhoeal diseases commonly transmitted through food estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for 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recognition by bacterial σs-rna polymerase effect of mutations in the cyclic amp receptor protein-binding site on arabad and arac expression systematic mutational analysis revealing the functional domain organization of escherichia coli nucleoid protein h-ns single nucleotide polymorphisms that cause structural changes in the cyclic amp receptor protein transcriptional regulator of the tuberculosis vaccine strain mycobacterium bovis bcg alter global gene expression without attenuating growth analysis of global transcriptional profiles of enterotoxigenic escherichia coli isolate e a regulated proteolysis in gramnegative bacteria-how and when? bacterial proteases and virulence publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -jy km fr authors: al kassaa, imad title: antiviral probiotics: a new concept in medical sciences date: - - journal: new insights on antiviral probiotics doi: . / - - - - _ sha: doc_id: cord_uid: jy km fr in recent decades, probiotics have shown beneficial effects on animal and human health. probiotics can protect the host against several health threats, including infectious diseases. before , researchers believed that the effect of probiotics was only on gut microbiota which can restore the gut flora and thus prevent pathogenic bacteria from triggering gastroenteritis. recent studies have shown that the immunomodulatory activity is the most important mechanism of action of probiotics. from this information, researchers started to evaluate the effect of some immunobiotics, not only on pathogenic bacteria but also on viruses, including enteric and respiratory viruses. several studies have confirmed the potential antiviral activity of some probiotics due to the immunomodulatory effect. these studies were conducted on humans (clinical trials) and in animal models. in this chapter, probiotics with antiviral effect against respiratory and enteric viruses will be presented and discussed, as well as their mechanisms of action. antimicrobial respiratory infections and gastroenteritis constitute the major causes of mortality and morbidity worldwide, both in developing and developed countries [ ] . despite the widespread adoption of vaccines strategies, some pathogens remain a threat to public health worldwide. the us national institutes of health (nih; bethesda, md, usa) declared the emergence of new infectious diseases, six of which have been considered reemerging infections [ ] . in the united states of america (usa), mortality caused by infectious diseases (ids) was amounted to , deaths in [ ] . an increase in immunocompromised patients plays a crucial role in the emergence and/or reemergence of ids; therefore, post-infection complications can lead to death. public health is faced with two major obstacles to eradicating ids: ( ) antibiotic therapies, which have been saving infected patient for several years. unfortunately, the rapid emergence of resistant bacteria is occurring worldwide, endangering the effi cacy of antibiotics, which have transformed medicine and saved millions of lives [ ] . ( ) the lack of antiviral agents against infectious viruses, which leads to a high treatment level between populations even in the presence of some vaccines covering a few virus types [ ] . several strategies have been developed to overcome this crisis, e.g., (i) the use of bacteriophages as antibacterial agents [ ] , (ii) the extraction and purifi cation of antimicrobial peptides [ ] , and (iii) the prevention of ids by using vaccines and/or recombinant vaccine strategies [ ] . preventing infectious diseases occurring seems to be the perfect method of avoiding id complications, since all of the abovementioned strategies have inconveniences such as side effects and stability in the host. immune system boosting is the essential key factor in id prevention. dietary balance in meals, administration of supplements such as fi ber, and probiotics are three methods to enhance and stimulate the immune system, thus protecting the mucosa against the entry of pathogens. probiotics have demonstrated their capacity to stimulate and modulate the immune system [ ] . in addition to the antibacterial activity of probiotics, some strains showed an effective antiviral activity which can be a solution to the lack of antiviral agents [ ] . in this chapter, we focus on probiotics which have been shown to be effective as antiviral agents against respiratory and enteric viruses. in addition, we give details of some clinical trials and both in vitro and in vivo experiments which have confi rmed this effi cacy. lactic acid bacteria (lab) can be found in many ecosystems, including human and animal fl ora. lab, as well as their metabolites such as bacteriocins, are generally recognized as safe (gras) [ ] . the antibacterial activity of probiotics has been confi rmed in a large number of research studies. this activity may occur through several mechanisms: ( ) pathogens exclusion: probiotic strains have a high affi nity for adhesion to epithelial cells. thus, probiotics will saturate the receptors and exert a barrier effect against the pathogens involved in infections. ( ) nutrient competition: probiotic strains can ingest many essential molecules, and consequently pathogens cannot grow in this ecosystem. ( ) production of antimicrobial compounds, such as lactic acid, hydrogen peroxide, bacteriocin-like inhibitory substances (bliss), nrps, and bacteriocins [ - ] (fig. . ). in addition to food applications, the use of lab is growing, in particular as probiotics for controlling, for example, gastroenteritis, infl ammatory pathologies of the digestive tract [ , ] and to stimulate the local and systemic immune response [ , ] . in recent decades, some probiotics have shown an antiviral activity and several mechanisms have been demonstrated. in respiratory tract infections (rtis), the majority of probiotics can inhibit the most important respiratory viruses (rvs) by immunomodulatory mechanisms [ ] (fig. . ) . this antiviral mechanism might be explained due to the entry routes of rvs. rvs infect the mucosal cells of the rt, and for this reason, probiotic strains and their antimicrobial compounds cannot directly shows the antiviral mechanisms of some probiotic strains used against viral respiratory infections. although there is a difference between the probiotic colonization ecosystem and the target rv ecosystem, several studies have showed that there is a relationship between gut microbiota and other tissues. probiotics can inhibit viruses and/or help the immune system defend itself against rvs. first, the rvs interact with the respiratory epithelium, which generates an innate immune response by activating the ifn signaling and other proinfl ammatory cytokines. once cytokines have been secreted, macrophages and nk cells will be recruited to phagocytize and kill both viruses and viral-infected cells. to trigger a specifi c immune response, the immune system needs proinfl ammatory cytokines, energy, and some cofactor elements. hence, probiotics can provide some elements to boost the immune response: a . probiotics interact with the gut epithelium and are recognized by intestinal dcs (idcs); this interaction results in the production of il- and ifnγ by idcs, which can modulate both the respiratory and gut immune response. b . secretion of ifnγ and il- by intestinal dcs; these two proinfl ammatory cytokines have dual functions: ifnγ and il- can circulate in the bloodstream to reach the respiratory epithelium and therefore help alveolar macrophages and nk cells eliminate rvs. c . the proinfl ammatory cytokines (ifnγ and il- ) secreted in the gut ecosystem after colonization of some probiotic strains help the immune system to generate a specifi c th /th immune response; the number of cd + and cd + increases and becomes more effi cient. in addition, cd + will secrete il- , which enhances the innate immune response. d . some probiotic strains, via induction of ifnγ and il- production, can stimulate the overexpression of innate immunity-related genes such as the overexpression of tlr , even in the lung. this overexpression of tlr amplifi es the innate immune responses. e . probiotics can help b lymphocytes differentiate and become plasma cells, which can secrete specifi c siga. in our case, some studies showed the impact of some probiotics in increasing siga in lung tissues. however, until now there is no explanation of the real mechanisms of how intestinal probiotics can help secretion of siga which are specifi c to elimination of rvs. this effect can be explained by the capacity of some probiotics to enhance cytokine production, which can improve the rapid differentiation of b lymphocytes to plasma cells in lung tissues. interact with viruses by physical contact. probiotic strains can fi nally reduce or eradicate virus infectivity by immunomodulatory activity, which has led scientists to call them "immunobiotics" [ ] . in this chapter, the majority of anti-rv probiotics will be mentioned with other information, such as the source of probiotic strain, target virus, experimental model, and mode of antiviral activity (table . ). lactobacillus is the most studied genus of anti-rvs probiotics, followed by the bifi dobacterium genus. it was reported that lactobacillus plantarum l- ( l. plantarum l- ) isolated from fermented foods showed proinfl ammatory activity which can decrease the titer of infl uenza virus type a (iva-h n ) in mouse lungs [ ] . another strain, l. plantarum yu, isolated from japanese fermented foods, showed anti-h n activity by activating the th immune response [ ] . recently, several studies have reported that l. plantarum species reduce the signs of infl uenza-like symptoms and even increase body weight and survival rate in a mouse model [ , , ] . in addition, mice infected by a lethal pneumovirus survived when they were protected using a combination of two lactobacilli strains, l. plantarum ncimb isolated from human saliva and l. reuteri f isolated from the human gastrointestinal tract (git) [ ] . it has not been reported that l. plantarum probiotic strains were assessed in a human experimental model, which may be due to the undesirable acid or metabolites secreted by this species. l. rhamnosus strains are the most important probiotics for human applications. furthermore, the majority of l. rhamnosus strains are immunobiotics, due to their ability to stimulate and enhance the host immune system. in animal experiments, l. rhamnosus gg (lgg), a famous probiotic strain, was evaluated and showed an anti-infl uenza virus activity on intranasal and oral administration [ , ] . in mice infected with respiratory syncytial virus (rsv), heat-killed l. rhamnosus crl and crl showed a good inhibitory effect and increased the body weight of mice [ ] . these two strains were considered good immunobiotics in rsv infection due to their ifn-α stimulation, which decreases the viral load in mouse lungs [ ] . the majority of clinical trials in children were performed using lgg [ , ] . lgg reduces the number of upper and lower viral rtis in children, reduces the days of absence from daycare and decreases antibiotic use [ ] . the administration route of lgg in clinical trials in children was often by drinking probiotic-inoculated milk [ , - ] . the antiviral activity of lgg was also assessed in adults and the elderly. several clinical trials were conducted in order to improve the benefi cial effect of this strain in the treatment and prevention of viral infections. kekkonen et al. studied marathon runners ( - years old) who drank two bottles of milk daily. the probiotic group drank a milk inoculated with lgg ( . cfus). the results showed that the ingestion of lgg did not decrease rti episodes or the severity of symptoms [ ] . however, the combination of lgg with bifi dobacterium animalis ssp. lactis bb ( b. animalis ssp. lactis bb ) decreased the duration and symptom severity of urtis signifi cantly [ ] . [ ] lgg, l. rhamnosus lactobacillus casei ( l. casei ) is a benefi cial bacterium found naturally in both the mouth and intestines of humans. l. casei may be found in "raw or fermented dairy and fresh or fermented plant products" [ ] . l. casei shirota (lcs) is the major probiotic strain among this species. this strain has been isolated from mouth fl ora [ ] . the intranasal administration of lcs in h n -infected mice showed a decrease in the viral titer in a nasal wash. moreover, lcs increases the secretion of antiviral cytokines such as interferon alpha (ifn-α). furthermore, lcs stimulates the innate immune response [ ] . lcs has shown an immunomodulatory activity against rvs. however, in clinical trials, in particular in the elderly group, lcs has shown insignifi cant results in comparison with the placebo group [ , ] . another probiotic strain, l. casei dn- , , showed good antiviral activity in clinical trials. l. casei dn- , was evaluated in children, adults, and the elderly in separate studies. in children clinical trials, l. casei dn- , decreased the symptoms and duration of rtis signifi cantly [ , ] . in the adult and elderly groups, the administration of l. casei dn- , decreased the duration of rtis and common infectious diseases (cids) [ - , ] . l. paracasei , in particular l. paracasei ssp. paracasei (lpp), was evaluated for its antimicrobial activity in animal models [ , ] . after oral administration in mice, the lpp tca and lpp tca strains, isolated from fermented camel milk, showed a signifi cant decrease in tnf-α in bronchoalveolar lavage fl uid (balf). this effect led to an increase in the mice's survival and a decrease in the macrophage and neutrophil concentrations in balf [ ] . l. fermentum is a species which can be found in human and animal fl ora [ ] . this species is usually used as a probiotic in humans. in rtis, l. fermentum was evaluated in both human clinical trials, in particular in children and adults [ , ] , and in animal models in order to investigate the mechanism of viral inhibition [ , ] . l. fermentum - and l. fermentum cjl- were assessed in h n -infected mice. the results have shown an important reduction in the viral load, with high stimulation of iga and il- secretion which allows an increase in the mice's survival [ , ] . l. fermentum cect was evaluated only in human clinical trials [ , ] . two hundred and fi fteen healthy infants ( months old) took . cfus/daily with galactooligosaccharides as prebiotics. this trial showed a signifi cant decrease in the incidence of urtis and lrtis in infants [ ] . l. fermentum vri and l. fermentum pcc are two probiotic strains which showed a signifi cant decrease in the duration of rti symptoms in healthy, physically active adults. however, these two strains did not reduce the incidence of rtis [ , ] . l. acidophilus is a famous probiotic strain used in pharmaceutical supplements [ ] . a few studies evaluated the antiviral activity of l. acidophilus , because this species is usually used for gastrointestinal problems [ , ] . all antiviral clinical trials used in humans were conducted using a combination formula with other probiotic strains, while one animal experiment was conducted using l. acidophilus l- , isolated from a healthy japanese volunteer, which showed an anti-ifv a (h n ) activity by increasing active nk cells in lungs. moreover, l. acidophilus l- showed an increase in ifn-α secretion [ ] . l. salivarius resides in the mouth and small intestine. it mainly plays a role in protection against several kinds of pathogens [ ] . sixty-six endurance athletes participated in a clinical trial; of them have taken . cfus of l. salivarius probiotic strains daily for weeks, while the control group ( n = ) took a placebo. the results showed a nonsignifi cant change in comparison with the placebo group [ ] . these results can lead to the conclusion that the infl uence of probiotics in rtis could be directly related to the species or to a new characteristic presented in a specifi c strain. l. brevis kb- (isolated from a traditional japanese pickle called "suguki"), l. gasseri tmc (isolated from the intestine of healthy adults), l. pentosus s-pt (isolated from kyoto pickles), and l. pentosus b (isolated from fermented tea leaves) are probiotic strains evaluated in mouse model experiments [ , - ] . these strains showed a strong anti-ifv activity, in particular against the h n strain. the anti-h n activity of l. brevis kb- was reported to increase ifn-α and iga secretion in mouse lungs after oral administration of this strain [ ] . the oral administration of l. gasseri tmc in intranasally h n -infected mice showed a positive effect on infl uenza symptoms. l. gasseri tmc can decrease the viral titers by interacting with the intestinal immunity system, in particular in peyer's patch, resulting in high production of il- , il- , ifn-c, and iga [ ] . kiso et al. reported that the oral administration of l. pentosus b increased protection in mice against a lethal dose of h n . the primary mechanism of this effect was by upregulation of antiviral genes such as egr (a critical regulator of host infl ammatory chemokines) and rsad (an interferon-stimulated gene (isg)) [ ] . izumo et al. reported a new antiviral activity mechanism in a mouse experimental model infected by the h n strain. they showed that the antiviral activity was created by activation of lung nk cells after intranasal administration of l. pentosus s-pt in balb/c mice. moreover, this strain can increase the production of ifn-α and iga and decrease the allergic reaction by modulating the th /th balance [ ] . bifi dobacterium is a very important bacterium in animal and human fl ora. this genus has benefi cial effects for the host, and it was used for the fi rst time as a commercial probiotic [ ] . bifi dobacteria promote good digestion, boost the immune system, and inhibit almost all intestinal pathogens [ ] . in viral rtis, the bifi dobacterial strains were used in combination in several human clinical trials to assess antiviral activity and investigate the mechanism of such activity [ , ] . the combination was conducted using lactobacilli probiotic strains [ , , ] . in a few studies, the antiviral mechanisms of bifi dobacteria were investigated in animal models, in particular mouse experiments. bifi dobacterium longum ( b. longum ) bb , isolated from japanese healthy infants, showed an anti-ifv a/pr/ / (h n ) activity after oral administration for weeks before infection. this antiviral activity was created by decreasing proinfl ammatory cytokines, such as ifnγ and il- , in balb/c mice. furthermore, this strain showed the capability to signifi cantly decrease the symptom score and body weight loss [ ] . in another balb/c mouse experiment, wu et al. reported that the administration of a bifi co® strains mixture, in particular b. longum , led to upregulation of the expression of several genes involved in antiviral responses, such as tlr , myd , irak , traf , and nf-kb [ ] . in a double-blind, placebo-controlled study, healthy newborns aged month participated; candidates were subjected to cfus/day of b. animalis ssp. lactis bb and the control group ( n = ) received control tablets as a placebo. taipale et al. reported that the b. animalis ssp. lactis bb strain reduced the number of viral rti episodes, while there was no effect on the occurrence of acute otitis [ ] . several studies investigated the combination of b. animalis ssp. lactis bb with other probiotic strains to determine the possibility of the strongest antiviral activity. the combination of l. reuteri atcc dsm (isolated from peruvian mother's milk) with b. animalis ssp. lactis bb was evaluated in a double-blind, placebocontrolled, randomized trial of healthy infants aged between and months. this combination reduced the viral rti symptoms, fever, and antibiotic consumption [ ] . rautava et al. showed that the combination of b. animalis ssp. lactis bb and the lgg strain reduced antibiotic consumptions. moreover, this combination reduced the incidence of acute otitis media in the fi rst months of life [ ] . in another double-blind randomized controlled trial, a combination of b. bifi dum with l. acidophilus probiotic strains (infl oran, bern, switzerland) was administered to healthy children aged between and years old. reduced viral rti symptoms and a decrease in the school absence rate were the main outcomes of this combination [ ] . b. animalis ssp. lactis b - reduced viral urti episodes in a clinical trial of physically active adults ( - years old) [ ] . lehtoranta et al. reported the effi cacy of a combination of lgg, l. rhamnosus lc , b. breve , and propionibacterium freudenreichii js in nasopharynx bocavirus infection, in particular in otitis-prone patients [ ] . this study was a randomized, double-blind, placebocontrolled trial on otitis-prone children (aged months to . years) with months of probiotic intervention. the authors showed the specifi c antiviral activity of this combination against bocavirus but not picornavirus. moreover, this combination seems to be effective in children, but not in the elderly [ ] . each of the abovementioned probiotic strains seems to have one or many specifi c antiviral mechanisms. furthermore, the viral specifi city is directly related to the strain used or the combination of several probiotic strains in the same or different genus types. moreover, the antiviral effect of probiotics by immunomodulatory mechanisms depends on the immune system status, which can be explained in the study conducted by lehtoranta et al., who showed that the combination of four probiotic strains worked very well in children but not in the elderly [ ] . lactobacillus and bifi dobacterium genera have the strongest antiviral activity against respiratory viruses, in particular against infl uenza virus type a. this antiviral activity depends on the strain's specifi city and the situation of the host immune system. clinical trials (cts) are the most used evaluation method in these studies. the alleviation of symptoms was measured in these cts in order to investigate the impact of suggested probiotic strains against rvs. the main mechanism of such probiotics is immunomodulation. the use of probiotics in respiratory infections leads to the activation of many signals for innate immunity and the production of iga antibodies in respiratory tissue. anti-infl ammatory probiotics in respiratory viral infections are not welcome, since they block the immune responses against the virus. however, in respiratory infl ammation, probiotics that stimulate the production of anti-infl ammatory (il- , tgfβ) cytokines play a crucial role in suppressing infl ammation. we recommend avoiding antibiotic treatment for respiratory infections except in the case of a confi rmed bacterial infection. although the antibiotic treatment may prevent respiratory bacterial superinfection, this antibiotic therapy eradicates the microbiota, in particular gram-positive probiotic strains. anti-rov probiotics should be evaluated in depth in germ-free mice and/or antibiotic-treated mice in order to determine the complete mechanism of such probiotics. furthermore, the molecules responsible for this immunomodulatory activity should be investigated and purifi ed for in vivo experiments. in , elie metchnikoff observed the healthy effect of fermented dairy products in his population. recently, researchers have confi rmed the benefi cial effects of fermented foods, in particular the role of lactic acid bacteria (lab), such as lactobacillus , bifi dobacterium and other lab, which have probiotic properties [ ] . gram-positive lactobacillus and bifi dobacterium bacteria are not occasional contaminants, but they are two genera that colonize the primary microbiota of humans. the colonization, development, and maturation of the newborn's gastrointestinal tract that begins immediately at birth and continues for years is modulated by numerous factors, including mode of delivery, feeding regime, maternal diet/weight, probiotic and prebiotic use, and antibiotic exposure pre-, peri-, and postnatally [ ] . this microbiota plays a major role in the host's defense against pathogens [ ] . this microbiota can maintain the health of the gut ecosystem and preserve defensive readiness against enteric pathogens and sometimes prevent enteric chronic diseases [ ] . microbial concentrations are distributed along the digestive tract, with bacterial cells/mm in duodenum and stomach, to bacterial cells/mm in the fasting ileus and distal ileum, and to bacterial cells/mm in the colon [ ] . unfortunately, due to an incorrect feeding regime, humans are losing the primary microbiota which is related to an increase in diseases, including infectious diseases. to restore this primary microbiota, several health organizations suggested using probiotics as a dietary supplement. recent studies have demonstrated the symbiotic relationship between intestinal microorganisms that benefi ts their human host. intestinal microorganisms, referred to as intestinal microbiota, have several mechanisms of maintaining gut health. the intestinal microbiota degrades indigestible dietary substances, such as fi ber, and converts it into an energy source for gut cells and the immune system [ , ] . the other role of intestinal microbiota is to develop the gut immune system. hooper et al. showed that germ-free infected mice have severely compromised immune responses and a reduction in the level of secretory immunoglobulin a (iga) and the number of intestinal t cells compared with wild-type mice [ , ] . in addition, intestinal microbiota can protect the host against pathogens by inducing intestinal epithelial cells to secrete antimicrobial proteins, such as angionin and c-type lectin regiiiγ [ , ] . the abo groups or "blood group antigens" in human red cells were discovered by karl landsteiner in [ ] . subsequently, this kind of antigens, called histoblood group antigens (hbgas), has been found in other tissues and biological fl uids, such as gut and saliva [ ] . some hosts lack the function of the fut gene and are called "nonsecretory hosts" [ ] . the biological role of hbgas has not yet been completely defi ned. in infectious diseases, the presence of a and b hbgas can inhibit the in vitro motility of carcinoma cells, and their absence is associated with an unfavorable prognosis [ ] . returning to infectious diseases, hbgas play a crucial role in bacterial and viral pathogenesis. specifi c strains of pathogens bind to carbohydrates of the hbg family. several studies have shown that a large number of pathogens bind hbg as the fi rst step of pathogenesis, such as uropathogenic strain of e. coli r , s. pneumoniae , s. aureus , salmonella typhimirium , and campylobacter jejuni [ - ] . the hbgas may not only provide an attachment receptor to pathogens, since they may be present on the pathogens themselves. gram-negative bacteria can present this type of antigen (in some strains may can be on the lps molecules) [ ] ; humoral immune responses can thus be generated. for example, e. coli presents b hbga; thus, an anti-b response will be evoked in a and o hbga individuals. thus, b group individuals were more susceptible to infection caused by e. coli [ ] . in this chapter, the role of hbga in viral infection will be discussed. the gut microbiota contains a large number of microbes, forming the biggest ecosystem in humans and animals [ ] . gastrointestinal infections have a great impact on public health both in developing and developed countries [ ] . viruses are the most frequent causative agent involved in gastroenteritis, in particular in infants and children [ ] . the sources of enteric viruses (envs) are usually contaminated food and water through ingestion by the orofecal route [ ] . after the occurrence of infection, treatment of the symptoms is the only way to prevent infection complications. in addition to antipyretic drugs, rehydration therapy is the most common treatment for viral gastroenteritis. the absence of specifi c antiviral agents against envs requires scientists to fi nd an alternative which can prevent or help in the treatment of such infections [ ] . the defi nition of envs is viruses that are able to replicate in the intestinal epithelium, even if only several types are the causing of gastroenteritis [ ] . noroviruses (novs), rotaviruses (rovs), arboviruses (avs), enteric adenoviruses (advs), and enteroviruses (evs) are the viruses most frequently responsible for gastrointestinal infections worldwide [ ] . reoviruses are one of the many envs that replicate in the intestinal tract, but they are generally asymptomatic. another type of enteric virus, such as poliovirus, can cause severe disease after dissemination to peripheral tissues [ ] . certain retroviruses, including mouse mammary tumor virus (mmtv), can be transmitted orally from an infected mother through her milk, after which they infect the gastrointestinal tract [ ] . does "gut microbiota" enhance or inhibit viral gastroenteritis? upon envs ingestion, viruses will "communicate" with gut microbiota resident in the intestinal lumen, which vary from one host to another. the result of this interaction seems to be dependent on the composition of the microbiota. in some hosts, good microbiota (a high number of commensal bacteria) is an inconvenience; while in other hosts, a complex microbiota (containing a large variety of microbial genera and species) is benefi cial in defending against envs and preventing viral gastroenteritis. this hypothesis is supported by several studies that have demonstrated that the intestinal microbiota is important and plays various roles in reducing infection by enteric viruses [ ] . the role of commensal bacteria in the persistence of enteric viral infections has previously been shown in a series of recent studies published in , using poliovirus, reovirus and mouse mammary tumor virus (mmtv) as env models [ - ] . the replication of poliovirus in the intestine was reduced in antibiotic-treated mice, while the reconstitution of the intestinal microbiota restored poliovirus infection [ ] . moreover, kuss et al. showed that intraperitoneal infection with poliovirus was independent of the presence or absence of intestinal microbiota, which highlights the important role of the microbiota in reducing poliovirus infection [ ] . in a recent study [ ] , showed that the use of broad-spectrum antibiotics in a mouse model infected with rotavirus decreased the infectivity of the latter. they reported that viral antigens were reduced in feces, and there was delayed shedding of viruses compared with the control mice group [ ] . in another study, murine norovirus (munov) was used to investigate the importance of intestinal microbiota in viral infectivity [ - ] . jones et al. showed that depletion of intestinal microbiota reduced the replication of munov in the distal ileum, mesenteric lymph nodes, and colon compared with the control group [ ] . the munov was reduced in feces shedding in antibiotic-treated mice compared with colonized mice as shown by kernbauer et al. [ ] . a recent study conducted by baldridge et al. showed that the use of a broad-spectrum antibiotic in mice did not help munov to establish persistent infection, while the transplantation of intestinal microbiota from another healthy mouse rescued the infectivity of this virus. moreover, they reported that systemic infection with the munov was independent of the presence or absence of gut microbiota as shown by poliovirus infection [ ] . virion stabilization and promotion of virus attachment are the two direct mechanisms by which the intestinal microbiota enhance env infections [ , ] . these fi ndings were investigated using an in vivo poliovirus model (in mice) and an in vitro model (cell culture testing). kuss et al. studied the stability of the poliovirus virion in the presence or absence of intestinal microbiota. they found that the isolation of poliovirus (before progeny virion production) from colonized mice was more viable and resistant to high temperatures and became bleach resistant. these results were also seen when the poliovirus was incubated with dead gram-negative bacteria [ ] . robinson et al. conducted an in-depth study of the mechanisms of intestinal microbiota. they found that surface compounds of gram-negative bacteria played a crucial role in poliovirus stability. the authors showed that the bacterial lps bound the viral protein (vp ) at threonine . the lps-bound virus increases the thermostability and chlorine resistance as well as decreasing the viral genome release [ ] . moreover, the pretreatment of poliovirus particles with gramnegative bacteria/or lps molecules promotes poliovirus attachment to the host cells [ ] . the poliovirus receptor (pvr) seems to be an important element in the abovementioned host cell attachment. the authors showed that poliovirus cannot attach to the permissive cells which were pretreated with anti-pvr antibodies. this result was independent of the presence of lps binding to poliovirus. moreover, non-pvr-expressing cells also showed the same results [ ] . using another viral model and after a long history of in vitro culture diffi culties, b cells seem to be permissive cells for munov [ ] . tan and jiang showed that human and munov required commensal bacteria to infect human b cells. these fi ndings were supported by the reduction of norovirus (nov) infectivity when infected stool was fi ltered with a . μm fi lter. the infectivity was rescued when live/dead commensal bacteria were added to the stool [ ] . the authors showed that lps molecules were not the bacterial compound that played the cofactor role in norovirus infection. norovirus and b cells were incubated with lps, and the results showed that the lps did not initiate the norovirus infection. moreover, the authors showed that the histo-blood group antigen (hbga) glycan was a cofactor of the norovirus infection [ ] . a recent study showed that a variety of commensal bacteria express these glycans and can bind norovirus in a virus-strain-specifi c manner [ ] . the majority of gram-negative bacteria express this kind of glycan, as shown in several recent studies [ , ] . as shown in fig. . , the gut microbiota can inhibit and/or reduce the antiviral immune response via an indirect mechanism. the gut microbiota can sometimes create a tolerogenic microenvironment that helps the virus infect cells and suppress antiviral antibody production, and sometimes it can block virus-induced ifn signaling [ ] . the gut microbiota, in particular gram-negative bacteria , induces a tolerogenic microenvironment that allows persistent enteric virus infection [ , ] . briefl y, the capacity of enteric viruses to bind gram-negative bacteria by lps-vp (viral protein) binding can skew the immune response. the story begins when the enteric virus binds the microbiota lps. the lps will be recognized by the tlr , which induces the production of il- . the b cells have il- r; when the il- binds to the il- r of the b cells, the b cells produce il- , which is an anti-infl ammatory cytokine. this action blocks the antiviral immunity response and leads to viral persistence. this information is supported by several studies using an mmtv and norovirus model in solenocyte and b-cell culturing, respectively [ , , ] . in another study, the norovirus infection occurred in germ-free mice which were also defi cient in production of il- . this study supports and confi rms that the production of il- by the presence of gut microbiota, in particular gram-negative bacteria , was the essential key to viral persistence through the creation of a tolerogenic microenvironment [ , ] . (ii) viral antibody production [ ] in the case of rotavirus infection, showed that the fecal and serum iga titer was higher in germ-free mice compared with the control mice group. this data suggests that gut microbiota suppress the antiviral humoral response. in contrast to the rotavirus case, the munov infection of antibiotic-treated mice reduced the serum igg titer after days of infection compared with the colonized mice group [ ] . these fi ndings will be investigated in depth to identify exactly which bacterial compound is responsible for this mechanism and to determine if this interaction occurred in a virus-strain-specifi c manner. several studies have reported that the ifnλ, which is considered to be in type iii of ifns, activates the same intracellular signaling pathway and many of the same biological activities as other ifn types, including antiviral activity, in a wide variety of target cells [ ] . baldridge et al. reported the importance of ifn type i, ii, and iii responses in reducing munov infection as well as viral persistence. tlr is required for bacterial regulation of viral persistence. therefore, the presence of lps/gram-negative bacteria were dispensable in this regulation [ ] . the advantage here is the capacity of probiotics to colonize the gut ecosystem, which is the target of envs: a . some probiotic strains can colonize the gut ecosystem and then form a carbohydrate biofi lm which probably saturates host iec receptors as well as viral receptors. b. probiotics protect the host iecs against damage and lesions. several studies have confi rmed that some probiotic strains play a crucial role in tissue restoration, especially by inducing mucin secretion by iecs and strengthening cell tight junctions. c. the immunomodulatory effect is the principal mechanism of antiviral probiotics (avpr). these probiotics can stimulate the secretion of proinfl ammatory cytokines, especially from dcs such as il- , il- , and ifnγ. in addition, avpr can boost innate immune cells, such as macrophages and nk cells. the latter also produce ifn-α, which is an antiviral cytokine. d. avpr help the immune system to react with more rapid specifi c responses. the th response is essential for b lymphocytes to be able to differentiate into plasma cells with specifi c siga secretion. e. avpr can inhibit or decrease viral infectivity and spreading by superproduction of mucin and by changing the morphology of villi, which can skew viral attachment. f . tlr is the ppr of viral mamps, especially for rna viruses. hence, the overexpression of tlr induced by some avpr can amplify the innate immune response by catching a large number of viral particles. g . some avpr interact physically with viral particles. indeed, several studies have showed that some probiotic strains can bind or trap viral particles on their cell wall. moreover, these trapped viruses lose some pathogenic characteristics and consequently lose cell infectivity. h . finally, avpr can play an indirect role in preventing and/or decreasing viral infection, especially against enteric viruses, by excluding the colonization of gram-negative bacteria (see fig. . ) . moreover, a recent study showed that the type iii ifn response was essential in reducing munov infectivity in the colon [ ] . briefl y, envs are recognized by a variety of tlrs, such as tlr , tlr , etc. these tlrs stimulate the ifn production by the b cells or other secretory cells. the ifns, in particular type iii ifnγ, bind to the ifn receptor present on the enterocytes, which can reduce the viral persistence. several studies have reported that commensal bacteria, in particular gram-negative bacteria recognized by tlr , bind to the envs and then the immune system will be skewed. thus, the tlr s inhibit the production of ifnγ, allowing viral persistence. this data was confi rmed using munovs as an env model [ , - ] . pott et al. reported that ifnγ also controls rotavirus infection in mice; thus, it will be interesting to determine whether this response is similarly regulated by the interactions between the enteric virus and commensal bacteria [ ] . in addition to the immunomodulatory effect of probiotics, these benefi cial bacteria have several mechanisms to defend gut pathogens and infections. in the previous part, the studies have shown that the composition of the intestinal microbiota can help envs to persist and sometimes amplifi es their infectivity. remarkably, the presence of gram-negative bacteria in the microbiota is essential to save the infectivity of envs. therefore, changing the intestinal microbiota composition seems to be effective in preventing or inhibiting enteric viral infection. otherwise, a high percentage of gram-positive bacteria may be a solution in viral gastroenteritis treatment and/or prevention. from this hypothesis, the importance of gram-positive bacteria, in particular lactic acid bacteria (labs) -which is considered to be grasin preventing and even treating this type of infection will be discussed in this part. the implantation of probiotics in the digestive tube is clearly benefi cial, since they have the ability to form a biofi lm on the enterocytes and prevent the adhesion and proliferation of other bacteria such as gram-negatives [ ] . moreover, as shown in fig. . , probiotics can exclude commensal and pathogenic bacteria by several mechanisms such as the immunomodulatory effect (immunobiotic action), reduction of ph, production of antimicrobial compounds (hydrogen peroxide, lactic acid, nrps, bacteriocins, etc.), trophic competition, and biofi lm formation (receptor competing) [ ] . anti-env probiotics (aenps) are divided into two categories according to the direct or indirect antiviral mechanisms. in this section, direct mechanisms will be discussed in detail. the probiotics with antiviral effects, called further antiviral/anti-env probiotics, can inhibit viral infections with several direct mechanisms. the antiviral compound secreted by these probiotics will be discussed in chap. . in this section, the immunomodulation and physical interaction will be presented and discussed (table . ). the microbiota diversity and composition are directly related to the incidence of gastroenteritis, including viral infection [ ] . for example, the presence of bifi dobacteria genera in the fi rst months in the gut microbiota of infants prevents the majority of intestinal infections [ ] . therefore, almost all intestinal probiotics can play a crucial role in preventing or treating viral gastroenteritis by indirect mechanisms. these probiotics reduce the "viral infection cofactor," which is the lps and hbga molecules present in gram-negative bacteria and some commensal bacteria, respectively [ ] . otherwise, orally administered probiotics can change the composition of the gut microbiota by increasing the number of probiotic cells and decreasing commensal and gram-negative bacteria . the meaning of direct mechanism is when the envs interact directly with probiotic cells and/or their metabolic compounds. as shown in fig. . , probiotics can interact and inhibit envs by several mechanisms. indeed, it is depending to the specifi city probiotic strain and viral type. before talking about the direct mechanism or direct interaction of these probiotics, the viral infection steps should be presented. in general, envs can infect target cells by fi ve steps called the viral replication cycle. the viral replication cycle starts by viral attachment to host cells ( ), followed by penetration and uncoating ( ), viroplasm formation ( ), and fi nishing with virus particle maturation ( ) and release ( ) [ ] . each env has its own specifi city in infection mechanisms and/or the replication cycle. for this reason, the following information will discuss the direct mechanism of probiotics regarding the type of env. rovs are the major cause of diarrhea and acute gastroenteritis in infants and young children [ ] . rovs are naked viruses containing dsrna. the rov virion or particle consists of three protein layers called a triple-layered particle (tlp) [ ] . the viral protein (vp) and nonstructural protein (nsp) are the two main viral proteins found in rovs. for tlp, the main protein forming the external layer is vp , with vp which forms the viral spike. vp forms the second layer of the rov particle. thus, the vp layer constitutes the double-layered particle (dlp) of the rov. kam et al. ( ) showed that, in actively transcribing dlp, the middle vp layer order decreased, while the number of cores increased. thus, the transcribed mrnas released from these cores translated later to the viral protein (vp and nsp) in host cells [ ] . the rov replication cycle starts with the attachment to the host cells mediated by vp and vp molecules which play a role in the penetration and uncoating of rov. the third step consists of the synthesis of ssrna (mrna), which is mediated by vp , vp , and vp molecules. viroplasm formation (viral protein (nsp , nsp ) and viral rna interact with each other to form cytoplasmic inclusion bodies), rna packaging, minus ssrna synthesis (rna replication), and dlp formation constitutes the fourth step. finally, rov will be released from host cells after maturation of virus particles (from dlps to tlps) [ , ] . fig. . ) in the gut ecosystem, which were considered a cofactor in some enteric virus infections. thus, proinfl ammatory probiotics (which induce a proinfl ammatory response) are welcome in viral gastroenteritis because they can trigger proinfl ammatory immunity to eliminate envs. probiotic strains capable of binding host cells very well and then creating a microenvironment which prevents many kinds of commensal and pathogenic bacteria from proliferating, including gram-negative bacteria. probiotics have a stronger capacity to adhere to host cells than gram-negative bacteria (probably because of the high hydrophobicity of their cell walls), which can decrease the number of gram-negative bacteria. probiotics can act in different ways: a. biofi lm formation: this biofi lm can protect host cells against other commensal bacteria, because this biofi lm covers the majority of host cell receptors. b. by the immunomodulatory effect, probiotics can stimulate the innate immune response, especially of phagocytes. c. at the same time, probiotics induce the secretion of antimicrobial peptides (amps) such as β-defensins and cathelicidins which target commensal bacteria. however, there is no explanation of the resistance of some probiotic strains against these amps. d. overproduction of mucin can also prevent commensal bacteria adhesion. e. the co-aggregation capacity of probiotic strains leads to the trapping of other microbes, as well as commensal or gram-negative bacteria . f. probiotics can secrete several enzymes to compete with other commensal bacteria for nutrients present in the gut ecosystem. in addition, the majority of probiotic strains possess arginine dehydrogenase, which is important in this mechanism. g. probiotic strains can secrete a variety of antimicrobial substances, such as hydrogen peroxide, lactic acid, non-ribosomal peptide synthetase (nrps), bacteriocins, and bacteriocin-like inhibitory substances (blis). several studies have shown the effectiveness of probiotics in the treatment and prevention of acute diarrhea including rov infections. human, murine, and porcine rotaviruses were used in these studies. the majority of investigations were based on the symptoms, such as duration of diarrhea, duration of hospitalization, virus shedding in feces, and sometimes immunomodulation. a few studies conducted an indepth investigation of the mechanism of action of some probiotics, in particular the interaction between virus-probiotic-host cells. lactobacillus and bifi dobacterium strains were the most studied genera in rotavirus infections. lactobacillus rhamnosus gg (lgg) is the best studied probiotic which showed a signifi cant reduction of diarrhea duration and rotavirus infectivity [ , ] . effects of various probiotic strains on rotaviruses have been conducted using double-blind placebo-controlled randomized trials since [ , , ] . guandalini et al. showed that lgg administration reduced the diarrhea duration in neonatal patients with rotavirus infection [ ] . in children, the administration of - cfus/ml of lgg twice daily for days reduced the duration of acute diarrhea from . to . days, accompanied by an increase of iga-specifi c responses [ ] . in other rcts, lgg reduced the duration of diarrhea caused by rotavirus gastroenteritis and improved the health recovery of infected children [ - ] . the l. reuteri sd strain was administered in patients aged - months with watery diarrhea caused by rotavirus. this strain showed a strongly reduction of diarrhea duration up to days [ ] . saavedra et al., shornikova et al., and sugita and togawa showed the anti-rotaviral activity in clinical trials of the following probiotic strains: streptococcus thermophilus ( s. thermophiles ), l. reuteri dsm , and l. acidophilus la [ - ] . another study showed that lgg strains and l. casei shirota (lcs) have an antiviral activity against rotaviruses and transmissible gastroenteritis virus (tgev). the lgg strain showed the strongest activity, because of their strongest attachment capability to different cell lines. in addition to the attachment effect, the induction of reactive oxygen species (ros) release seems to play a role in such activity [ ] . teran et al. conducted a randomized single-blind controlled trial (rsbct) in bolivian children aged from days to months. a -gram mix of probiotic strains was administered to the probiotic group ( n = ) for days. the mix contained the following strains: l. acidophilus , l. rhamnosus , b. longum , and saccharomyces boulardii ( s. boulardii ) . the second group ( n = ) was given nitazoxanide (an antiparasitic agent) at the dose of mg/kg. the third group ( n = ) was subjected to the normal protocol of rehydration. the results showed that the duration of diarrhea was reduced to h compared with h and h for the nitazoxanide and rehydration groups, respectively [ ] . moreover, a study conducted by grandy et al. in rdbpc trial showed the effectiveness of probiotic strains against rotavirus infections using s. boulardii alone and s. boulardii with a mixture of probiotic strains. the results showed that the two probiotic prepa-rations reduced the infection symptoms ( p = . ) [ ] . l. reuteri , called probio- , showed antiviral activity against porcine rotavirus; this activity was poorly demonstrated [ ] . l. reuteri dsm was evaluated in rcts on children with rotavirus infection, the results showed a decrease in the number of patients with acute diarrhea [ ] . simakachorn [ ] . in a murine infected model, lgg has decreased both the barrier permeability in murine intestine and epithelium vacuolation in the jejunum. furthermore, lgg was able to reduce the duration of acute diarrhea, and fi nally lgg was able to stimulate the secretion of iga [ , ] . l. casei dn- , was administered in germ-free suckling rats infected further by rotavirus. the results showed that l. casei dn- , changed the morphology of the intestinal villi and decreased intestinal cell lesions [ ] . l. reuteri dsm was also evaluated in normal mice infected by rotavirus. the results showed that l. reuteri dsm has decreased the intestinal cell lesions and consequently reduced the duration of acute diarrhea [ ] . recently, mao et al. studied the effect of lgg on the intestinal physiology, morphology and primary immune-specifi c responses of weaned piglets infected by the porcine rotavirus. this study showed that lgg administration in the weaned piglets group enhances specifi c immune responses by increasing rotavirus-specifi c iga secretion. in addition, lgg decreased the nsp (rotavirus enterotoxin) -consid-ered an intracellular receptor essential for dlp particles to interact with viroplasms and modulate intracellular ca + and rna replication [ ] -in the jejunal mucosa induced by rotavirus infection [ ] . the production of mucin and mucin and morphological improvement of the jejunal mucosa were evaluated in the presence of lgg. the results showed that lgg enhanced the production of mucin and recuperated the integrity of both the villus and the tight junction by stimulation of occlusion and other gene expression assisting the morphological jejunal defense against rotavirus [ ] . e. coli nissle (ecn) -gram-negative probiotic strain -was evaluated alone or in combination with lgg in neonatal gnotobiotic piglets. the viral shedding titer was lower using ecn in comparison with lgg, lgg+ecn, and without probiotic strains. this result was correlated with the reduction of the specifi c iga responses in the small intestine in ecn colonized piglets. the in vitro investigation using mononuclear cell culture, ecn, showed stimulation effects on the production of antiinfl ammatory cytokines such as il- and il- [ ] . these fi ndings support the hypothesis conducted by stephanie karst in which showed that gram-negative bacteria improve viral infection by various direct and indirect mechanisms [ ] . yang et al. evaluated the impact of dietary rice bran (rb) on the human rotavirus vaccine (hrov) in vaccinated gnotobiotic pigs. they found that the rb-supplemented diet enhanced the vaccination responses in gnotobiotic pigs. in addition, the levels of ifnγ production from cd + and cd + were increased in intestinal and systemic lymphoid tissues [ ] . in , the authors showed that rb plays a role as a prebiotic for some probiotic strains. the lgg+ecn colonized gnotobiotic pigs were supplemented with rb daily, followed by human rotavirus (hurov) orally challenged. the rb showed a prebiotic effect promoting the growth of lgg and ecn in the gut. moreover, rb-fed pigs had a lower mitotic index and villus width. the rb and/or probiotic strains increased immunomodulation by enhancing the secretion of ifnγ and hurov-ab [ ] . l. ruminis species have shown antiviral activity for the fi rst time against the human rotavirus wa strain. l. ruminis spm showed an anti-hurov activity which was explained by an immunomodulatory effect enhancing the type i ifns immune response [ ] . to fi nish the last investigation of the antiviral mechanism of lgg, a new experimental model was developed in order to understand the benefi cial interaction between pathogens and probiotics. an ex vivo experiment called intestinal organoid (derived from lgr + stem cells) was conducted by aoki-yoshida et al. [ ] . the lgg strains showed an increase in tlr gene expression -tlr is the essential key in innate immune responses following the recognition of rotavirus -in murine intestine both in in vivo and ex vivo experiments, without alteration of other tlr gene expressions. moreover, lgg increased the mrna levels of interferon-α (ifn-α) and a neutrophil chemokine (cxcl ). furthermore, other probiotic strains, b. bifi dum and l. paracasei , failed to increase the tlr mrna levels ex vivo [ ] . these fi ndings confi rm the hypothesis about the specifi city of probiotic strains against viruses. thus the antiviral activity occurred in a "virus-strainspecifi c manner." bifi dobacterial probiotic strains were also evaluated against rovs using in vitro and in vivo experiments. b. longum spm and spm showed antiviral activity against the hurov wa strain in an infected neonatal mouse model and caco- cells. the two bifi dobacterial strains showed an immunomodulatory effect on the type i ifns immune responses [ ] . a complete genome sequence of b. longum subsp. infantis cect was conducted in by [ ] . this strain had previously showed, in a study conducted by muñoz et al. [ ] , a direct effect on rotavirus strains in both in vitro (ma- and ht- cell culture) and in vivo (mcn mouse model) experiments. the immunomodulatory mechanism was the main effect of this strain [ ] . after complete sequencing of the b. longum subsp. infantis cect strain, they reported that there were more elements (genes) in this strain compared with the complete genome sequence of b. longum f [ ] . thus, more in-depth research must be conducted on this strain to identify the detailed mechanism of antiviral activity, and more specifi cally the anti-hrov activity. noroviruses (novs) are naked rna viruses belonging to the calicivirus family. novs are transmitted via the fecal-oral route and cause gastrointestinal disease with vomiting and acute diarrhea lasting - h [ ] . novs cause million infections each year and over , deaths, mostly in infants and the elderly [ , ] . novs need host receptors to start the infection cycle. debbink et al. reported that hbga (see sec. i-b ) is a diverse family of carbohydrates expressed in mucosal surfaces, which are the main receptors of novs, in particular for the gii. genotype considered to cause the majority of human nov infection because they can bind to a, b, and o secretors which are the majority ( %) of the population [ ] . the expression of hbgas depend on the fut gene which codes for an enzyme called fucosyltransferase. the gi. genotype (norwalk virus) cannot infect patients with a nonfunctional fut gene (called a "nonsecretory host"). however, some nov strains are capable of binding other receptors such as lewis carbohydrates [ , ] . the immune responses are very important to blockade novs infection and viral spreading. the iga genogroup-specifi c secretion is the main humoral immune response against novs [ ] . the cd + th response is essential in the cellular immune response against novs which increases ifnγ and il- production [ ] . the development of antiviral treatments and vaccines to fi ght nov infection has been hindered because of their extreme genetic diversity. recently, the uncultivable nature of novs has been resolved by using a b-cell model. thereby, the pathogenesis and replication cycle have been understood deeply in cell cultures and animal models [ ] . the prevention strategies seem to be most effective mainly in infants and the elderly. to prevent and treat hunovs, several researchers have worked on the role of probiotics in such infection. the probiotic effectiveness in nov infections was evaluated using both in vitro and in vivo experiments and clinical trials. lcs introduced in fermented milk alleviated fever in nov-infected elderly patients. the probiotic group ( n = ) showed fast recuperation compared with the control group. moreover, the acetic acid concentration in feces has increased, and thereby bifi dobacterium and lactobacillus genera became dominant [ ] . takeda et al. reported that the administration of lcs improves the natural killer (nk) cell activity by producing the il- by macrophages in response to lcs [ ] . lactococcus lactis ssp. lactis lm ( l. lactis ssp. lactis lm ) -probiotic strains -were evaluated for antiviral activity against feline calicivirus (fcv), a hunov surrogate. this strain, "bacterial cell suspension (bcs)" and its metabolites "bacterial growth medium cell-free fi ltrate (bgmf)" were added to crandell-reese feline kidney (crfk) cells line. the results showed that crfk pretreated by bcs and bgmf caused nonsignifi cant decreases in the fcv titer. the pretreatment of fcv by bcs resulted in a decreased fcv titer after h. the co-incubation of fcv and bcs in crfk cells showed % virus titer reduction ( . log tcid / . ml) [ ] . the effect of bgmf will be discussed in chap. . in order to investigate the physical interaction between probiotic cells and nov particles, rubio-del-campo et al. used a p-particles model designed from the c-terminal protruding p-domain of the nov vp capsid protein. the p-particles exhibit the same surface conformation of viruslike particles (vlps), and therefore these p-particles can bind to the hbgas. in this study, probiotic strains were tested: e. coli nissle l. lactis mg , l. acidophilus la- , l. bulgaricus atcc t, l. plantarum v, l. plantarum v adh-(an isogenic derivative of v strain with decreased adhesion capacities), l. casei atcc , l. casei bl cect , l. casei vsl# , lgg atcc , and l. rhamnosus hn . the norwalk virus (gi. ) and gii. (hunov) were used in these experiments. the results showed that the probiotic strains possessed the capacity to bind to both gi. and gii. p-particles. furthermore, l. rhamnosus , l. casei bl cect , l. casei vsl# showed the highest binding effect of both p-particles. as unexpected results, the e. coli nissle -gram-negative probiotic -showed the poorest binding capacity to gi. and gii. , although other studies showed that gram-negative bacteria can bind enteroviruses via lps molecules or hbgas [ , ] . in contrast, in ht- culture cells, e. coli nissle was more effi cient in nov p-particles blocking, resulting in low host cell binding, while the other probiotic strains showed a low inhibition effect. the low adhesion capacity of probiotic strains to host cells did not affect p-particles binding; this suggestion was confi rmed by the l. plantarum v adh-(probiotic strain with low attachment capacity) which showed high gi. p-particles binding compared with l. plantarum v (normal attachment capacity). in order to investigate the interaction between probiotic strains and nov p-particles in more depth, an exclusion assay (ht- cells incubated with bacteria followed by p-particles challenge) and displacement test (ht- cells incubated with p-particles followed by bacterial challenge) were performed. the results showed that the probiotic strains enhanced the nov p-particle attachment of monolayer surfaces. these results are not clear, since they disagree with other studies. the probable hypothesis is that the attached probiotic strains can bind to the nov p-particles on their peptidoglycans (teichoic acid), which can lead to higher p-particle retention on the ht- surfaces [ ] . a recent study has evaluated an engineered probiotic strain of l. paracasei which can produce the d scfv protein (an antiviral protein that can penetrate into host cells and hydrolyze nucleic acid molecules) against munov. the results showed that l. paracasei d scfv retained its cell-penetrating effect, and therefore the intracellular nucleic acids have been hydrolyzed. the pretreatment of raw . cells with this engineered probiotic strain prevented the cell apoptosis caused by munov infection. moreover, l. paracasei d scfv has decreased mrna expression of the viral capsid protein (vp ) [ ] . recently, b. adolescentis showed an antiviral activity against munov as a hunov surrogate. the results showed that the inhibition did not occur in the viral binding step. using vlps as model, b. adolescentis decreased the attachment of hunov gi. vlps to both caco- and ht- cells, while no effect was shown in the presence of gii. vlps [ ] . astroviruses are nonenveloped viruses with positive-sense ssrna. the astroviridae family consists of two genera, mamastrovirus (mastv) and avastrovirus (aastv), based on mammalian and avian species, respectively [ ] . astroviruses can infect a wide variety of mammalian species, such as cats [ ] , dogs [ ] , mice [ ] , sheep [ ] , and cattle [ ] . these mammals are always in direct contact with humans. hastvs are one of the most important causes of acute gastroenteritis in newborn and infant patients [ ] . cross-species transmission is frequent, in particular in poultry as avian species [ ] and between pigs, cats, and humans as mammalian species [ ] . thus, the zoonotic potential of these viruses is high, and future nonhuman-to-human transmissions are likely to occur [ ] . some authors have speculated that probiotics, which may interfere with the biological cycle of enteric viruses at many different stages, may be useful as a measure to prevent and/ or treat intestinal viral infections [ , ] . e. faecium ncimb is the fi rst probiotic strain authorized by the european union (eu) as a probiotic feed additive for animals, including piglets. e. faecium ncimb has shown an immunomodulatory effect in several studies [ ] . transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus, causes % mortality in newborn piglets after severe gastroenteritis. tgev can also infect respiratory tissues in some cases [ ] . chai et al. showed the antiviral activity of e. faecium ncimb against tgev using in vitro swine testicle (st) cell lines. they showed that this strain has a double antiviral mechanism. first, the strain can trap virus particles on its cell wall and consequently prevent infection. the second mechanism is the stimulation of eukaryotic cells that produce no, il- , and il- [ ] . in addition to gastrointestinal infections, enteroviruses can cause extraintestinal infections. via the orofecal route, coxsackievirus type a strain (ca ) and enterovirus (ev ) cause hand, foot, and mouth disease (hfmd) [ ] . this viral infection results in morbidity and mortality in several regions, including asia pacifi c and europe [ ] . hfmd can lead to neurological complications and cardiopulmonary dysfunction resulting from acute ev infection [ ] . liu et al. evaluated a bivalent vaccine against ev , which has completed the phase iii clinical trials [ , ] . since ca and ev act by the orofecal route, ang yin et al. evaluated the impact of colonization of the probiotic strain on hfmd using in vitro human skeletal muscle and colon cell lines. the authors showed that the use of l. reuteri protectis (atcc ) [ ] , decreased the viral load. moreover, this antiviral activity is dose-dependent. the authors suggested that l. reuteri protectis interacted physically with ca , ca , and ev and impaired viral entry to eukaryotic cells. this antiviral activity seems to be virus probiotic strain specifi c, since no antiviral effect was shown using coxsackievirus b strain (target virus) in the presence of another probiotic strain lcs [ ] . probiotics exhibit direct and indirect mechanisms in eradicating enteric viruses. the effectiveness of probiotics in the gut ecosystem is more relevant, since they interact with viral infections by several mechanisms, including immunomodulation, which is almost the only mechanism available for probiotics in respiratory infections. the impact of enteric viruses can be decreased by changing the microbiota composition. otherwise, hbga and lps are molecules that can be presented by gramnegative bacteria and are considered a secondary receptor for enteric viruses such as novs and rovs. for this reason, using probiotics can change the microbiota to gram-positive dominant fl ora, which blocks the gram-negative cofactor of viral infection. furthermore, the physical interaction of probiotics has been confi rmed in several studies which confi rm the capacity of some probiotic strains to trap viruses. the use of antibiotics in viral gastroenteritis is a double-edged sword. broadspectrum antibiotic therapy kills probiotic strains or inhibits their multiplication. in contrast, using anti-gram-negative antibiotics such as polymyxin b or other nonbroad-spectrum antibiotics can be a crucial factor in blocking the viral cycle. moreover, using probiotic strains with antibiotic resistance should be taken into consideration when treating viral gastroenteritis to keep probiotics live and eradicate gram-negative resident fl ora. the antibiotic resistance of commercial probiotic strains can be found in a review conducted by sharma et al. 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resistance gene-carrying plasmids from lactobacillus reuteri atcc and characterization of the resulting daughter strain, l. reuteri dsm antibiotic resistance among commercially available probiotics key: cord- -n wq rq authors: arden, k.e.; faux, c.e.; o’neill, n.t.; mcerlean, p.; nitsche, a.; lambert, s.b.; nissen, m.d.; sloots, t.p.; mackay, i.m. title: molecular characterization and distinguishing features of a novel human rhinovirus (hrv) c, hrvc-qce, detected in children with fever, cough and wheeze during date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: n wq rq background: human rhinoviruses (hrvs) are associated with more acute respiratory tract infections than any other viral group yet we know little about viral diversity, epidemiology or clinical outcome resulting from infection by strains, in particular the recently identified hrvs. objectives: to determine whether hrvc-qce was a distinct hrv-c strain, by determining its genome and prevalence, by cataloguing genomic features for strain discrimination and by observing clinical features in positive patients. study design: novel real-time rt-pcrs and retrospective chart reviews were used to investigate a well-defined population of specimen extracts to observe the prevalence and the clinical features of each hrv-qce positive case from an in- and out-patient pediatric, hospital-based population during . an objective illness severity score was determined for each hrvc-qce positive patient. results: differences in overall polyprotein and vp binding pocket residues and the predicted presence of a cis-acting replication element in b defined hrvc-qce as a novel hrv-c strain. twelve additional hrvc-qce detections ( . % prevalence) occurred among infants and toddlers ( – months) suffering mild to moderate illness, including fever and cough, who were often hospitalized. hrvc-qce was frequently detected in the absence of another virus and was the only virus detected in three ( % of hrvc-qce positives) children with asthma exacerbation and in two ( %) toddlers with febrile convulsion. conclusions: hrvc-qce is a newly identified, genetically distinct hrv strain detected in hospitalized children with a range of clinical features. hrv strains should be independently considered to ensure we do not overestimate the hrvs in asymptomatic illness. acute respiratory tract infections (artis), a major cause of human morbidity and hospitalization, are most frequently viral in origin. the human rhinoviruses (hrvs) have for over a decade been associated, more than any other individual factor, with asthma - and chronic obstructive pulmonary disease exacerbations and the majority of cold and flu-like illnesses (cflis). , it has been shown that hrvs infect both upper and lower respiratory tract tissues. , classical serotypes (hrv- to ) or "strains", the term we use to indicate a molecular equivalent, form two accepted species within the heavily populated genus enterovirus, family picornaviridae. despite the considerable economic burden of hrv infections, deliberate and comprehensive investigation of strain-specific clinical data is lacking. in we proposed that a clade of divergent sequences belonged to a novel viral subgroup, hrv-a , which we later redefined as a putative third species, hrv-c, in agreement with lau et al. to date, nine complete polyprotein sequences of hrv-c strains have been determined from australia (hrvc-qpm hrvc-ny ) , hong kong (hrvc-c , hrvc-c and hrvc-c ) and shanghai (hrvc-n and hrvc-n ). many hrv strains have distinct genetic, antigenic, immunogenic and epidemiologic profiles which are similar in nature to those of other respiratory virus species including human respiratory syncytial virus (hrsv). the prospect of variation in sensitivity to antivirals is also an important reason to investigate each novel strain comprehensively, as is the chance that infection by specific hrv species could be associated with more severe illness or particular pre-existing conditions. disappointingly, the possibility that discrete hrv strains exist as distinct viral entities is not accounted for in asymptomatic control populations because strain identification is not routinely conducted. as a consequence elevated prominence is, by default, assigned to hrvs as if to a single virus. we sought to genomically characterize hrv-qce, an apparently distinct hrv-c strain whose novel subgenomic sequences were first detected in a child with exacerbated asthma and to define distinct molecular features among the hrv-cs that are useful in discriminating strains for future studies and observe the clinical features amongst hrvc-qce positive patients. clinical specimens (n = ; three additions were made to the previously described hrvc-qpm population ) were predominantly nasopharyngeal aspirates (npa; %) from patients ( % male) aged day to years (mean . years, median . years) presenting to queensland hospitals with symptoms of arti during . extracts were stored and tested as previously described. a clinical illness severity scoring system was applied as previously described. this study was approved by the royal children's hospital and university of queensland medical research ethics committees (specific approvals # / and # / and # ). primers for genome deduction were first designed using early hrvc-qce sequences in a/ b (eu and eu ). all oligonucleotide sequences are available upon request. additional primers supplemented the process. , , to confirm our primary sequence belonged to a distinct virus and was not the result of mixed hrv template, a second round of rt-pcr and sequencing of overlapping fragments was performed on rna from the original patient specimen using newly designed hrvc-qce-specific primers. discrepant sequence results were confirmed by additional rounds of rt-pcr and sequencing. two specific diagnostic real-time rt-pcrs (rt-rtpcrs) were performed on the rotor-gene tm (corbett research) for diagnostic screening. polyprotein protease cleavage sites were sought using the net-picorna server. cis-acting replication element (cre) sequences were sought using the rnaalifold server and structures were predicted using mfold. the polyprotein encoding sequence (genbank #gq ) of hrvc-qce, a new member of the species human rhinovirus c, spanned nt; shorter than all hevs and hrvs except hrv-ny . , , it had a g + c content of % and predicted amino acids terminating in an hrv-c-specific isoleucine. the hrvc-qce polyprotein shared % average amino acid identity with hrv-a strains, % with hrv-bs and % with other hrv-cs. ten protease cleavage sites were predicted to reduce the hrvc-qce polyprotein into typical picornavirus structural (vp - ) and non-structural proteins ( a-c and a-d , , ). hrvc-qce shared a translation initiation site (mgaqvs) with most other hrvs and three motifs crucial for rna polymerase binding (ygdd, tflkr and sirwt) shared by hrvs, and hevs , but lacked two of three exposed vp motifs previously conserved among hrv polyproteins were absent in hrv-qce. phylogeny inferred from a discontinuous alignment of amino acids in vp and vp which comprise the predicted intercellular adhesion molecule (icam)- footprint in major group hrv strains, placed hrvc-qce amongst other hrv-cs on a branch with hrv- (a minor group hrv which employs a different receptor) but distinct from the known major (employing the icam- molecule as receptor) or remaining minor group hrv strains (fig. ) . key residues on the vp bc loop involved in receptor contact with the vldlr are missing in hrvc-qce and there are differences in the hi loop compared to the classical strains. antigenic site a is absent in hrvc-qce and the sequence at site b is unique to hrvc-qce. as with other hrv-c strains identified to date, the conserved loop sequence motif constituting a cre, r nnna a r nnnnnnr was identified (gcucaagcaaauca) in b of hrvc-qce in the context of an appropriate predicted rna stem-loop structure (not shown). all classical hrv-a strains and / hrv-b strains are susceptible to the antiviral compound, pleconaril. among the key contact residues lining the antiviral binding pocket, there were or hrvc-qce-specific differences compared to hrv-a or hrv-b consensus sequences, respectively (fig. ) ; differences exist between hrv-as and bs and two differences between the consensus sequence of hrv-b sensitive versus resistant strains. changes at two hrvc-qce residues historically important for identifying naturally resistant strains were identified as tyr (in resistant strains) to phe and val to thr . his , conserved in nearly all other classical hrv strains, varied in hrvc-qce and among other hrv-cs. other conserved hrv residues including ile/leu and tyr/phe/ala , differed in hrvc-qce (fig. ) . to examine whether hrvc-qce was a variant of a previously identified hrv strain, we first compared the full polyprotein against public sequence databases. the nearest matches were hrvc-c ( . % identity, identical amino acids) and hrvc-qpm ( . %, amino acids). the inferred amino acid sequences from complete d regions of eight hrvc-qce-positive extracts (variants - , , , , ; intra-strain amino acid identity, . %; genbank accession number gq and bankit numbers, , , , , , , ) were compared to those from known hrv and hev serotypes and to previously described hrvc-qpm variants (n = ; intra-strain identity of . %; fig. ). the amino acid identity between variants of the hrvc-qpm and hrvc-qce strains ( . %) was lower than among variants of the two strains (≥ . %). at least % polyprotein-length pairwise identity is also shared by % (n = ) of antigenically distinct and officially recognised hrv-a strains ( strains occur in at least one pairing in which sequence identity is ≥ %; hrv- and , hrv- a and b and hrv- and sc were not considered discrete viruses). the hrvc-qce vp ( aa) is - amino acids shorter than that of other hrv serotypes. the hrvc-qce residues aligning with the residue icam- footprint differed by one from those of the most similar strain (hrvc-c ). in vp , two of amino acids from antigenic site b, four of from the ef loop, one of from the fg loop, two of from the gh loop, three of from the hi loop, one of constituting the binding pocket and two of nt in the b cre differed from the most similar hrv-c strain. since dq = ny ). the complete polyprotein of most of these strains has not been described and for the one that has (hrvc-nat ), no additional variants were sought and only preliminary clinical studies were performed. hrvc-qce rna was detected in . % (n = ) of specimen extracts collected from january to december using a b targeted rt-rtpcr and confirmed by an assay targeting the c region. no control patients were sampled in . hrvc-qce prevalence was bimodal with the major peak in summer when . % of specimens were positive ( . % of specimens from february were positive representing . % of all hrvc-qce positives and . % of all virus detections (n = ) that month). hrvc-qce was the only hrv detected in january. the remaining . % of hrvc-qce positives were detected in winter ( . % of specimens, peaking at . % of all viral detections in june). no other viruses were detected in . % (n = ) of hrvc-qce positives (table ) leaving five extracts in which another viral sequence was identified. respiratory picornaviruses were the most frequently detected virus at . % of detections (n = ) followed by hbov ( . %, n = ), hrsv ( . %, n = ) and hmpv ( . %, n = ). no viruses were found in . % (n = ) of extracts. isolation of hrvc-qce in culture was not attempted because of the age of the clinical specimens. chart review of the hrvc-qce positive cases revealed presentation with lower respiratory tract illnesses ( / ; . %), most commonly involving wheeze and administration of bronchodilators (table ) . antibiotics were not usually given to wheezers compared to non-wheezers. eleven of the cases ( . %) were admitted to hospital and hrvc-qce was the sole detection in . % ( / ) of patients. the average severity score among single hrvc-qce detections was higher ( . ) than that for co-detections ( . ) and was higher in summer ( . ) than in winter ( . ). average scores were highest among lower respiratory tract presentations ( . ) and scores of were obtained from an upper respiratory tract illness and a cystic fibrosis patient with an hrsv co-detection. there were no adult cases, despite adults comprising . % of the screened population. most ( . %) patients were years of age or younger. we have described the detection, polyprotein coding region and distinguishing genetic features of a newly identified and evidently genetically distinct hrv-c strain. we have summarised the clinical features of a predominantly pediatric population from whom hrvc-qce variants were detected. the relatively shortened genome with truncations and sequence diversity in the d capsid region may affect receptor usage, antigenicity and sensitivity to antivirals. phe in vp is a distinctive feature of pleconaril resistant clinical hrv-b strains, and was present in the vp of hrvc-qce as was thr , which is found in both resistant and sensitive strains. the predicted hrv-c-specific cre motif in b, the terminal isoleucine, the comparatively elevated g + c content and other features we have previously defined, identified hrvc-qce as a candidate for the proposed new species hrv-c. hrvc-qce appears to be a distinct global pathogen however no consensus molecular criteria exist which define strains or provide identity thresholds below which a clinical detection may be defined as a novel strain nor above which it may best be called a variant of a previously described strain. the hrv literature notes infections which apparently have not elicited a cross-protective immune response as demonstrated by patients infected by distinct strains of the same or different species such over a defined period which share - % amino acid identity in vp . , , these biological data together with strain-determining genetic criteria permit the proposal of a practical molecular threshold, for example ≤ % amino acid identity, that could be used as a surrogate to define the biological distinctiveness of hrvs that cannot be cultured. strain-defining criteria are especially useful to determine whether it is more accurate to consider "the hrvs" as a single group or as a collection of related but mostly antigenically discrete viruses, as is the norm for hrsv or hmpv for example. applying strain-defining criteria to control populations may very likely redefine our concept of the hrvs in asymptomatic illness, since the proportion of specimens positive for any distinct hrv strain in controls will be less than that which is positive for the hrv super-group when considered as a whole. approximately half of hrvc-qce detections were from patients with lower respiratory tract symptoms and % of hrvc-qce positives were detected from hospitalized children. hrvc-qce was the sole virus detected in over half of all cases. the paucity of sequence data, strain-defining criteria and clinical investigations of the novel hrvs or of individual classical strains parallel our limited knowledge of the extent and clinical impact of hrv diversity. the most recent significant healthcare impact attributed to hrvs was the h n v influenza pandemic when hrvs were the most common viruses detected among patients qualifying, but laboratory-negative for, pandemic virus testing. similar findings were described during the sars-cov outbreak. a lack of viral sequence data weakens our differential diagnostic and overall detection capabilities which limits our ability to define epidemiological features as well as the occurrence of strain-, or species-specific clinical outcomes and potentially differentiating genetic features. due to the retrospective nature of our study, no temporally comparable control population was available for testing. this is a limitation among most other hrv 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children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup a variety of respiratory viruses found in symptomatic travellers returning from countries with ongoing spread of the new influenza a(h n )v virus strain sars surveillance during emergency public health response mega : integrated software for molecular evolutionary genetic analysis and sequence alignment we are grateful to patrick woo and susanna lau for primer and hrv strain sequences and to daniel gerlach for bioinformatics support. we thank pathology queensland central for the provision of clinical specimens.funding. this work was supported by an australian nhmrc project grant . key: cord- -ma oz o authors: ma, tianxin; xu, liwen; ren, mengting; shen, jie; han, zongxi; sun, junfeng; zhao, yan; liu, shengwang title: novel genotype of infectious bronchitis virus isolated in china date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: ma oz o recombination events are known to contribute to the emergence of novel infectious bronchitis virus (ibv) genotypes. in this study, we carried out detailed phylogenetic analysis and sequence comparisons based on complete nucleotide sequences of the ibv s gene, including strain i / and representative sequences from each genotype and lineage. the results showed that strain i / represented a novel genotype, designated as lineage within genotype vii (gvii- ). further comparative genomic analysis revealed at least two recombination sites that replaced the spike gene in a lineage within genotype i (gi- )-like virus with an as-yet-unidentified sequence, likely derived from another ibv strain, resulting a novel serotype with a lower affinity to the respiratory tract in chickens. to the best of our knowledge, this provides the first evidence for recombination leading to replacement of the complete spike gene and the emergence of a novel genotype/serotype with a lower affinity to the respiratory tract in chickens comparing to one of its parental virus ck/ch/lgx/ . these results emphasize the importance of limiting exposure to novel ibvs that may serve as a source of genetic material for emerging viruses, as well as the importance of ibv surveillance in chicken flocks. avian infectious bronchitis, caused by infectious bronchitis virus (ibv), was first described in north dakota, usa in the s (schalk and hawn, ) . it is a highly contagious viral respiratory disease that is considered as one of the most important causes of heavy economic losses to the poultry industry worldwide (cavanagh and gelb, ) . all ibv strains replicate primarily in the respiratory tract and cause respiratory diseases in birds. however, some virus strains are also able to replicate in many other epithelial surfaces, including kidneys and oviducts, and infection with these strains may thus result in nephritis, decreased egg production and quality, and significant mortality. numerous different ibv strains have been reported to date, with pathologies ranging from mild respiratory symptoms to severe kidney and oviduct diseases (cavanagh, ) . ibv is an avian coronavirus with a single-stranded, positive-sense, rna genome of approximately kb. the ′ end of the genome encodes four structural proteins, including spike glycoprotein (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, and four non-structural accessory proteins, a, b, a, and b. the ′ end of the genome encodes two polyproteins ( a and ab) that contain proteins necessary for rna replication (boursnell et al., ) . similar to other coronaviruses, genetic diversity in ibv is created by both recombination events (kottier et al., ) and by mutations, including substitutions, deletions, and insertions (shi et al., ) , that occur during replication of the viral genome. the high mutation rates are attributed to the limit of replication fidelity although it has been shown that coronaviruses possess proofreading enzyme ( ′-to- ′ exoribonuclease) which is essential for replication fidelity (denison et al., ) , while recombination events are thought to result from a unique templateswitching copy-choice mechanism during rna replication (simon-loriere and holmes, ) . the spike protein comprises about amino acids and undergoes post-translational cleavage to form s and s subunits (cavanagh, ) . the s subunit contains virus-neutralizing epitopes and carries serotype-specific determinants. furthermore, the s gene has demonstrated the highest variability in the whole viral genome (niesters et al., ) , with mutations and recombination events in the s gene considered as critically important for the emergence of new virus genotypes, serotypes, and variants. many different ibv types have been https://doi.org/ . /j.vetmic. . . received october ; received in revised form january ; accepted january found worldwide, and new variants continue to emerge in different parts of the world. a new classification based on analysis of the whole s gene has recently been proposed (valastro et al., ) , including grouping and naming lineages, comprising six genotypes (gi- -gi- , gii-gvi), and a number of inter-lineage recombinants. some of these lineages are distributed in several continents, countries, or regions, such as gi- (formerly massachusetts; mass), / , or cr ) , , gi- (ck/ch/ldl/ i (ldl/ i) or q ), gi- (italy ), and gi- (var ) (de wit et al., ; valastro et al., ) , while others are geographically confined to specific regions. among the widely distributed ibv lineages, gi- , gi- , gi- , and gi- are currently circulating in china (de wit et al., ; valastro et al., ) , while strains assigned to some of these lineages, such as gi- and gi- , were also found to originate in china. in addition, ibv variants of indigenous lineages were also found to be circulating in chicken flocks in china. new lineages, gi- , gi- , and gi- (chen et al., ; jiang et al., ) , were recently found in chickens suffering from respiratory and urinary health problems. these results indicated that novel ibv lineages have been emerging continuously in china in recent years. an epidemiological study conducted in china from january to december based on the s gene sequence isolated and characterized a new variant, designated i / , in guangxi province in (xu et al., ) . despite the fact that most heterogeneity of ibvs occurs in the s gene, sequence analysis of this gene alone was not sufficient to characterize this novel strain. we therefore carried out a detailed investigation of the s gene and the complete genomic sequence of the i / strain to clarify its genetic characteristics and gain further insights into the origin of the strain. we also investigated the antigenicity and pathogenicity of the ibv strain i / in the present study. fertile white leghorn specific pathogen-free (spf) eggs and chickens were purchased from harbin veterinary research institute, chinese academy of agricultural sciences. ethical approval to carry out this study was obtained from the ethical and animal welfare committee of heilongjiang province, china (license no. hsy-iacuc- - ). we used the virus γcov/ck/china/i / , referred to as i / , in this study. this virus was previously isolated in spf chicken eggs from a disease outbreak associated with respiratory disease in a layer flock. the virus underwent six passages in spf eggs and was tentatively classified as a variant (xu et al., ) . a further eight ibv strains (h (gi- ) (chen et al., ) , / (gi- ) , ldl/ (ldl/ ) (gi- ) (liu et al., ) , ldl/ i (gi- ) (liu et al., ) , ck/ch/lsc/ i (lsc/ i) (gi- ) (liu et al., ) , ck/ ch/lgx/ (lgx/ ) (gi- ) , i / (gi- ) , and i / (gvi- ) (xu et al., ) ), representative of different ibv lineages circulating in china in recent years, were used in cross virus-neutralization (vn) tests. viral stock was prepared by inoculating each of the viruses into -day-old spf embryonated chicken eggs by the allantoic cavity route, followed by incubation for - h and chilling at °c overnight. allantoic fluid was harvested from the inoculated eggs and clarified by low-speed centrifugation at × g for min. cleared supernatant was stored in aliquots of . ml at − °c until further processing. virus titration was performed using -day-old spf chicken eggs, as described previously (liu et al., ). the eggs were examined for ibv lesions including curling and dwarfing up to days post-inoculation. viral titers were calculated according to reed and muench ( ) and expressed as the % egg infective dose per milliliter (eid /ml). . . rna extraction, reverse transcription-polymerase chain reaction, sequencing, and sequence determination viral rna was extracted from the allantoic fluid using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's protocol. the genomic sequence was amplified by reverse transcriptionpolymerase chain reaction (rt-pcr) using a primescript™ one-step rt-pcr kit ver. (takara bio inc., shiga, japan) according to the manufacturer's instructions. the strategy and primers used for amplifying, cloning, and sequencing the complete genome of strain i / have been described previously (liu et al., ) . the primers were previously designed for ibv complete genome sequencing (liu et al., ) , and if the primers failed to work due to sequence differences, new primers were designed based on the newly determined sequences flanking those genome regions. the ′/ ′ ends of the genome were determined using a ′/ ′ race kit (takara bio inc.) according to the manufacturer's protocol. the pcr products were cloned into the pmd -t vector (takara bio inc.) according to the manufacturer's instructions. each genome fragment was sequenced three times. chromatograms were analyzed using the program chromas (http:// technelysium.com.au/wp/chromas/) and sequences were aligned using bioedit (http://www.mbio.ncsu.edu/bioedit.htm). open reading frame (orf) predictions were carried out using the orf-finder program (https://www.ncbi.nim.nih.gov/orffinder/), and orfs were compared with the beaudette strain (genbank accession number: nc_ ). the complete genomic sequence of i / has been deposited in the genbank database, with the accession number mh . the full s gene sequence generated in this study was subjected to blast searches using the national center for biotechnology information database, and then analyzed phylogenetically using a dataset consisting of sequences (supplemental table ), including representative sequences for each genotype and lineage, as recently recommended (valastro et al., ; chen et al., ; jiang et al., ) , and the gx-nn strain, which was closely related to i / by blast analysis. the s gene sequences of the ibv strain i / and the selected viruses were aligned using clustalw, and phylogenetic analysis was carried out using mega version . software (http://www. megasoftware.net/) using the maximum likelihood method with the tamura-nei substitution model and bootstrap replicates to assess the robustness of the branches. only one ibv representative was selected from each lineage based on the phylogenetic trees to calculate the percentage identities between strain i / and the representative strains at both the nucleotide and amino acid levels. recombination events and the probable parental genotypes of strain i / were analyzed by selecting and downloading the complete genomes of ibv strains (supplemental table ) from genbank (www.ncbi.nlm.nih.gov/genbank/), representing sequences of ibv strains from all continents and each lineage if possible, three turkey coronavirus (tcov) strains, including north american and european strains, and the only available strain of gfcov. the complete genomic sequences of the ibv strain i / and the other selected viruses were aligned using clustalw, and phylogenetic trees were constructed using the above-mentioned method to reduce the database size, by collapsing the sequences into clusters with different sequence identities. sequence representative(s), including the complete genome sequences of ibv strains ck/ch/gd/gz and gx-yl , which showed close sequence identity to the i / strain, were examined using simplot version . . (http://sray.med.som.jhmi.edu/scropftware/simplot/) t. ma, et al. veterinary microbiology ( ) - to identify likely recombination breakpoints (lole et al., ) .the ibv strain gx-yl was used as a query virus. the window width and step size were set to bp and bp, respectively. to confirm these recombination breakpoints, three phylogenetic trees were constructed on the basis of the results of similarity plot (simplot) analysis for the nucleotide fragments - , ( ′ untranslated region (utr) to ′ end of gene ), , - , ( ′ end of gene to ′ end of gene ), and , - , ( ′ end of gene to ′ utr), from nine ibv strains, including i / , five strains (lgx/ , ck/ch/gd/gz , γcov/ck/china/i / (i / ), gx-yl , and gx-yl ) showing close relationships with i / based on the complete genomic sequence analysis, and three mass strains (beaudette, h , and m ) as an outgroup. in addition, the nucleotide identities between i / and the eight ibv strains were calculated. finally, blastn (http://blast.ncbi.nlm.nih.gove/blast.cgi) analysis using the fragment ( , - , ) of the recombined region was conducted using the genbank database. antisera against the viruses h , / , ldl/ , ldl/ i, lsc/ i, lgx/ , and i / chen et al., ; jiang et al., ) were used in this study. antisera against strains i / and i / were produced in this study following a standard protocol . briefly, -month-old spf chickens were inoculated intratracheally with approximately eid per bird, followed by an intravenous injection of the same dose weeks later. blood samples were collected after a further weeks, and serum was harvested, pooled, and inactivated for min at °c before being used in vn tests. we investigated the antigenic relationships between i / and the other reference strains by reciprocal β vn tests, using a fixed concentration of virus and serial dilutions of serum (ducatez et al., ) . two-fold serial dilutions of each antiserum were mixed with an equal volume of virus dilution containing eid in . ml and incubated for h at °c. each serum-virus mixture was then inoculated into embryonated spf chicken eggs via the allantoic sac route. the eggs were evaluated for non-specific mortality h after inoculation and for the presence of specific lesions at week after inoculation. end points corresponded to the serum dilutions that neutralized % of the virus. end-point titers were calculated by the method of reed and muench ( ) . vn end-point titers were used to calculate the percentage of antigenic relatedness (r) according to the method of archetti and horsfall ( ) . isolates with r values between % and % were considered to be antigenically related (gelb et al., ) . forty-five -day-old spf chickens were separated in three groups of birds each and housed in negative pressure isolators with ad libitum food and water. birds in groups and were inoculated via the ocular and nasal routes with eid /per bird of strain i / and lgx/ in μl volume, respectively, while birds in group served as a negative control. oropharyngeal and cloacal swabs and blood were collected from all birds in all groups at , , , , , , and days post-inoculation. the oropharyngeal and cloacal swabs were used to recover virus using -day-old spf chicken eggs via the allantoic cavity route (liu et al., ) and the allantoic fluids were used for rt-pcr to detect the presence of challenge viruses , to measure the duration of excretion of the strains i / and lgx/ during the course of the experiment. the sera were used to detect specific antibodies against ibv using a commercial enzymelinked immunosorbent assay (idexx corporation, westbrook, me, usa) according to the manufacturer's instructions. morbidity and mortality were recorded daily. five birds from each group were selected randomly and killed humanely at days post-inoculation. trachea, lung, proventriculus, cecal tonsil, and kidney tissues were collected from these birds and used for virus titration in day-old spf chicken eggs, as described previously . trachea and kidney were considered as the two targeted organs for ibv strains. lung is the representative of respiratory tract of chickens. proventriculus and cecal tonsil were representatives of upper and lower digestive tracts which were often used for ibv isolation (alvarado et al., ; yu et al., ) . data are expressed as mean ± standard deviation. virus titers were analyzed by a student's t-test using graphpad prism for windows version (graphpad software, la jolla, ca, usa). differences were considered significant if the p value was < . (*p < . , **p < . , ***p < . ). based on blast analysis using the complete nucleotide sequence of the s gene, the i / strain was found to be closely genetically related to gx-nn , which was isolated in guangxi province in . a phylogenetic tree comparing reference strains representing the well-established genotypes and lineages, strain gx-nn and the current strain i / are illustrated in fig. . the ibv strain i / , which was isolated in guangxi province in , was closely related to the reference strain gx-nn . strains i / and gx-nn were clearly separated from other ibv reference strains, including the six well-established genotypes and lineages. homology analysis of the ibv strains from the different lineages revealed similarities between isolate i / and the established lineages of . %- . % and . %- . % at the nucleotide and deduced amino acid levels, respectively. the deduced amino acid substitutions were scattered across the s subunit of the spike proteins in i / , compared with the other lineages (data not shown). however, strains i / and gx-nn shared % and . % nucleotide and amino acid identities, respectively, with each other. alignment of the s deduced amino acid sequences identified substitutions between i / and gx-nn , of which only two were located in the hypervariable region (hvr) (supplemental fig. a) . a consensus sequence of strain i / was obtained and the fulllength genome consisted of , nucleotides excluding the ′ poly a tails. orf analysis predicted ten orfs, resulting in a typical ibv genome organization of ′utr- a- b-s- a- b- c(e)-m- a- b-n- ′utr. in addition, there was also an orf with the potential to code for a protein of amino acids between gene m and a of strain i / which was identified previously in some ibv strains (bentley et al., ) . phylogenetic analysis of complete genomes was conducted to investigate the relationships between the obtained strain i / and different avian coronaviruses downloaded from genbank. strain i / was grouped together with all the ibv strains used in this study, which had been isolated from different regions at various times but was grouped apart from coronaviruses isolated from other avian species (fig. b) . within ibv strains, i / showed close relationships with ibv strains lgx/ ( . %), ck/ch/gd/gz ( . %), i / ( . %), ck/ch/ /jt- ( . %), gx-yl ( . %), and gx-yl ( . %), most of which were isolated from south china, particularly from guangxi province. we investigated the impact of probable recombination events on the origin of strain i / by conducting an analysis using simplot software ( fig. a) . two major recombination events were observed, one in the ′ end of gene (at approximately nucleotide , ) and another in the ′ end of gene (at approximately nucleotide , ). the phylogenetic trees (fig. b ) clearly supported the simplot results, with strain i / showing identities of > . % with lgx/ , ck/ch/gd/gz , i / , ck/ch/ /jt- , gx-yl , and gx-yl for the genomic region - , , compared with an identity of < . % with the mass-type strains. similar to this genomic region, i / showed an identity of . % with lgx/ for the genomic region , - , , compared with < . % with the mass-type strains. these results suggested that the lgx/ (gi- lineage) virus, circulating in chicken flocks in china , was a potential parent of recombinant i / . in contrast, in the second phylogenetic tree, the branch (genomic region , - , ) that included the spike gene separated the strain i / from all reference strain sequences, and shared no more than % nucleotide identity with any of the reference strains, making difficult to assess the origin of the fragment , - , . blastn analysis using this fragment of i / showed that this strain was closely related to strain gx-nn ( % nucleotide identity), but shared no more than % nucleotide identity with any other ibv strain in the genbank database. overall, these data suggest that gvii- viruses emerged as a result of replacement of the spike gene in strain lgx/ -like viruses (gi- ) through recombination. the antigenic properties of i / were compared to those of reference strains by vn tests. the calculated antigenic relatedness values, r, of strain i / against homologous and heterologous strains are listed in fig. . the r values confirmed the absence of relationships between i / and h , / , ldl/ , ldl/ i, lsc/ i, lgx/ , i / , and i / (all < %). the value of r for lgx/ was slightly higher but did not reach the % threshold for antigenic relatedness between strains, suggesting that the i / strain represented a novel serotype. respiratory signs started on day in four birds post-challenge, followed by the development of symptoms in nearly all birds. the t. ma, et al. veterinary microbiology ( ) - respiratory signs varied from mild respiratory distress to severe rales. generally, the respiratory signs of chickens challenged with strain lgx/ were more severe than those of chickens challenged with i / . one bird died on day after challenge with strain i / . in contrast, three birds died on days , and after challenge with strain lgx/ . macroscopic lesions in the dead birds were mainly confined to the kidneys, which showed pale, swollen, and mottled kidney parenchyma, and slightly distended tubules and urethras with uric acid crystals. petechiae were also observed in the trachea, pharynx, and larynx in most challenged birds. the clinical signs continued up to day in one bird that showed mild respiratory distress. no clinical signs were observed in the control birds throughout the experiment. no birds seroconverted before day post-challenge, . % and % birds seroconverted on day after challenge with i / and lgx/ , respectively. nearly all birds seroconverted on day after challenge with the two virus strains. as expected, no birds in the negative control group showed an antibody response against ibv. virus recovery from oropharyngeal swabs using -day-old spf chicken eggs challenged with i / revealed that % of birds were positive on day , % were positive on day , and . % were positive on day post-challenge. in contrast, using cloacal swabs, % were positive on days and , and . % were positive on day post-challenge. no birds tested positive on day or thereafter postchallenge. comparing with those of chickens challenged with i / , prolonged virus shedding were observed from both the respiratory tract and cloaca of chickens challenged with the lgx/ strain (fig. a) . no birds in the negative control group tested positive for virus recovery at any timepoint. the titers of the challenge viruses in trachea, lung, kidney, proventriculus, and cecal tonsil tissues were determined in five challenged chickens from each of the three groups, at days post-inoculation using -day-old spf chicken embryos . infection with strain lgx/ resulted in significantly higher production of infectious viruses in trachea, lung, kidney, proventriculus, and cecal tonsil tissues than those of corresponding tissues of chickens infected with strain i / (fig. b) . comparatively, infection with strain i / resulted in lower production of infectious virus in the trachea than those in the kidneys and cecal tonsils. no infectious viruses were detected in the lungs in the five chickens challenged with strain i / . no viruses were detected in any tissues in the five control chickens. some studies have reported that genotyping based on sequencing the hvrs of the s gene or partial sequencing of the s gene is representative of the grouping based on the complete s gene (lee et al., ) ; however, others disagree with these findings (moreno et al., ; schikora et al., ) , and this discrepancy is considered to be due to the presence of recombination events involving the s hvr. in the present study, we used the complete s gene for ibv typing, as suggested previously (manswr et al., ; valastro et al., ) , and found no evidence of recombination events in the i / s gene sequence. we did identify a novel genotype consisted by ibv isolate i / and the reference strain gx-nn , which clustered together in the phylogeny and did not fall within established lineages. in agreement with this result, the two strains shared high nucleotide and amino acid identities ( % and . %), compared with low identities with representative ibv strains from different lineages. based on these results, these two ibv strains should be grouped into a novel genotype, designated as gvii- , although the designation of lineage/genotypes should be assigned to monophyletic groups of at least three viruses sampled from at least two different outbreaks (valastro et al., ) . generally, it is considered that the chance of having the same serotype is higher between strains with the same genotype (wang and huang, ) , though these data showed that this relationship was not very strong, given that a change in a small percentage of the amino acids in the s protein could result in a change of serotype (cavanagh et al., ) . the current virus cross-neutralization results showed that gvii- was not only a novel genotype, but also antigenically different from the currently tested genotype lineages. strain gx-nn was isolated in guangxi province, china, in , suggesting that the novel gvii- had been circulating in china over a -year period. however, the source of this new virus introduction into the chicken population in china remains unknown. full-length genome analysis showed that the emergence of the i / strain involved a double crossover event that replaced the spike gene by that from an as-yet-unidentified ibv strain. however, the remaining parts of the genome originated from commonly known gi- ibv strains, suggesting that the i / strain was a mosaic, and that one of its putative parents was descended from gi- lineage viruses. the antigenic differences among ibv strains have been investigated, and previous work showed that s sequences had a stronger correlation with protective relatedness than with antigenic relatedness between strains (ladman et al., ) . the as-yet-unidentified parental virus of strain i / , which shared the same spike gene with strain i / t. ma, et al. veterinary microbiology ( ) - , might thus be antigenically different from vaccine strains used in china, such as h and / ; therefore vaccination with these live vaccines might not offer complete protection against infection with the parental virus of strain i / . similarly, considering the high prevalence of the gi- lineage in south china where the novel gvii- viruses were isolated, it is reasonable to speculate that the gi- lineage may co-infect birds more frequently than other lineages, although the current commonly used vaccines cannot offer complete protection against gi- viruses . vaccination with these vaccines is therefore likely to induce non-sterilizing immunity, which may allow prolonged replication, shedding, and circulation of both the deduced parental strains of gvii- viruses. consequently, although vaccination with attenuated vaccines is carried out intensively in china, this procedure may produce an environment where co-infection with gi- and the as-yet-unidentified parental viruses can enhance the likelihood of recombination. theoretically, the short persistence and shedding, and limited numbers of i / -like viruses isolated so far in china suggest that the probability of recombination between the as-yet-unidentified parental virus and gi- viruses may not be high, similar to the hypothesis suggesting that the shorter persistence and shedding of mass-based vaccines (bijlenga et al., ) and the low percentage of m -like viruses (chen et al., ; moreno et al., ) lgx/ fig. . infection of chickens with strains lgx/ and i / . virus recovery from oropharyngeal (upper) and cloacal (lower) swabs from chickens challenged with ibv strains lgx/ and i / (a). virus recovery was performed by inoculating -day-old embryonated, specific pathogen-free eggs through the allantoic route with supernatant from the swabs. replication of strains lgx/ and i / in trachea, lung, kidney, cecal tonsil, and proventriculus in chickens (b). one-dayold spf layer chickens in groups and were inoculated via the ocular and nasal routes with × eid /per bird of strains lgx/ and i / in . ml, respectively, and trachea, lung, kidney, cecal tonsil, and proventriculus tissues were collected from five birds at days post-challenge for virus titration in eggs. data are expressed as mean ± standard deviation. virus titers were analyzed by a student's t-test using graphpad prism for windows version (graphpad software, la jolla, ca, usa). differences were considered significant if the p value was < . (*p < . , **p < . , ***p < . ). t. ma, et al. veterinary microbiology ( ) - the ibv genome undergoes mutations and recombination, and the exchange of a long region of the genome by recombination may allow viruses to rapidly explore ample areas of the sequence space, potentially leading to the emergence of novel strains with different features in terms of virulence and disease pathogenesis, antigenicity, and cell and tissue tropisms (cavanagh et al., ; kant et al., ; simon-loriere and holmes, ) . the genome of strain i / was closely related to gi- viruses, except in the spike gene. this phenomenon was similar to that of tcov, the genome of which was also closely related to ibv, except in the spike gene. replacement of the spike gene of tcov was responsible for the host shift from chickens to turkeys and a pathogenicity shift from upper-respiratory disease to enteric disease (jackwood et al., ) . even though the recombination events that replaced the spike gene did not result in a tissue tropism shift in the kidneys of chickens after challenge, the i / strain showed low affinity to the respiratory tract compared with the parental virus lgx/ . in contrast, the ability of i / to reach the cecal tonsils and subsequently to replicate in this tissue was slightly higher compared with that of respiratory tract. the replicase gene of avian coronavirus ibvs is known to be a determinant of pathogenicity (armesto et al., ) , and we were therefore unable to compare the pathogenicity of i / with one of its deduced parental virus until a source has been identified for the replicase gene because it is as-yetunidentified, though another deduced parental virus (lgx/ ) showed more severe pathogenicity than i / in spf chickens. however, we cannot conclude that this difference in pathogenicity was due to replacement of the spike gene until the source of the gvii- spike gene has been identified, and alternate explanations for the observed differences between i / and gi- spike should be considered. the frequency of successful inter-typic genetic changes may depend on the viability of the recombinant progeny able to establish in the host population, and on the increase in fitness of the recombinant offspring in the host population compared with their non-recombination parents (smits et al., ) . the recombinant i / was only detected sporadically and persisted for a short time, and thus probably represented strains that were not sufficiently competitive with respect to one of their progenitor strains, lgx/ -like viruses . however, compared with the unidentified parental virus, i / -like strains may be sufficiently competitive, and the parental virus may have been circulating at below cut-off levels for detection, due to its lower replicative capacity and lower percentage. we isolated the i / ibv strain and passaged it in spf chicken eggs . the number of passages of the virus in eggs was high (six passages), and the possibility that some genetic changes were introduced as 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comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s gene genetic diversity of avian infectious bronchitis coronavirus strains isolated s gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives altered pathogenicity, immunogenicity, tissue tropism and '- kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos characterization of a recombinant coronavirus infectious bronchitis virus with distinct s subunits of spike and nucleocapsid genes and a ' untranslated region full-length human immunodeficiency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination evaluation of full s gene sequencing of classical and variant infectious bronchitis viruses extracted from allantoic fluid and fta cards a novel variant of the infectious bronchitis virus resulting from recombination events in italy and spain the peplomer protein sequence of the m strain of coronavirus ibv and its comparison with beaudette strains a simple method of estimating fifty per cent endpoints an apparently new respiratory disease of baby chicks genetic diversity of avian infectious bronchitis virus california variants isolated between and based on the s subunit of the spike glycoprotein genetic relationships of infectious bronchitis virus isolates from mississippi broilers why do rna viruses recombine? phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks genetic diversity of avian infectious bronchitis virus in china in recent years characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens sequencing and serologic identification of s genes of infectious bronchitis viruses isolated during - in guangxi province, china the authors declare that they have no competing interests. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- -sdg zdc authors: lin, huixing; chen, lei; gao, lu; yuan, xiaomin; ma, zhe; fan, hongjie title: epidemic strain yc of porcine epidemic diarrhea virus could provide piglets against homologous challenge date: - - journal: virol j doi: . /s - - -z sha: doc_id: cord_uid: sdg zdc background: porcine epidemic diarrhea virus (pedv) is the main causative agent of porcine epidemic diarrhea (ped). since december , a large-scale outbreak of diarrhea has been observed in swine farms in china. accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent pedv variants. methods: a pedv strain, yc , was isolated from intestinal samples of suckling piglets with acute diarrhea in . the complete genomic sequence of yc and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega . software. the immune protective efficiency of yc were determined by testing pedv neutralizing antibodies in sera, the colostrum and the milk on th day after farrowing of the immunized sows. the diarrhea symptoms of piglets after challenge were also observed. results: phylogenetic analysis of the complete genomic sequence of yc and the nucleotide sequence of s gene demonstrated that the yc pedv strain was clustered with the pedv epidemic strains, with > % nucleotide identity to these pedv strains. the s gene sequence of yc shared only . % ~ . % identities with classical cv , dr and js strains, with nucleotide insertion in three sites and three nucleotide deletion in one site. the amino acid (aa) sequence of s gene of yc shared only . % ~ . % identities with classical cv , dr and js strains, with aa insertion in two sites and aa deletion in one site. in the immune protective efficiency tests, the neutralizing antibody titers in sera, the colostrum and the milk on th day after farrowing of the inactivated yc pedv strain immunized group were significantly higher than the inactivated cv immunized group and the inactivated dr immunized group (p < . ). the traditional inactivated pedv vaccines made from cv or dr could not protect piglets from yc challenge, while inactivated yc could provide piglets with % protection against yc challenge. conclusions: the results showed that, great antigenicity variation had occurred to this yc pedv strain. the yc pedv strain could provide piglets against homologous challenge. it is critical for future pathogenic and antigenic studies, as well as for the development of effective preventive and control vaccines against pedv. porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded, positive-sense rna virus that is taxonomically classified within the family coronaviridae, genus alphacoronavirus. pedv is the main causative agent of porcine epidemic diarrhea (ped), a devastating enteric disease that is characterized by watery diarrhea, vomiting, dehydration and significant mortality in piglets. approximately to % of pedv-infected piglets die within h of being infected with virulent pedv strains, resulting in tremendous economic losses to the swine industry [ , ] . since december , a large-scale outbreak of diarrhea, characterized by watery stool, dehydration, and vomiting, with to % morbidity and to % mortality in suckling piglets, has been observed in swine farms in china [ , ] . accumulated evidence indicates that this large-scale outbreak of diarrhea may be caused by highly virulent pedv variants [ , ] . in the present study, a pedv strain, yc , was isolated from intestinal samples of suckling piglets with acute diarrhea in , the evolutionary characteristics and the immune protective efficiency of yc were also determined. in the pedv isolation and propagation experiment, the partial gene of nucleocapsid protein was analyzed by rt-pcr using primers n /n , which amplified an approximately kb nucleocapsid gene fragment present in the isolated yc infected vero cells, but not in the blank control vero cells (fig. a) . the isolated yc pedv strain was detected in the cytoplasm of infected vero cells by an anti-pedv n protein polyclonal antibody. red fluorescence could be observed in the yc strain-infected vero cells (fig. b) . the isolated yc pedv strain was confirmed to be negative for other porcine enteric viruses, such as rotavirus groups a, b and c, tgev, prcv, calicivirus and porcine deltacoronavirus by rt-pcr. the growth kinetics study showed that yc replicated rapidly and efficiently in vero cells, reaching a maximum titer > tcid /ml by hpi (fig. ) . a total of , nucleotides were sequenced in the isolated yc pedv strain, including the polyprotein, s, orf , e, m, and n protein-encoding genes. the sequence of yc was submitted to genbank (accession no. ku ). the complete genomic sequence of yc and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega . . phylogenetic analysis demonstrated that the yc pedv strain was clustered with the pedv epidemic strains (g cluster, fig. a and b), with > % nucleotide identity to these strains. the complete genome nucleotide sequence shared . %~ . % identities with classical cv , dr , js , ah-m and sd-m strains (g cluster). the s gene sequence of yc shared only . %~ . % identities with the pedv g cluster, such as cv , dr and js strains, with nucleotide insertion in three sites ( bp, ~ bp, ~ bp) and three nucleotide deletion in one sites ( ~ bp). the amino acid (aa) sequence of s gene of yc shared only . %~ . % identities with classical cv , dr and js strains, with aa insertion in two sites (aa ~ , aa ) and aa deletion in one site (aa ). nucleocapsid protein specific antibodies tests showed that a antibody response was detected in all the inactivated pedv strains immunized groups days after the first immunization (fig. a) . the antibody levels of all the inactivated pedv strains immunized groups maintain high levels at all the testing time points, but the antibody titers of these three groups were not significantly different (p > . ). the results of pedv neutralizing antibodies tests showed that the neutralizing antibody levels of the inactivated pedv strains immunized groups gradually increased until days after the first immunization, and still maintain high level at days after farrowing. the neutralizing antibody titer in sera samples of the yc pedv strain immunized group was significantly higher than the other three groups (p < . , fig. b ). the neutralizing antibody titer in the colostrum and the milk on th day after farrowing of the yc pedv strain immunized group was also significantly higher than the other three groups (p < . , fig. c ). after yc challenge, piglets in group one, group two and group four showed significant acute diarrhea. weight gain was reduced, loss of appetite and mental uneasiness persisted, while piglets in group three, group five and group six showed no obvious diarrhea symptoms. two days after challenge, mortality of group one, group two and group four were %. no mortality or obvious clinical symptoms were observed in group three, group five and group six ( table ). ped can generally be controlled using a vaccine strategy. vaccination with killed or attenuated pedv vaccine has been widely carried out in china and other swine raising countries, where ped usually manifests as a mild and enzootic pattern (lower mortality) some years ago. however, severe acute diarrhea outbreaks associated with high morbidity ( - %) and mortality ( - %) were observed in suckling piglets in most areas of china since december , although most sow herds had previously been vaccinated with traditional inactivated pedv vaccines based on cv or dr . in april , ped was diagnosed in the eastern midwest region of the united states, subsequently, it spread rapidly to neighboring states by june . accumulative evidence indicates that this large-scale outbreak of diarrhea may be caused by highly virulent pedv variants [ ] [ ] [ ] . the s protein makes up the large surface projections of the virion and plays a pivotal role in determining viral-cellular fusion activity and activating the immune system [ ] [ ] [ ] . the variations in amino acid sequence likely changed the immunogenicity of the s protein and led to immunization failure of current commercial vaccines. in this study, we successfully isolated the yc pedv strain from porcine intestinal samples in dead piglets during outbreaks of acute diarrhea. the s gene nucleotides analysis of yc indicated that it was clustered with the pedv epidemic strains, with nucleotide insertion in three sites and three nucleotide deletion in one site compared to classical pedv vaccine strains cv and dr . the variations of the s gene sequence and deduced amino acid sequence of the yc strain compared to traditional pedv vaccine strains may be the reason why some swine farms had well pedv vaccine immunizations but still had sustained epidemic diarrhea which caused huge economic losses. vaccination is one of the most effective ways in preventing ped infection. immunization of sows with pedv vaccines at - days before production will provide substantial passive immunity to the newborn piglets [ ] . in this study, the nucleocapsid protein specific antibody levels of three different inactivated pedv strains immunized groups gradually increased at all the testing time points. however, the antibody titers of these three groups are not significantly different. immunization with inactivated yc could protect piglets from acute diarrhea against homologous strain challenge. since yc was clustered with the pedv epidemic strains, with > % nucleotide identity to most of these epidemic strains, the inactivated yc may protect other variant strains challenge in some extent. however, it needs further refine animal studies to assess this suppose. the results showed that, great antigenicity variation had occurred to this yc pedv strain under the selective pressure of vaccines. therefore, it is important to investigate pedv variants currently circulating in sow herds to assess their ability to allow for cross-protection against highly virulent pedv strains and prevent pedv epidemics. the results showed that, great antigenicity variation had occurred to this yc pedv strain. the yc pedv strain could provide piglets against homologous challenge. it is critical for future pathogenic and c neutralizing antibodies detection in the colostrum and the milk on th day after farrowing. * indicates the neutralizing antibody titer of the yc pedv strain immunized group was significantly higher than the other three groups (p < . ) antigenic studies, as well as for the development of effective preventive and control vaccines against pedv. in may , porcine intestinal tracts, intestinal contents and fecal samples were collected from dead piglets during outbreaks of diarrhea on a breeding farm. this farm keeps more than sows and located in yancheng city within the jiangsu province. clinical signs were characterized by acute vomiting, anorexia, and watery diarrhea, with high mortality in piglets less than days old. the diarrhea outbreaks occurred throughout the year and re-occurred at - weeks interval. virus isolation was performed as described previously [ ] . the rna of the isolated yc pedv strain cultures were extracted using the viral rna mini kit (geneaid biotech, taiwan) according to the manufacturer's instructions. the presence of pedv in the vero cell culture was confirmed by reverse transcription pcr (rt-pcr) with one pairs of primers to amplify approximately kb partial sequence of nucleocapsid protein, n : gcaaacggg tgccattatctc, n : ctagctcacgaacagccac attac. the samples of pedv in the vero cell culture were confirmed to be negative for rotavirus a, b and c, transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), caliciviruses and porcine deltacoronavirus via rt-pcr as previously described [ ] [ ] [ ] [ ] [ ] . the pedv yc strain was then identified with immunofluorescence assay (ifa). briefly, vero cells grown on a -well plate were infected with the pedv yc strain. at h post-infection, cells were washed twice with pbs and fixed with cold methanol for min at − °c. cells were then washed three times with pbst and blocked with % bovine serum albumin (bsa) at °c for h. preparations were incubated for h at °c with mouse anti-pedv nucleocapsid protein polyclonal antibody in dilution buffer ( % bsa in pbst), this mouse anti-pedv nucleocapsid protein polyclonal antibody, prepared by our laboratory, was collected from serum of icr mice immunized with purified prokaryotic expressed n protein. after three washes with pbst, cells were treated with a rhodamineconjugated goat anti-mouse igg (cwbio, china) at a : dilution with pbs for min at °c. after a final four washes with pbst, all wells were examined using fluorescence microscopy (axio observer z , zeiss, germany). after ten passages on vero cells, the one-step growth curve of yc strain in vero cells was monitored at h interval after infection. based on the sequence of the cv pedv strain (genbank: af . ), seven pairs of oligonucleotide primers ( table ) were designed to amplify the different regions of the yc genome. the pcr products were cloned into the puc vector using clonexpress entry one step cloning kit (vazyme) and sequenced by invitrogen biotechnology (shanghai, china). the ′ and ′ ends of the genome of yc were validated using the rapid amplification of cdna ends (race) cdna amplification kit (clontech, japan). all fragments were sequenced in both directions in triplicate. the complete genomic sequence of yc and the nucleotide sequence of s gene were aligned with sequences of published isolates using mega . software. phylogenetic trees were constructed using the maximum likelihood method and supported with a bootstrap test of replicates. genomic sequences of the isolated yc pedv strain were submitted to genbank under accession no. ku . all experimental protocols were approved by the laboratory animal monitoring committee of jiangsu province and performed accordingly. twelve commercial high-health sows (large white) were randomly divided into four groups (table ) after immunization, sera samples were collected from sows at -day intervals until days after immunization ( days after farrowing) for detection of nucleocapsid protein specific antibodies with commercial indirect elisa kits (biovet, canada) and pedv neutralizing antibodies using yc as indicator virus as described previously [ ] . briefly, μl of pedv strain yc ( . × tcid /ml) was added to an equal volume of the sera samples and incubated for h at °c. the mixture was then inoculated to a -well plate containing confluent vero cells. h later, the culture plate was washed with d-hank's three times, and then added μl of dmem containing % fetal bovine serum (fbs). h later, the culture plate was fixed with cold methanol for min at − °c, and incubated with mouse anti-pedv nucleocapsid protein polyclonal antibody for h at °c, and then stained with fitc-labeled rabbit antimouse igg (santa cruz biotechnology). the serum titers were determined as the reciprocal of the last serum dilution at % or greater fluorescent focus reduction in the infected cell cultures under a fluorescent microscope. the colostrum and the milk on th day after farrowing of each sow were also collected and used for the detection of pedv neutralizing antibodies. the average number of sows farrowing was , piglets in each group was coded and chosen randomly, respectively. on th day after farrowing, thirty piglets were chosen (table ), ten from group one (inactivated cv immunized group), ten from group two (inactivated dr immunized group), five from group three (inactivated yc immunized group), and five from group four (saline treated group). these piglets were divided into six groups (table ) , each group were housed in a separate room and were artificial feeding with milk. piglets in group one to group four were challenged orally with . × tcid of yc . group five were challenged orally with . × tcid of cv . group six were challenged orally with . × tcid of dr . piglets were observed daily after challenge about the diarrhea symptoms. all data were analyzed using one-way anova and values of p < . were considered statistically significant. submit your next manuscript to biomed central and we will help you at every step: pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/ / ) in -week-old weaned pigs epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy new variants of porcine epidemic diarrhea virus, china molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in gansu complete genome sequence of a chinese virulent porcine epidemic diarrhea virus strain sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (pedv) strains in china origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states complete genome sequence of porcine epidemic diarrhea virus strain usa/colorado/ from the united states emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus the gprlqpy motif located at the carboxyterminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr strain propagation of the virus of porcine epidemic diarrhea in cell culture detection and genetic diversity of porcine group a rotaviruses in historic ( ) and recent ( and ) swine fecal samples in ohio: predominance of the g p[ ] genotype in nursing piglets detection of group b and c rotaviruses by polymerase chain reaction detection and differentiation of porcine epidemic diarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex rt-pcr detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus pcr-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in us swine construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus (pcv ) in mice this study was supported by program from the jiangsu province science and technology support program (be ), the jiangsu agriculture science and technology innovation fund (cx ( ) the authors declare that they have no competing interests.authors' contributions hxl and hjf designed the study. lc and lg performed the virus isolation. hxl and zm performed the animal test. all authors read and approved the final manuscript. key: cord- -zxerb de authors: liu, xiaoli; shao, yuhao; ma, huijie; sun, chuyang; zhang, xiaonan; li, chengren; han, zongxi; yan, baolong; kong, xiangang; liu, shengwang title: comparative analysis of four massachusetts type infectious bronchitis coronavirus genomes reveals a novel massachusetts type strain and evidence of natural recombination in the genome date: - - journal: infect genet evol doi: . /j.meegid. . . sha: doc_id: cord_uid: zxerb de four massachusetts-type (mass-type) strains of infectious bronchitis coronavirus (ibv) were compared genetically with the pathogenic m and h vaccine strains using the complete genomic sequences. the results revealed that strains ck/ch/lnm/ and ck/ch/ldl/ were closely related to the h vaccine, which suggests that they might represent re-isolations of vaccine strains or variants of vaccine strains that have resulted from the accumulated point mutations after several passages in chickens. in contrast, strains ck/ch/lhlj/ vii and ck/ch/lhlj/ had a close genetic relationship with the pathogenic m strain. in addition, molecular markers have been identified that distinguish between field and vaccine (or vaccine-like) mass-type viruses, which may be able to differentiate between field and vaccine strains for diagnostic purposes. phylogenetic analysis, and pairwise comparison of full-length genomes and the nine genes, identified the occurrence of recombination events in the genome of strain ck/vh/lhlj/ vii, which suggests that this virus originated from recombination events between m - and h -like strains at the switch site located at the ′ end of the nucleocapsid (n) genes. to our knowledge, this is the first time that evidence for the evolution and natural recombination under field conditions between mass-type pathogenic and vaccinal ibv strains has been documented. these findings provide insights into the emergence and evolution of the mass-type ib coronaviruses and may help to explain the emergence of mass-type ibv in chicken flocks all over the world. in , schalk and hawn described ''an apparently new respiratory disease of chicks'' in north dakota in the united states, which was considered to be infectious bronchitis (ib) by later researchers of avian respiratory diseases (schalk and hawn, ) . currently, ib still occurs in nearly all poultry-producing countries; it is a highly contagious, acute, and economically important viral disease of chickens. the etiology of ib, which was first demonstrated by beach and schalm ( ) , is infectious bronchitis virus (ibv). ibv is grouped in the genus gammacoronavirus of the family coronaviridae in the order nidovirales (de groot et al., ) . the coronavirus genomes are the largest among the known rna viruses and are polycistronic, generating a nested set of subgenomic rnas with common and sequences (masters, ) . like those of all other coronaviruses, the two-thirds of the ibv genome consists of two large replicase open reading frames (orfs), orf a and orf b. the orf a polyprotein (pp a) can be extended with orf b-encoded sequences via a À ribosomal frameshift at a conserved slippery site (brierley et al., ) , which generates the polyprotein pp ab, comprising more than amino acids, which includes the putative rna-dependent rna polymerase (rdrp) and rna helicase (hel) activity (ziebuhr et al., ) . the pp a and pp ab of ibv are processed autocatalytically by two different viral proteases, encoded by a papain-like protease (plp) and a c-loke protease ( cl pro ) (lee et al., ; ziebuhr et al., ziebuhr et al., , . other putative domains, presumably associated with a -to- exonuclease (exon) activity, a poly(u)-specific endo-rnase (xendou) activity, and a -o-methyltransferase ( -o-mt) activity, have been predicted in pp ab (ivanov et al., ; snijder et al., ) . the end of a coronavirus genome includes the viral structural and accessory protein genes: a spike (s) glycoprotein gene; an envelope (e) protein gene; a membrane (m) glycoprotein gene; a nucleocapsid (n) phosphoprotein gene; and several orfs that encode putative non-structural accessory proteins (masters, ) . of the virus-encoded proteins, the s subunit of the s protein carries virus-neutralizing activity, determines the serotype of ibv and is responsible for viral attachment to cells. it is also a major determinant of cell tropism in culture (casais et al., ) . the accumulation of point mutations, deletions, insertions and recombination events that have been observed in multiple structural genes, especially the s gene, of ibv recovered from naturally occurring infections have been considered to contribute to the genetic diversity and evolution of ibv, and consequently, to a number of ibv serotypes (cavanagh, ) . the occurrence and emergence of multiple serotypes of the virus have complicated control by vaccination because many serotypes and variants do not confer complete cross-protection against each other (cavanagh and gelb, ) . the originally discovered massachusetts (mass) type of ibv was identified in the united states, beginning in the s (fabricant, ; johnson and marquardt, ; mondal et al., ) . mass-type strains have been isolated in europe and asia since the s and up to the present day (cavanagh and gelb, ) , together with dozens of other serotypes that have been isolated in africa, asia, india, australia, europe, and south america (cavanagh, (cavanagh, , (cavanagh, , . the first mass-type ''h'' vaccines were developed in about . they include h and h (bijlenga et al., ) , and are used very commonly and widely around the world. however, virus of this type is occasionally isolated from massachusetts-vaccinated and -unvaccinated flocks with respiratory clinical signs. some of the viruses have shown close genetic relationships with pathogenic mass-type, rather than vaccine, strains by s gene analysis. however, conclusions based on the genetic analysis of a single gene sequence, and sometimes even a partial gene sequence, require caution because the true phylogeny can only be demonstrated by analyzing complete genomic sequences. herein, we sequenced the complete genome of four ibv mass-type strains that showed s gene diversity (liu et al., ; ma et al., ; sun et al., ) , and we present evidence for in-field recombination between pathogenic and vaccinal strains. furthermore, we characterized the molecular variability of the four mass-type strains to gain insight into the emergence and evolution of these viruses. four mass-type ibv strains were used for complete genomic sequence comparison and analysis in this study. strain ck/ch/lhlj/ vii was isolated in from the kidney of a layer hen vaccinated with h and / in heilongjiang province, china (liu et al., ) . strain ck/ch/lnm/ was isolated in from the swollen proventricular tissues of a broiler vaccinated with h in neimenggu province, china . strains ck/ ch/ldl/ and ck/ch/lhlj/ , both of which were isolated in , were obtained from laying hens in dalian and heilongjiang provinces, respectively, in china; the birds were suffering from nephropathogenic lesions and respiratory signs, respectively. in addition, the diseased birds in both flocks were suffering from proventriculitis (ma et al., ) . all of the ibv strains have been associated with various ib outbreaks in recent years in china and were assigned to the mass-type strains by s sequence analysis. to avoid the possible mutation in the viral genome after serial passages in specific-pathogen-free (spf) embryonated chicken eggs, the first passage of each original virus stock was used and purified once by propagating in -to -day-old spf chicken eggs with a dose of l -fold dilutions per egg, and the presence of viral particles in the allantoic fluids of inoculated eggs was confirmed with a negative contrast electronic microscope (jem- , ex) and reverse transcriptase-polymerase chain reaction (rt-pcr) as described previously . in addition, since these viruses were isolated from chickens vaccinated with h , it is possible that mixed ibv infections are present in one chicken flock. to exclude this, nine clones of s gene of each virus obtained from three independent pcr reactions were sequenced and compared. sequences of each virus identical to the previously results were obtained (liu et al., ; ma et al., ; sun et al., ) . fertile white leghorn spf chicken eggs were obtained from the laboratory animal center, harbin veterinary research institute, the chinese academy of agricultural sciences, china. to determine the full-length genomic sequences of the four viruses, pairs of overlapping primers encompassing the entire genome were used. the primers were designed in regions that are conserved among most of the ibv strains available in the gen-bank database. the sequences and locations of the primers used in rt and pcr in this study are presented in table . viral rna was extracted from ll of infectious allantoic fluid using trizol reagents (invitrogen, grand island, usa), following the manufacturer's protocol. complementary dna (cdna) was synthesized using ll of the first strand mixture (invitrogen) containing lm of primers n (À), . mm each of dntp (takara, dalian, china) and ll of total rna. the mixture was incubated at °c for min and then quick-chilled on ice for min. the rt master mix was composed of ll  rt buffer (invitrogen), ll mm dtt, u of m-mlv reverse transcriptase (invitrogen), and u rnase inhibitors (invitrogen). this rt master mix was incubated at °c for h. the reaction was terminated by heating at °c for min then chilling on ice for min. the pcr was performed in a ll reaction containing ll first strand cdna; nmol each of downstream and upstream primers; ll of  pcr buffer (mg + plus, takara); ll of . mmol dntps; u taq polymerase (takara); and ll of water. the reaction was conducted at °c for min, and cycles of °c for min; °c for min; °c for min, and a final extension step of °c for min. a product, detectable by ethidium bromide staining, of the expected size was generated. . . the -and -ends of the genome a cdna clone representing the and ends of the genome of the four ibv strains was synthesized according to the race and race system for rapid amplification of cdna ends (takara). pcr was performed according to the instructions accompanying the kits. the sense and antisense primers used to amplifying the and -ends of the genome had been designed on the basis of the sequences obtained above that were constant in the four ibv strains, respectively. the outer and inner primers used to amplify the -end of the four ibv strains were -cagctatggcaatgcg cag- and -catctttggtgtctca/tcc- , respectively. the primer used to amplify the -end was -gaggagaggaacaatgc aca- . the dna generated by pcr amplification was cloned using a ttailed vector, pmd -t (takara), and transformed using jm competent cells (takara) according to the manufacturer's instructions. at least five clones of each fragment in each strain were sequenced and the consensus sequence was determined. the sequences were analyzed using the sequencher . sequence analysis program, and a single contiguous sequence comprising the entire ibv genome of each of the four ibv strains was constructed. the nucleotide and amino acid sequences of the entire genome of the four ibv strains were assembled, aligned, and compared with those of other reference ibv and turkey coronavirus (tcov) strains using the megalign program in dnastar (version , lasergene corp, madison, wi). the orfs were determined using the gene runner program version . (http://www.generunner.com) by comparison with those of other reference ibv and tcov strains. a total of ibv and tcov reference strains, for which entire genomic sequences were available in genbank database, were selected for phylogenetic analysis of full-length genomes. the selected avian coronavirus reference strains and their accession numbers are provided in table . phylogenetic analysis, accurate estimation and comparison of the -utr, gene , s , s , gene , m, gene , n and -utr of the four ibv strains was conducted with those of the mass-type strains selected in this study using the clustal v method of dnastar software and mega (liu et al., ) , and the alignments were edited manually and adjusted to remove mistakes. deletion, insertion and gene recombination were determined according to the results of the phylogenetic analysis and pairwise comparisons. the full genomic sequences of the four mass-type ibv strains described in this report have been deposited in the genbank database with accession numbers ck/ch/lnm/ jf , ck/ ch/lhlj/ vii jf , ck/ch/ldl/ jf and ck/ ch/lhlj/ jf . four mass-type ibv strains were subjected to genome sequencing and phylogenetic analysis in this study. the sequences of each the four strains were assembled into one contiguous sequence to represent the entire viral genomes. sequences of , , and nucleotides were obtained from strains ck/ch/ lnm/ , ck/ch/lhlj/ vii, ck/ch/ldl/ and ck/ch/ lhlj/ , respectively, excluding the polyadenylation tail at the end. the genomes of the viruses were similar overall in their coding capacity and genomic organization to those of other ibvs. the genome of each of the viruses contained two large slightly overlapping orfs in the two-thirds of the genome and multiple additional orfs in the one-third of the genome. both termini were flanked with untranslated regions (utrs). ten orfs were identified within the genome. gene contained motifs common to all coronaviruses, including ribosomal frameshifting and slippery sequences, because orf b is translated in the À frame. the typical coronavirus structural genes encoding the s, e, m and n proteins were identified following gene (fig. ) . the genome organization was determined to be as follows: -utr-gene (orf a, b)-s-gene (orfs a, b, e)-m-gene (orfs a, b)-n-utr- . the analysis of the complete genome showed that strains ck/ch/ lnm/ and ck/ch/ldl/ possessed . % and . % nucleotide identity with h , respectively. however, they shared . % and . % identity with m , respectively. phylogenetic analysis using the full-length genome and the -utr, gene , s , s , gene , m, gene , n and -utr showed that the ibv strains ck/ ch/lnm/ and ck/ch/ldl/ consistently formed the same clade with vaccine-related strains of mass-type (figs and ). the analysis of the s gene showed that strains ck/ch/lnm/ and ck/ch/ldl/ had high nucleotide identities ( . % and . %, respectively) with h , while they had . % and . % identity with m . multiple alignments revealed that there were and nucleotide mutations within the s gene between strains ck/ch/lnm/ and ck/ch/ldl/ and h ; however, there were and mutations between strains ck/ch/lnm/ and ck/ch/ldl/ and m . all these results suggest that strains ck/ch/lnm/ and ck/ch/ldl/ are closely related to the h vaccine strain. the percent nucleotide similarity between strain ck/ch/lhlj/ and h for the full-length genomes was . %; however, the percent similarity was up to . % between strain ck/ch/lhlj/ and m . in addition, in all of the nine trees constructed for the -utr, gene , s , s , gene , m, orf , n and -utr, the strain ck/ch/lhlj/ constantly fell into the same clusters as the pathogenic m strain, and both belonged to the mass-type. pairwise comparison of the s protein gene revealed that strain ck/ ch/lhlj/ had and nucleotide mutations with respect to m and h , respectively. taken together, these results demonstrate that strain ck/ch/lhlj/ exhibits a close genetic relationship to pathogenic m . pathogenic and non-pathogenic mass-type strains were clustered into different clades by phylogenetic analysis of full-length genomic sequences and the -utr, gene , s , s , gene , m, gene , n and -utr. in addition, insertions and deletions were also observed that distinguished between the genomes of pathogenic and non-pathogenic mass-type strains, as illustrated in fig. and supplementary material . in non-pathogenic strains, five deletions: of nucleotides, nucleotides, nucleotides, nucleotides, and nucleotides, respectively, were observed to be located in the nsp of gene . they were found to occur between genomic positions - , - , - , - , and - , respectively , by comparing the sequences with the homologous regions of pathogenic strains. in contrast, a -nucleotide and a nucleotide insertion were found in nsp and between the m gene and gene , respectively. additionally, a cluster of insertions was found at the -utr region in the non-pathogenic strains. these changes might not only account, at least partly, for viral fitness when the pathogenic virus has become adapted to egg embryos hewson et al., ) , but may act also as molecular markers, able to differentiate between vaccine and field strains, for diagnostic purposes. comparative sequence analysis based on full-length genomic sequences and the sequences of the -utr, gene , s , s , gene , m, gene and n showed that strain ck/ch/lhlj/ vii clustered with pathogenic mass-type strains. the exceptions were the trees constructed using the -utr and s gene, in which ck/ch/lhlj/ vii was grouped with non-pathogenic strains; this suggests that a possible recombination event may have occurred. thus the n and -utr of ck/ch/lhlj/ vii were carefully compared pairwise with those of strains ck/ch/lnm/ , ck/ch/ldl/ , ck/ch/ lnm/ , h and m . parallel to the result of the phylogenetic analysis, ck/ch/lhlj/ vii showed high similarity with m at the -end of the n gene; however, it showed high similarity with vaccine strain h at the -end of the n gene (supplementary material ). the data strongly suggest that ck/ch/lhlj/ vii arose from a homologous rna recombinant event that involved a template switch between massachusetts pathogenic m -like and non-pathogenic h -like strains. we located the switch site at the -end of the n gene (supplementary material ), which implies that the template switch occurred within the n gene. the percent nucleotide similarity between strain ck/ch/lhlj/ vii and h , and ck/ch/lhlj/ vii and m , for the full-length genomes was . % and . %, respectively. alignment revealed that a -nucleotide insertion was located in nsp of the ck/ch/ lhlj/ vii strain between genomic positions and (supplementary material ). in addition, the s gene of strain ck/ ch/lhlj/ vii showed extensive mutations by pairwise comparison (supplementary material ) though it was grouped with h by s gene phylogenic analysis. these and our previous results (liu et al., ) showed that, with the exception of the occurrence of recombination events, ck/ch/lhlj/ vii has experienced multiple mutations and deletions in the genome over time. understanding the evolution of mass-type ibv is important because not only is this virus circulating worldwide but information on virus genomics will aid our understanding of the evolution and emergence of ibv with infectious potential in vaccinated chicken flocks. in this study, we focused on the full-length genomic sequences of four ibv isolates which had been shown to be of the mass-type by s gene analysis (liu et al., ; ma et al., ; sun et al., ) . based on the high degree of similarity in the full genomic sequence, it could be concluded that two ibv strains, ck/ ch/lnm/ and ck/ch/ldl/ , were very similar to the vaccine strain h . they might therefore represent re-isolations of vaccine strains, although they were isolated from vaccinated chickens with respiratory disease. similarly, ibv strains that showed a very close relationship to the h vaccine strain have been isolated from unvaccinated broiler flocks in slovenia with respiratory problems (krapež et al., ) . alternatively, these strains might be variants of vaccine strains that have resulted from accumulated point mutations after several passages in chickens. a few key mutations in the s subunit of the spike protein might result in a change to a new serotype, which is defined as a lack of cross-neutralization with specific sera against different ibv serotypes (cavanagh et al., ) . the point mutations found in the genome that distinguish between the two isolates and vaccine strain h might be the result of adaptive evolution driven by the host immune response when the vaccine strain was transmitted among chickens. adaptive evolution is the process by which genetic changes in the viral genome leading to a more fit virus population become fixed over time, and it has been reported to occur in many coronaviruses (hasoksuz et al., ; lee and jackwood, ; shi et al., ; tang et al., ; zhang et al., ) . as shown in this study, and as also occurs in other countries (dolz et al., ; rimondi et al., ; roussan et al., ) , the isolation of mass-type ibv is expected, because attenuated vaccine strains are used extensively in chicken flocks in china. however, vaccination is not likely to be the only explanation for the circulation of mass-type virus, because ck/ch/lhlj/ and ck/ch/ lhlj/ vii were most closely related to a massachusetts pathogenic type strain, m . the isolation of a massachusetts pathogenic strain from h -vaccinated chicken flocks may be due to vaccination failure in these flocks . alternatively, molecular studies have shown that only a few changes in the amino acid composition of the s spike protein can result in immune failure, even when the majority of the virus genome remains unchanged (cavanagh et al., ) . our findings showed that mutations had occurred in the genomes of both ck/ch/lhlj/ and ck/ch/ lhlj/ vii, especially in the s genes of ck/ch/lhlj/ vii though it was in the same group with h strain in the phylogenetic analysis, implicating that strain ck/ch/lhlj/ vii has experienced evolution over time. it has been reported that amino acid changes may result from immunological pressure caused by the widespread use of vaccines (cavanagh et al., . the occurrence of recombination events is another process that allows new strains to emerge, and this has been well documented in ibv (hughes, ; jia et al., ; kottier et al., ; kusters et al., ; wang et al., ) and other coronaviruses baric, , ; makino et al., ) . it is believed that the conditions for recombination amongst ibv strains in the field are as follows: an extremely large number of chickens, most maintained at high density; the ease of spread of the virus; and serotype cocirculation, including proof of co-infection with more than one serotype in a given flock (cavanagh, ) . in china, intensive chicken farms are concentrated in many provinces, including heilongjiang, where ck/ch/lhlj/ vii was isolated (liu et al., ) . almost all the chickens in china receive mass-type vaccines at a very young age and subsequently receive this vaccine a couple more times during the rest of their life span. therefore the vaccine virus exists constantly in chickens; it may persist in various internal organs for days or longer (cavanagh and gelb, ) . generally, vaccination using the h vaccine provides full protection against pathogenic mass-type pathogenic ibvs and prevents the same type of pathogenic strain from being replicated and spreading in the flocks. however, a single amino acid substitution at position of the s subunit of the spike has resulted in escape mutants of mass (cavanagh et al., ) . this may have occurred in the case of the ck/ch/lhlj/ vii s gene (liu et al., ) , which might have made it possible for both pathogenic and vaccine strains to co-exist in a given flock, leading to the occurrence of recombination. similarly, an escape mutant could be a result of adaptive evolution driven by the host immune response. consequently, it is likely that genetic changes due to adaptive evolution and recombination both contributed to the origin and evolution of strain ck/ch/lhlj/ vii: it is possible that adaptive evolution created a mutant, followed by recombination between mass -and h -like strains to create a novel virus. the recombination events from which the ck/ch/lhlj/ vii virus resulted can be explained by a scenario in which the recombination may have involved two parental viral strains, with initiation of rna replication in a m -like template of either negative or positive polarity (liao and lai, ) . this would be followed by switching of the polymerase-nascent crna complex to an h like virus template. the switch may have occurred at the -end of the n gene. in general, for a recombinant virus to emerge and establish itself in the field, it must be viable and have selective advantages. it has been reported that uptake of canine coronavirus (ccv) sequences by type ii feline coronavirus (fcov) may have led to increased viral fitness when compared with type i fcov (herrewegh et al., ) . recombination can also result in the emergence of new strains with distinct characteristics, such as pathogenicity and tissue tropism (worobey and holmes, ) . in addition, in the cov genome, as with most rna viruses, the -utrs usually harbor important structural elements that are involved in replication and/or translation (chang et al., ; raman et al., ; raman and brian, ; goebel et al., ; züst et al., ) . in ibv, the -utr-binds to the n protein, which is essential for the synthesis of negative-strain viral rna. perhaps the acquisition of the end of the n gene and the -utr from h -like virus by an m -like virus (e.g. ck/ch/lhlj/ vii) can alter the efficiency of viral replication. this alteration may in turn affect pathogenicity. however, it remains unknown whether this is the true origin of ck/ ch/lhlj/ vii, and therefore this strain is of particular importance to the surveillance of 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pseudoknot, replicase gene products, and the extreme end of the mouse coronavirus genome supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.meegid. . . . key: cord- -bmdpbkni authors: tabares, paula; pimentel-elardo, sheila m.; schirmeister, tanja; hünig, thomas; hentschel, ute title: anti-protease and immunomodulatory activities of bacteria associated with caribbean sponges date: - - journal: mar biotechnol (ny) doi: . /s - - - sha: doc_id: cord_uid: bmdpbkni marine sponges and their associated bacteria have been proven to be a rich source of novel secondary metabolites with therapeutic usefulness in cancer, infection, and autoimmunity. in this study, strains belonging to genera of the order actinomycetales and seven strains belonging to two genera of the order sphingomonadales were cultivated from different caribbean sponges and identified by s rrna gene sequencing. seven of these strains are likely to represent novel species. crude extracts from selected strains were found to exhibit protease inhibition against cathepsins b and l, rhodesain, and falcipain- as well as immunomodulatory activities such as induction of cytokine release by human peripheral blood mononuclear cells. these results highlight the significance of marine sponge-associated bacteria to produce bioactive secondary metabolites with therapeutic potential in the treatment of infectious diseases and disorders of the immune system. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. infection, cancer, and autoimmunity pose serious problems to mankind (who ) , and thus, there is a clear and continued need for new therapeutic agents against human diseases (caspi ) . natural product discovery programs have yielded an exhaustive supply of biologically active secondary metabolites from terrestrial plants and microorganisms (challis ) . in the last four decades, drug discovery efforts have shifted to the marine environment where invertebrates (sponges, soft corals, ascidians), and plants have been subject to elaborate screening programs. in recent years, marine microorganisms, either free living or as symbionts, have moved into the forefront of attention (gulder and moore ) fueled largely by the recognition of untapped actinomycete diversity in the marine environment. many marine sponges are associated with dense and phylogenetically diverse microbial consortia that can account for nearly half of the animal's biomass (hentschel et al. ; taylor et al. ) . of special interest are members of the phylum actinobacteria which have been identified in sponges both by cultivation and cultivation-independent approaches (abdelmohsen et al. ; hentschel et al. ; jiang et al. ; kim et al. ; montalvo et al. ; pimentel-elardo et al. ; webster et al. ; . actinobacteria and specifically electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. members of the order actinomycetes are in fact quite abundant in the marine environment (maldonado et al. ; mincer et al. ; stach and bull ) . a number of obligately marine actinomycete species and genera have been described which indicates true adaptations to the marine environment rather than them simply being washed into the ocean as spores from terrestrial soils. the actinomycetes are of particular relevance due to their unmatched capacity to produce novel and bioactive secondary metabolites. about , compounds have been isolated from this bacterial taxon alone lam ). the anticancer compounds salinosporamide and sporolide from the actinomycete salinispora tropica (buchanan et al. ; fenical et al. ), as well as the antitumor antibiotic marinomycin (kwon et al. ) from the obligately marine genus marinispora, are just two examples of metabolites from marine actinomycetes. in this study, we aimed to isolate taxonomically novel actinomycetes and sphingomonads from diverse caribbean sponges. sphingomonads were included in this study because of their reported immunomodulatory activities due to specific glycosphingolipids (long et al. ) . organic extracts of the bacterial cultures were then subjected to two screenings: (a) anti-protease assays using human, parasitic, and viral proteases as targets. proteases are relevant drug targets in cardiovascular, inflammatory, and infectious disease areas with about protease inhibitors currently approved for clinical use (turk ) . one prominent example is ritonavir, an aspartic protease inhibitor of hiv- virus which has been in clinical use since for the treatment of aids (danner et al. ) , and (b) immunomodulatory activities in terms of cytokine release in cultures of human peripheral blood mononuclear cells (pbmc) and induction of cell proliferation. immunoadjuvants from bacterial cells and their fractions have been used in vaccination and immune therapy (azuma and seya ) . for example, immunostimulatory cpg oligonucleotides have been found to induce maturation, differentiation, and/or proliferation of multiple cell types (klinman et al. ) . the first group of sponges (aplysina fistularis, plakortis sp., amphimedon compressa, aiolochroia crassa, agelas clathrodes, agelas cerebrum, ircinia felix, scopalina ruetzleri, erylus formosus, chondrilla nucula, and aplysina archeri) was collected by scuba diving at depths of - m in bahamas in july (gps- ° ′ . ″ n, ° ′ . ″ w). the second group of sponges (a. clathrodes, aplysina insularis, agelas tubulata, biemna cribaria, a. crassa, discodermia dissoluta, s. ruetzleri, dragmacidon reticulata, i. felix, monanchora arbuscula, and plakinastrella onkodes) was collected at depths of - m including sediments and seawater in el morro, santa marta bay, colombia (gps- ° ′ . ″ n, ° ′ . ″ w) in december . sponges were transferred to plastic bags containing seawater and immediately transported to the laboratory. sponge specimens were rinsed in sterile seawater, cut into pieces of ca. cm , and then thoroughly homogenized in a sterile mortar with volumes of sterile seawater. the supernatant was diluted in ten-fold series ( − , − , − ) and subsequently plated out on agar plates. the same protocol was repeated for sediment and seawater samples. six different media were used for the isolation of actinobacteria, such as m ( l artificial sea water (asw) containing g starch, g yeast extract, g peptone, g bacto agar, and l asw), m ( l asw was made up of ml of % glycerol, g arginine, g k hpo , . g mgso , and g bacto agar; mincer et al. ) , isp medium ( l asw containing g yeast extract, g malt extract, g dextrose, and g bacto agar; shirling and gottlieb ) , m ( l asw containing g peptone, . g asparagine, g sodium propionate, k hpo , . g mgso , . g feso , g glycerol, g nacl, and g bacto agar; webster et al. ) , nast cx (solution a ml of artificial seawater containing g k hpo and g bacto agar and solution b ml artificial seawater containing g kno , g mgso , g cacl . h o, . g fecl , and . g mnso · h o; magarvey et al. ) , and oligotropic medium ( l asw containing . g tryptone, . g sodium glycerophosphate, . g yeast extract, and g bacto agar; santavy et al. ). heat shock ( °c for min) and incubation with . % phenol at °c for min were applied to the sponge homogenates to select for spore-forming actinomycetes and rare actinomycete genera. all plates were incubated and observed for growth of colonies for - weeks. all media were supplemented with the following antibiotics: cycloheximide ( μg/ml), nystatin, and nalidixic acid ( μg/ml) to inhibit the growth of fungi and gram-negative bacteria. sphingomonads were isolated on m , isp medium , and oligotropic medium. the isolates from the bahamas are abbreviated as "ba" and those from colombia as "co". the s rrna genes from all bahamian sponge isolates were amplified, cloned, and sequenced according to hentschel et al. ( ) using the universal primers f and r (lane ) . isolates from the colombian sponges were first sorted into groups according to their morphological characteristics. restriction length fragment polymorphism (rflp) analysis was then performed on all isolates to reduce strain replication. following s rrna gene amplification using the universal primers f and r (lane ) and f and r (muyzer et al. ) , the pcr products were digested with the restriction enzymes haeiii and mspi for h. one to two isolates from each rflp group were selected for s rrna gene sequencing. chimeric sequences were identified using the pintail program (ashelford et al. ) . sequence alignment and phylogenetic analysis were performed using the arb software package (ludwig et al. ) . the genus-level affiliation of the isolates was validated using the ribosomal database project classifier (wang et al. ). tree construction was conducted using neighbor-joining algorithm (jukes-cantor correction) with bootstrap values based on , replications. the s rrna gene sequences of the putatively novel isolates were deposited in genbank under the accession numbers indicated in parentheses: ba (hm ), ba (hm ), co (hm ), co (hm ), co (hm ), co (hm ), and co (hm ). all other s rrna gene sequences (suppl. tables and ) were deposited in genbank under the accession numbers: hq -hq . sixteen strains were selected based on phylogenetic novelty (four actinomycetes, three sphingomonads, fig. a , b) or their affiliation to clades for which secondary metabolites had not been reported (nine strains). the isolates were cultured in ml erlenmeyer flasks each containing ml liquid m medium. the cultures were grown for - days depending on their growth rate at °c while shaking at rpm, and -ml culture aliquots were taken at different time points (days , , , and ); . ml methanol was added to each culture aliquot for cell lysis, and shaking was continued at rpm for h at room temperature (shaker sm , e. bühler). the broths were centrifuged in -ml falcon tubes at , rpm for min at room temperature (megafuge . r, heraeus), and the supernatants were dried using a rotary evaporator (heidolph, germany). media control using uninoculated m was extracted using the same method above. eleven strains were grown on m agar plates (three square plates per strain with approximately mm diameter each) at °c for - days. biomass together with the agar was removed from the plates by cutting it into small pieces and macerated overnight with sufficient volume of ethyl acetate to submerge the agar pieces. extraction with ethyl acetate of the macerated agar was repeated, and the macerations were subsequently filtered by gravity using a whatman filter paper no. . the filtrates were combined and dried by rotary evaporation. media control using uninoculated m agar was extracted using the same method above. screening for anti-protease activity cathepsins b and l and rhodesain protease inhibition assays were performed according to vicik et al. ( a, b) . briefly, assays were done at °c in a -mm tris-hcl buffer ph . (cathepsins) or in a -mm acetate buffer ph . (rhodesain) in a total volume of μl. the final substrate concentration was . and . mm for cathepsins b and l and . μm for rhodesain. the final enzyme concentration was ng/ml for cathepsins b and l and nm for rhodesain. the falcipain- inhibition assay was performed as described previously by breuning et al. ( ) . shortly, the enzyme was incubated with crude extracts for min prior to substrate addition in a total volume of μl. the following buffer was used: mm acetate, ph . supplemented with mm , -dithiothreitol. substrate (cbz-phe-arg-amc for all four enzymes) and inhibitor stock solutions were prepared in % final concentration dimethyl sulfoxide and were diluted with assay buffer. crude extracts were tested in duplicates at a final concentration of μg/ml. protease inhibition assays were carried out on a cary eclipse fluorescence spectrophotometer (varian, darmstadt, germany) using a microplate reader (excitation nm, emission nm). the fluorometric sars m pro and pl pro protease inhibition assays were performed according to kaeppler et al. ( ) . briefly, assays were performed at °c in a mm tris-hcl buffer ph . in a total volume of μl. the final substrate (h n-abz-ser-val-thr-leu-gln-ser-gly-(no )tyr-arg-(mts)-tfa-salt for m pro and z-arg-leu-arg-gly-gly-amc-acetate salt for pl pro ) concentration for inhibition assays was μm, and the final enzyme concentration was . μg/ml. crude extracts were tested in duplicates at a final concentration of μg/ml. assays were carried out at nm excitation and nm emission. screening for immunomodulatory activity pbmc from healthy donors were prepared as a by-product of platelet concentrates obtained with leukoreduction system chambers. the cell concentrate was then diluted in versene (ethylenediaminetetraacetic acid). pbmcs were isolated from this preparation using density gradient centrifugation with lymphocyte separation medium (paa laboratories gmbh, pasching, austria) and washed with ice-cold buffered salt solution/bovine serum albumin. cells were counted and cultured in roswell park memorial institute medium plus l-glutamine, supplemented with mm mercaptoethanol, mem non-essential amino acids ( ×), mm sodium pyruvate (gibco brl, gaithersburg, md, usa), u/ml penicillin (grünenthal gmbh, aachen, germany) and u/ml streptomycin sulfate (riemser arzeimittel, greifswald, germany), mm ( -hydroxyethyl)- -piperazineethanesulfonic acid (applichem gmbh, darmstadt, germany), and % abpositive heat-inactivated ( min at °c) human serum (paa laboratories gmbh, pasching, austria). cells were stimulated in triplicates using -well cell culture plates (greiner bio-one, kremsmünster, austria; × cells in μl per well) in a humidified incubator at °c with % co . to increase sensitivity of t cells to stimulation, pbmc were precultured for h at . × cells in . ml medium in -well plates (t.h., unpublished). supernatants were collected after h and stored at − °c. a panel of cytokines (tnf, ifn-γ, il- , and il- ) was measured by cytometric bead array (bd biosciences, san jose, ca, usa), using an lsr ii flow cytometer (bd biosciences, san jose, ca, usa) following the manufacturer's instructions. results were analyzed using fcap array software (soft flow, inc., usa). forty-eight hours after the stimulation, cell proliferation was measured as radioactivity incorporated into dna from tritiated thymidine during a -h pulse, using a liquid scintillation counter (perkinelmer, waltham, ma, usa). a total of actinomycetes were recovered from different caribbean sponge species, sediment, and seawater, of which were identified based on nearly complete s rrna gene sequence information (supplementary table ). the highest number of actinomycete isolates was recovered from s. ruetzleri ( isolates), followed by a. insularis ( ), d. dissoluta ( ), a. fistularis ( ), d. reticulata ( ), a. clathrodes ( ), p. onkodes ( ), i. felix ( ), plakortis sp. ( ), m. arbuscula ( ), a. crassa ( ), and e. formosus ( ). actinomycetes were not recovered from the remaining six sponges. additionally, five isolates were recovered from marine sediments and one isolate from natural seawater. to our knowledge, this is the first report on the isolation of actinomycetes from the sponges a. cerebrum, a. tubulata, a. compressa, a. archeri, b. cribaria, and c. nucula. in terms of actinomycete diversity, the cultivated strains are represented by different genera namely microbacterium ( isolates), rhodococcus ( ), streptomyces ( ), mycobacterium ( ), micromonospora ( ), knoellia ( ), gordonia ( ), curtobacterium ( ), arthrobacter ( ) salinispora ( ), saccharopolyspora ( ), nocardioides ( ), citromicrobium ( ), sanguibacter ( ), lapillicoccus ( ), kocuria ( ), dietzia ( ), cellulosimicrobium ( ) cellulomonas ( ), and agrococcus ( ; supplementary table ) . several recent publications have shown that streptomyces, micromonospora, and rhodococcus are among the dominant genera commonly isolated from marine sponges (abdelmohsen et al. ; schneemann et al. ; sun et al. ; ). in our study, microbacterium ( %) was the most dominant strain followed by rhodococcus ( %) and streptomyces ( %). nevertheless, we also present here the isolation of rare actinobacteria genera namely cellulosimicrobium, citromicrobium, sanguibacter, and lapillicoccus. moreover, this is the first report on the cultivation of the genera citromicrobium, sanguibacter, and lapillicoccus from marine sponges and of the genus lapillicoccus from the marine environment in general. remarkably, four actinomycete strains exhibited less than . % s rrna gene sequence similarities to validly described species. these low similarity values suggest that the strains belong to novel taxa (stackebrandt and ebers ) . phylogenetic analysis (fig. a ) revealed that these strains belong to the order actinomycetales under the following genera: lapillicoccus, microbacterium, rhodococcus, and nocardioides. phenotypic and genotypic characterization is further required to confirm the taxonomic affiliation of these strains. moreover, the isolation of novel actinomycete taxa has previously yielded new chemistry (feling et al. ; fiedler et al. ; pimentel-elardo et al. ) , and thus, this targeted approach offers a more productive route to natural products discovery. seven isolates were identified belonging to the order sphingomonadales and the genera sphingobium (six isolates) and sphingomonas (one isolate). nearly complete s rrna gene information is provided for four isolates (supplementary table ). the low s rrna gene sequence similarities (< . %) of two of these strains also suggest the taxonomic novelty at the species level (fig. b) . while members of the genus sphingomonas have previously been isolated from marine organisms such as from a bivalve (heindl et al. ; romanenko et al. romanenko et al. , and sponges ( dieckmann et al. ; laroche et al. ), we report here on the isolation of members of the genus sphingobium from a marine source for the first time. the crude extracts of isolates cultivated in m medium and extracted at four different time points were tested for their anti-protease activities. this involved testing against the human cysteine proteases and cathepsins b and l, which are involved in tumor metastasis (calkins and sloane ) . extracts were furthermore tested against proteases from the human parasites plasmodium falciparum (falcipain- ) and trypanosoma brucei rhodesiense (rhodesain) which are responsible for causing malaria and african trypanosomiasis (sleeping sickness), respectively. moreover, crude extracts were tested for their capacity to inhibit the papain-like (sars-cov pl pro ) and the main protease (sars-cov m pro ) of the sars coronavirus. these enzymes are essential for the replication of the severe acute respiratory syndrome (sars) coronavirus (anand et al. ; ratia et al. ) . cathepsins b and l, rhodesain, and falcipain- enzymes belong to the cathepsin l subfamily of cysteine proteases (clan ca, family c ; cac ). sars-cov pl pro also belongs to the clan of cysteine proteases ca but is affiliated to the family c , which contains polyprotein endopeptidases from coronaviruses. the protease sars-cov m pro belongs to the clan pa (family ) with a catalytic type of mixed cysteine, serine, and threonine (rawlings et al. ) . the extracts of eight isolates showed a certain specificity to the clan ca (family c ; cac ) group of proteases, whereas no protease inhibition was observed against viral proteases (table ) . extracts were considered active when, at a concentration of μg/ml, an inhibition of at least % was observed. the strains nocardioides sp. ba , saccha-ropolyspora shandongensis strain co and sphingobium sp. co inhibited rhodesain. the isolates agrococcus jenensis strain ba and sphingomonas mucosissima strain co inhibited cathepsin b and falcipain- . micromonospora coxensis strain co was active only against falcipain- , and rhodococcus sp. co was only active against cathepsin l. extracts of m. coxensis strain co were most active, inhibiting all four proteases tested. in fluorometric assays, the enzyme activity is measured by the hydrolysis rate of a fluorogenic or chromogenic substrate (ludewig et al. ) . this means that substrate and inhibitor compete for the enzyme's active site and that depends on the substrate concentration and on the affinity of the substrate to the target enzyme. the inhibition of rhodesain, to which the substrate (cbz-phe-arg-amc) has a particularly high affinity, by extracts of nocardioides sp. ba , s. shandongensis co , m. coxensis strain co , and sphingobium sp. co , is thus remarkable. future efforts will be directed at structure elucidation of the antiprotease secondary metabolites from these isolates. most of the protease inhibitors reported to date are synthetic molecules developed by structure-based design (turk ) . nevertheless, protease inhibitors have been also found in natural sources. miraziridine a, a pentapeptide inhibitor of cathepsins b and l, which was isolated from the marine sponge theonella mirabilis, is one such example (nakao et al. ) . a family of aeruginosin inhibitors is active against human serine proteases and was isolated from marine sponges and cyanobacterial waterblooms (ersmark et al. ). only few protease inhibitors have so far been reported from actinomycetes, leupeptin being a notable exception (hozumi et al. ) . in this study, we report on six actinomycete and two sphingomonad isolates as potential sources of novel protease inhibitors. human pbmc were stimulated with crude extracts prepared from bacterial isolates at four different time points as well as grown on solid media. the cd -specific monoclonal antibody okt , which addresses the t cell antigen receptor complex, was used as a positive control. after h, a panel of cytokines (tnf, ifn-γ, il- , and il- ) was measured in the culture supernatants, and after h, cell proliferation was determined by incorporation of tritiated thymidine. human pbmc contain monocytes and lymphocytes, the latter consisting of b cells, t cells (cd + t cells, cd + t cells, and regulatory t cells), as well as nkt cells. cytokines are proteins which are secreted by the cells of innate and adaptive immunity and which mediate many cellular functions. in the present study, we chose the following cytokines which are key pro-and antiinflammatory factors and which are characteristic of particular types of immune responses to pathogens: tnf, the principal mediator of the acute inflammatory response to gram-negative infectious microbes, is responsible for many of the systemic complications of severe infections. ifn-γ is the principal macrophage-activating cytokine and serves critical functions in innate and in adaptive cellmediated immunity. il- is a cytokine responsible for t cell clonal expansion after antigen recognition. il- , a cytokine with anti-inflammatory properties, has a central role in infection by limiting the immune response to pathogens and thereby preventing damage to the host. when culturing pbmc in the presence of our crude extracts, different patterns of cytokine release were observed. even though this assay does not distinguish which cells are responsible for cytokine production, we can make some assumptions about their cellular source. this is facilitated by the fact that individual extracts induced quite distinct patterns of cytokine release. for example, a. jenensis strain ba was very active in inducing tnf and il- , suggesting that this preparation addresses monocytes. in contrast, m. coxensis strain co was a potent inducer of ifn-γ, il- , and, to a lesser extent, il- . since the main sources of ifn-γ are t h cd + t cells and cd + t cells, this preparation appears to have t cell activating properties. furthermore, the induction of il- but not of ifn-γ by s. shandongensis strain co suggests a further specificity for cd + t cell activation. isolates a. jenensis strain ba , arthrobacter oxydans strain ba , and a. oxydans strain ba were effective in inducing tnf and il- release (fig. a, d) , cytokines induced mainly by monocytes and t cells (tnf: t h , il- : t h and treg). strains lapillicoccus sp. ba and m. coxensis strain co induced ifn-γ, il- , and il- ( fig. b-d) . ifn-γ and il- can be released by t helper cells and il- by t helper and regulatory t cells as well as monocytes. s. shandongensis strain co effectively induced the release of il- and il- (fig. c, d) . il- is mainly released by t h cells and il- is mostly produced by macrophages and t helper and regulatory t cells. sphingobium sp. co induced the release of tnf, ifn-γ, il- , and il- ( fig. a-d) , indicating a stimulatory effect on both t cells and monocytes. the remaining crude extracts from liquid and solid cultures from bacterial isolates did not induce cytokine release in pbmc culture supernatants. fig. cytokine responses of precultured human peripheral blood mononuclear cells to crude extracts from bacterial isolates. a tnf, b ifn-γ, c il- , and d il- . crude extracts were tested in triplicates at three different concentrations (preparations from liquid cultures- , . , and . μg/ml, from solid cultures- , , and . μg/ml), and the most active one is shown. the isolates a. jenensis strain ba , a. oxydans strain ba , a. oxydans strain ba a, and m. coxensis strain co were grown on m agar plates for - days. crude extract from isolate a. oxydans strain ba b was obtained after days of liquid culture, and active extracts from strains lapillicoccus sp. ba , s. shandongensis strain co and sphingobium sp. co were prepared after days of liquid culture fig. pbmc proliferation, measured as radioactivity incorporated into dna from tritiated thymidine, in response to stimulation with crude extract from strain sphingobium sp. co . crude extract was tested in triplicates at a concentration of ng/ml with regard to the induction of proliferation, crude extract prepared from solid culture of strain sphingobium sp. co exhibited the strongest mitogenic activity, reaching % of the positive control (okt , activating all t cells; fig. ). when strain sphingobium sp. co was tested for its capacity to induce cytokine release, a weak induction of il- was observed (data not shown), which suggests that t cells are the responsive cell type. the remainder of the samples did not stimulate pbmc proliferation. sphingomonads are well-known for their production of glycosphingolipids. for example, the glycosphingolipid gsl- was found to be produced by a sphingomonas strain isolated from the sponge plakortis simplex (laroche et al. ). the immunomodulatory activities of the two sphingomonads presented in this study also do not come as a surprise. glycosphingolipids are a class of compounds that have been shown to serve as immunomodulatory agents (long et al. ). the substance krn , a synthetic analog of the natural product agelasphin b from the sponge agelas mauritianus, is one such example and is currently in clinical trials for its capacity to stimulate nkt cells (park et al. ) . to our knowledge, we present here, for the first time, immunomodulatory activities from actinomycetes. the cultivation of strains belonging to different actinomycetales genera as well as seven strains belonging to two sphingomonadales genera represents considerable diversity of culturable bacteria in caribbean sponges as shown in this study. the isolation of rare actinomycete genera which have not previously been reported from marine organisms and the identification of seven putatively novel actinomycete and sphingomonad species based on the phylogenetic analysis of their s rrna gene sequence interestingly adds to this caveat. these results further prove that marine sponges still remain a relatively untapped resource for actinomycetes. moreover, anti-protease activities against cathepsins b and l, rhodesain, and falcipain- , as well as immunomodulatory activities specifically the induction of cytokine release by pbmc and induction of cell proliferation, were found to be exhibited by the crude extracts of selected strains. the anti-protease and immunomodulatory activities exhibited by these strains further warrant the need for a bioassay-guided purification and identification of the bioactive secondary metabolites. furthermore, it will be necessary to identify the responsive immune cell types and to obtain a more detailed mechanistic understanding of the processes underlying their modulation. isolation, phylogenetic analysis and anti-infective activity screening of marine sponge-associated actinomycetes coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs at least in s rrna sequence records currently held in public repositories is estimated to contain substantial anomalies development of immunoadjuvants for immunotherapy of cancer michael acceptor based antiplasmodial and 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complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for s rrna bioactive marine metabolites. part . miraziridine a, a novel cysteine protease inhibitor from the marine sponge theonella aff. mirabilis syntheses and biological activities of krn analogues having aromatic residues in the acyl and backbone chains with varying stereochemistry cebulactams a and a , new macrolactams isolated from saccharopolyspora cebuensis, the first obligate-marine strain of the genus saccharopolyspora anti-parasitic compounds from streptomyces sp. strains isolated from mediterranean sponges severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme merops: the peptidase database sphingomonas molluscorum sp. nov., a novel marine isolate with antimicrobial activity arenicella xantha gen. nov., sp. nov., a novel gammaproteobacterium isolated from a marine sandy sediment from 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taxonomy phylogenetic diversity of bacteria associated with the marine sponge rhopaloeides odorabile a comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species acknowledgments we gratefully acknowledge h. angermeier (u. würzburg) and j. pawlik (u. north carolina, wilmington) for sponge collection from the bahamas, c. heindl, s. berr, and c. gernert for technical assistance in the laboratory and p. römer for the introduction into the immunological techniques (u. würzburg). we furthermore thank s. zea (invemar, santa marta, colombia) for help in sponge identification as well as l. veloza and l.e. ramírez (universidad tecnológica de pereira, colombia) for providing the laboratory conditions for the cultivation of the colombian strains. financial support was provided by a doctoral fellowship (graduate college "immunomodulation gk " funded by the dfg) to p.t. and the dfg (sfb tp a ) to u.h. key: cord- -kb fnbgy authors: nan title: oral presentations date: - - journal: clin microbiol infect doi: . /j. - . . .x sha: doc_id: cord_uid: kb fnbgy nan [ primary immunodeficiency diseases are a heterogeneous group of disorders, caused by inherited defects in the immune system, and characterised by wide spectrum of clinical manifestations, particularly an increased susceptibility to infections and a predisposition to autoimmune diseases and malignancies. recurrent infections or infection with unusual organisms are the most commonly presentation of primary immunodeficiency diseases. although recurrent respiratory tract infections and gastrointestinal manifestations are the most common features of these diseases, especially in predominantly antibody deficiencies and combined immunodeficiencies, other organs can be involved as well. recurrent cutaneous abscesses with unusual organisms or deep abscesses may represent infections with an association with immunodeficiencies, particularly in phagocytes defects. meningococcal infections could have an association with complement deficiencies. meanwhile other bacterial infections, mainly streptococcus pneumoniae and staphylococcus aureus, as well as infections with viruses, fungi and parasites are also common in several primary immunodeficiency diseases. autoimmune diseases such as idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia, systemic lupus erythematosus, juvenile arthritis, sclerosing cholangitis, and vasculitis are common in primary immunodeficiency diseases. whilst some syndromic immunodeficiencies (e.g., wiskott aldrich syndrome, di george syndrome) have a strong association with autoimmunity, there are a group of disorders (e.g., alps, apeced, ipex) that the autoimmune manifestations are typically the first and most significant findings. malignancies are also common in some primary immunodeficiency diseases (e.g., cvid, alps, xlp, and dna repair defects). other manifestations such as dysmorphic features, associated anomalies, skeletal dysplasia, and oculocutaneous hypopigmentation can be unique characteristics of some cases with primary immunodeficiency diseases. the clinical manifestations of these diseases are often helpful in guiding the appropriate evaluation of the patients. prompt and precise diagnostic laboratory evaluation should be performed in the patients with such features, whereas early diagnosis and successful management of these patients prevent irreparable organ system damage and improve the prognosis. immunodeficiency specialists from all over europe have composed a multistage diagnostic protocol that is based on their expert opinion, in order to increase the awareness of pid among doctors working in different fields. the protocol starts from the clinical presentation of the patient; immunological skills are not needed for its use. a list of relevant symptoms and signs from the history and physical examination that should alert any physician to potential pid is given. these are grouped together to form eight typical clinical presentations of pid: recurrent ent and airway infections; failure to thrive from early infancy; recurrent pyogenic infections; unusual infections or unusually severe course of infections; recurrent infections with the same type of pathogen; autoimmune or chronic inflammatory disease, or lymphoproliferation; characteristic combinations of clinical features in eponymous syndromes; and angioneurotic edema. these presentations lead the user towards different algorithms, which in fact represent the traditional division into antibody, complement, lymphocyte, and phagocyte deficiencies, respectively. the algorithms each are comprised of several steps. this multistage design allows cost-effective screening for pid within the large pool of potential cases in all hospitals in the early phases, while more expensive tests are reserved for definitive classification in collaboration with an immunologist at a later stage. g. schmid°(geneva, ch) in , articles suggesting that male circumcision (mc) decreased the risk of hiv infection appeared. over the next years, studies of two epidemiologic types − ecologic and observational − increasingly supported this contention. ecologic studies showed strong correlations between prevalences of mc and hiv, e.g., tribes with low prevalences of mc had high prevalences of hiv infection. observational cross-sectional studies showed that uncircumcised men had higher rates of hiv than circumcised men. observational cohort studies confirmed these weaker study design findings. a systematic review of observational studies in found a relative risk (rr) of . ( % ci, . − . ), a % protective effect. in and , results from three randomised controlled trials, all from sub-saharan africa, were reported. results were consistent, and the pooled rr of . ( % ci, . − . ) was identical to that of the observational studies. the protective effect in the three trials, found at about − months' follow-up, has been extended in one trial to a protective effect of % at months of follow-up. who and unaids have strongly endorsed mc as an effective hiv prevention strategy in generalised hiv epidemics where mc is uncommon. what about europe? mc is uncommon with an adult male prevalence of < %. hiv incidence is low enough that mc for hiv prevention purposes is unlikely to have much impact. no public health authority recommends routine neonatal circumcision. increasingly, however, data are showing benefits of mc in addition to hiv prevention. lessened risk of urinary tract infection in infants (rr . , % ci . − . ) and lifetime avoidance of phimosis and associated conditions occur when mc is performed neonatally. other benefits occur in males circumcised at any age. mc protects against acquiring sexually transmitted infections characterised by genital ulcers-syphilis, chancroid and herpes-and possibly trichomoniasis. circumcised men may be less likely to acquire hpv and are more likely to clear the infection. through the protective effect against hpv, mc halves risk of penile cancer (rr . , % ci . − . ) and partners of circumcised men are at lessened risk of cervical cancer. other issues must be considered in making public health decisions about mc. cultural objections may occur, but mc in the developing world is readily accepted in non-circumcising societies. studies of sexual pleasure and function have found no relationship to circumcision status. mc may be advised for subgroups, even if not for the entire population. and, surgical risk and cost must be considered. while many sub-saharan african countries are scaling up mc services to prevent hiv infection, public health agencies in many industrialised countries are reconsidering mc policies-the outcomes of both efforts are being followed with interest. acute otitis media (aom) is generally considered a bacterial infection that is treated with antibiotics. however, despite extensive use of broadspectrum antibiotics for this condition, the clinical response to the treatment is often poor. this fact, together with vast clinical experience connecting aom with viral respiratory infections, has prompted research into the role of viruses in aom. to date, ample evidence from studies ranging from animal experiments to large clinical trials supports a crucial role for respiratory viruses in the aetiology and pathogenesis of aom. in most cases, viral infection of the upper respiratory mucosa initiates the whole cascade of events that finally leads to the development of aom as a complication. the pathogenesis of aom involves a complex interplay between viruses, bacteria, and the host's inflammatory response. recent studies indicate that with sensitive techniques viruses can be found in the middle-ear fluid in most children with aom, either alone or together with bacteria. viruses appear to enhance the inflammatory process in the middle ear, and they may profoundly impair the resolution of otitis media. it is important to understand, however, that our increasing knowledge of the importance of viruses in the etiopathogenesis of aom does not diminish the central role of bacteria in aom. therefore, while viruses may explain many of the problems encountered in treating aom, the ultimate decision on whether or not to treat aom with antibiotics cannot be based solely on the degree of viral involvement in aom. the non-judicious use of antibiotics has lead to an epidemic in antimicrobial resistance. acute otitis media (aom) is the most common indication for use of antibiotics in children in the united states (us). despite available evidence that supports a wait and see approach, most us physicians immediately prescribe antibiotics for the treatment of aom. the american academy of pediatrics published a guideline in that addressed the diagnosis and treatment of aom. this guideline recommends the use of observation as a potential strategy for the treatment of aom. the key components of this published guideline will be discussed, as well as the evidence and rationale that supports the use of observation as an initial strategy to treat aom. otitis media (om) is the most common bacterial infection in children aged < years for which antibiotic treatment is prescribed worldwide. although most of the time this entity resolves spontaneously it is associated with morbidity, family dysfunction, antibiotic use and burden on the medical system. efforts to reduce the burden of om by vaccination have not been extremely rewarding, but some progress has been made. the first obvious step would be to reduce viral infections leading secondarily to om. in the modern era, the only viral vaccine with proven effect on aom is the influenza virus vaccine. both the inactivated and the live virus showed some effect, but since influenza virus has only a limited season yearly the effect on the overall om rate is far from being remarkable. haemophilus influenzae (hi) b vaccine did not reduce om since most hi causing om are nontypable (nthi) and not hib. the newly developed pneumococcal conjugate vaccines (pcvs) have all been shown to reduce > % of the om caused by the serotypes included in the vaccines, but some replacement with serotypes not included in the vaccines and non pneumococcal organisms was demonstrated to reduce the overall effect of pneumococcal vaccines. the effect of pcv on the reduction of recurrent om, om with effusion, the need for ventilation tubes and frequent visits for aom has been suggested, and the real impact is still being studied. aiming with pcv at those with established recurrent om has proved disappointing. pcvs can reduce om caused by antibiotic-resistant s. pneumoniae but the continued overuse of antibiotics is responsible for the increase in antibiotic resistance in non-vaccine serotypes. a newly developed pcv with an outer membrane protein for hi (pnpd) is suggested to reduce also om caused by hi, but confirmation studies are needed. the expansion of the serotypes included in the current licensed pcv to or more serotypes may add to the prevention of om in the near future. in the next decade, om will continue to be an important disease in children. however, we can expect it to be modified in terms of bacteriologic aetiologies, antibiotic resistance and hopefully short and long term consequences. v. korten°(istanbul, tr) infectious consequences of an earthquake mainly involve several types of communicable diseases and crush related infections. water-borne and food-borne illnesses often result from the disruption of the public water and sewage systems and contamination of water supply. overcrowding, poor hygiene and sanitation in temporary shelters also may be factors. the type of infectious diseases are associated with the epidemiology of communicable diseases in the area where the earthquake occurred. the most common outbreaks associated with earthquakes are gastroenteritis, infectious hepatitis and pulmonary infections. in unvaccinated populations, there are reports of increased measles. tetanus can be seen in populations where vaccination coverage levels are low. the risk for diarrhoeal disease outbreaks following earthquakes is higher in developing countries than in industrialised countries. an outbreak of acute watery diarrhoea involved > cases occurred in a camp after the earthquake in pakistan. acute respiratory infections, hepatitis e clusters and measles (> clinical cases in the months) also occurred among the displaced victims after the same earthquake. contamination of drinking water led to an outbreak of rotavirus after the earthquake in kashmir, india. an unusual outbreak of coccidiomycosis associated with exposure to increased levels of airborne dust occurred after the southern california earthquake. persons who have been trapped by rubble for several hours or days may develop compartment syndromes requiring fasciotomy or amputation. infectious complications were common in renal victims of the marmara earthquake in turkey and were associated with increased mortality when complicated by sepsis. of renal victims, ( . %) had infectious complications, mainly sepsis and wound infections. most of the infections were nosocomial in origin and caused by gram-negative aerobic bacteria and staphylococcus spp. multivariate analysis of the risk-factors for nosocomial infections revealed a significant association with fasciotomy and length of hospital stay in a back up university hospital. the most frequent pathogens isolated from pus and/or wounds culture in wenchuan earthquake survivors were s. aureus, e. coli, a. baumannii, e. cloacae, and p. aeruginosa. disaster-preparedness plans, focused on trauma and mass casualty management and also on health needs of the surviving affected populations may decrease the health impact of earthquakes. s infections in the disaster setting: famine. experience from darfour, sudan clinic malnutrition is a known risk factor for id worldwide. subsaharan africa and india is at higher risk due to vegetarian habits on absolute absence of animal meat proteins, resulting to depletion of micronutritients (zinc, iron, selenium), responsible for recovery of postmalarial anaemia. in addition, depletion of proteins results to immunoglobulinaemia and to delayed response to many bacterial pathogens causing id in topics (pneumococci, salmonella, etc.) . third problem is absence of vitamins dissolved in oil and fat, resulting to delayed phagocytic activity. therefore proteinocaloric malnutrition results to significant adverse outcome in hiv, tb (diarrhoea, pneumonia), the major killers of children under five. st. elizabeth university tropical programme runs antimalnutrition centres: in sudan, darfour and in kenya amaong upcountry refugees from major conflict areas (sudan − turrana border) and in uganda trying to rehabilitate malnourished children under and helping them to combat disease, responsible for . million deaths in children mean a year − malaria ( . mil), tb ( . mil), hiv ( . mil), pneumonia ( . mil) and diarrhoea ( . mil. children deaths approximately a year). h. giamarellou°(athens, gr) for the last six years greece has faced a large number of infections, mainly in the intensive care units (icu), due to carbapenemsresistant klebsiella pneumoniae. the proportion of imipenem-resistant k. pneumoniae has increased from less than % in , to % in isolates from hospital wards and to % in isolates from icus in . likewise, in , these strains were identified in only three hospitals, whereas now they are isolated in at least of the hospitals participating in the greek surveillance system. until this situation was due to the spread of the blavim- cassette among the rapidly evolving multiresistant plasmids and multiresistant or even panresistant strains of mainly k. pneumoniae and also other enterobacterial species. however, the fact that most strains display mic values below or near the clsi resistance breakpoint create diagnostic and therapeutic problems, and possibly obstruct the assessment of the real incidence of these strains. as of , the emergence of kpc-producing k. pneumoniae has been noted in icus of some greek hospitals and has now spread to most hospitals throughout the country creating a countywide outbreak in . in attikon university hospital we recently described the icu outbreak of kpc-producing k. pneumoniae. twenty-nine patients (admitted from february to december ) were colonised mainly in gi tract. fifteen patients were male ( %) and the median apache ii was . patients had already long hospital stays preceding icu admission with a median of ( − ) days. in twenty-two of these patients ( %) kpc-producing k. pneumoniae colonisation was definitely icuacquired while in ( %) acquisition in other wards or other hospitals was hypothesized. five of these patients are still hospitalised in the icu and, of the remaining , died (icu mortality %). ten of the colonised patients were clinically infected. fifteen infections were documented, mostly bsi ( / ), followed by vap ( / ) and ssi ( / ). only patient died from this infection ( / , . %). an evidence-based consensus on the therapeutic strategy for these infections has been reached by keelpno and the greek ministry of health which proposed the use of high dose meropenem ( − g/day) combined with an active aminoglycoside or colistin for strains with an mic mg/ml whereas for strains with a higher mic the use of carbapenems is contraindicated and active alternatives (monotherapy with tigecycline, colistin, or an aminoglycoside or aztreonam-based combinations) could be used. antibiotic stewardship is of great importance in such a dismal situation but stringent adherence to infection control measures is probably of even greater importance for the effective containment of these pandrugresistant strains. the presentation of clostridium difficile infection (cdi) varies from mild diarrhoea to a potentially fatal pseudomembranous colitis. the recent emergence of types and of c. difficile has been associated with increased virulence. c. difficile takes advantage of disruption of the normal intestinal flora as caused by antibiotic therapy. the antibiotical class and the antimicrobial resistance pattern of c. difficile influence the development of disease. in the netherlands, significantly more patients with cdi due to type used fluoroquinolones (or, . ; % ci, . − . ) compared with those who were infected with other pcr ribotypes. similar as type cdi, patients infected with type also more frequently received fluoroquinolones therapy (or, . ; . the risk to develop cdi due to type was particularly high in persons receiving a combination of cephalosporin and fluoroquinolone (or . , ). this association was also strongly dependent on the duration of therapy. the use of clindamycin was found as a protective factor. however, the recent detection of clindamycin-resistant c. difficile type strains in other european countries is an important and worrying development. since the association of cdi with fluoroquinolones has only been investigated at patient level, a study was performed to investigate the relationship between cdi incidence and the preceding use of different antibiotic classes at hospital level in the netherlands. comparisons were made between hospitals where type caused an epidemic, hospitals where only isolated cases of type were observed and hospitals where no outbreak of cdi or type were encountered. in the pre-epidemic period, the total use antibiotics was comparable between affected and unaffected hospitals. higher use of secondgeneration cephalosporins, macrolides and all other studied antibiotics were independently associated with a small increase in cdi incidence, but the effect was too small to predict which hospitals might be more prone to -associated outbreaks. despite the fact that the netherlands is known by its restrictive and conservative use of antibiotics, outbreaks of cdi due to new emerging types have been recognized. this is probably associated with the use of antibiotics at patient level and hospital department level rather than the use of antibiotics at the level of the healthcare institute. m. peiffer, j. bulitta, h.a. haeberle, m. kinzig-schippers, m. rodamer, v. jakob, b. nohé, f. sörgel, w.a. krueger°(trier, de; albany, us; tubingen, nuremberg, constance, de) piperacillin-tazobactam (pip-tazo) is a broad spectrum antibiotic, used for treatment of severe infections such as ventilator-associated pneumonia (vap). the effectiveness of betalactams is best predicted by the duration of free drug concentrations above the minimal inhibitory concentration (t > mic) of infecting pathogens [ ] . animal experiments suggest that more than % of t > mic should be reached. continuous infusion (ci) of pip-tazo may enhance the therapeutic performance, but there is little data on pharmacokinetic/-dynamic (pk/pd) parameters, when ci is used in critically ill patients. objectives: the aim of our study was to determine concentrations of pip-tazo in plasma and broncho-alveolar epithelial lining fluid (elf) at steady state during ci. based on these results, the penetration ratio (plasma/elf) and pk/pd parameters for pip-tazo are derived. methods: after approval by the ethics committee, mechanically ventilated critically ill patients were enrolled during treatment in intensive care units. each patient received a loading dose of g/ . g of pip-tazo, followed by ci of g/ . g over h. at steady state ( . + . h after loading dose), a total of blood samples were drawn and bronchoalveolar lavage (bal) was simultaneously performed in cases ( sample discarded for technical reasons). samples were stored at − ºc until analysis by liquid chromatography coupled with mass-spectrometry (lc-ms). elf-concentrations were calculated from bal-samples using the relation of ureaplasma:ureabal as dilution factor. results: plasma concentrations of pip and tazo (n = in pts.) amounted to . + . mg/ml, and . + . mg/ml, respectively. elflevels (n = ) were . + . mg/ml, and . + . mg/ml. elf-levels were + %, and + % of corresponding plasma levels (n = ) for pip and tazo, respectively. the ratio pip:tazo was . : in plasma, and . : in elf. conclusions: using advanced analytical techniques, elf concentrations were higher compared to traditional bolus administration [ ] . ci yielded steady state plasma concentrations in excess of mics of susceptible bacteria (< mg/ml, according to eucast) in . % of measurements, respectively, but elf levels exceeded mg/ml in all cases. taken together, our data provide further arguments for ci being the preferred mode of administration for pip-tazo in critically ill patients with suspected vap. [ objectives: staphylococcus aureus is a potential pathogenic microorganism and a causative agent of~ % of infections in intensive care patients. an optimal empiric choice for the treatment of these infections will result in a reduction in morbidity and mortality. therefore, it is essential to provide the clinician with resistance data of the bacterial population to be treated. to optimise the empiric choice and to monitor the emergence of microbial resistance, a national surveillance program of the swab was started in the netherlands in .this study describes the results of the resistance development of s. aureus from icu's of hospitals all over the netherlands over a ten year period. methods: in the first months of each year, the participating hospitals collected clinical isolates from among others blood and respiratory samples. in total isolates were collected: from hospitals in the north, from in the east, from five in the west and from four in the south. the antimicrobial susceptibility was determined as a micro broth dilution method according to the clsi guidelines. results: an increase in resistance to ciprofloxacin was observed from % until to % from in , which dropped again to % in . the resistance to moxifloxacin was rather constant over time, i.e. %, only in % resistance was found. resistance to clarithromycin increased to % in , but decreased in to % the level before . resistance to penicillin, clindamycin and tetracycline fluctuated over time at~ %, − % and − % respectively. during the study period seven methicillin resistant s. aureus were isolated, no resistance to vancomycin, teicoplanin and linezolid was observed. resistance to gentamicin and rifampicin was sporadicly found. regional differences were observed for ciprofloxacin, being the highest in the western and southern part and tetracycline being the lowest in the northern part. conclusion: during the year study period only an increase in resistance to ciprofloxacin was observed. the data presented justify the empiric choice of flucloxacillin, (with rifampicin or gentamicin depending on the indication) in case of an infection in icu patients probably caused by s. aureus. j.j. lu°, p.r. hsueh, s.y. lee (taichung, taipei, tw) objectives: to investigate the prevalence of visa in hospitalised patients with mrsa infections or colonisations at a teaching hospital in taiwan and to evaluate the possible clonal spread of visa in the hospital. methods: from september to august , consecutive mrsa isolates were collected from various clinical specimens of patients hospitalised at a teaching hospital in taiwan. minimum inhibitory concentrations (mics) of vancomycin for all mrsa isolates were determined by the broth microdilution method in accordance with clsi guidelines. molecular characteristics and antimicrobial susceptibilities of visa isolates were investigated and pulsed-field gel electrophoresis was used to evaluate the clonality of the isolates. results: among the mrsa isolates, ( . %) were visa. of the visa isolates, had vancomycin mics of microgram/ml and had vancomycin mics of microgram/ml. all isolates were inhibited by tigecycline at . microgram/ml, linezolid at microgram/ml, and ceftobiprole at microgram/ml. five ( . %) isolates had reduced susceptibility to daptomycin (mics of − microgram/ml). six of the visa isolates had decreased susceptibility to autolysis in . % triton x- . the visa isolates were recovered from patients; of these patients had received glycopeptide treatment prior to the isolation of visa. five ( . %) patients died despite vancomycin therapy. all visa isolates carried sccmec type iii and agr group i but were negative for pvl gene (luks-lukf). none of the enterococcal van genes were detected in the visa isolates. results of pfge analysis revealed that one major clone of visa isolates ( . %, clone a exhibiting sccmec type iii, agr group i, and absence of pvl gene) had disseminated in the hospital. conclusion: this retrospective study demonstrated that clonal dissemination of visa had occurred in the hospital. rapid and correct detection of visa and proper use of antibiotics are the most effective approaches for preventing its emergence and spread. x. zheng°, c. qi, a. o'leary, m. arrieta, s. shulman (chicago, us) objectives: vancomycin remains one of the major options for treating methicillin-resistant s. aureus (mrsa) related infections. some but not all studies have shown an increase in prevalence of mrsa isolates with elevated vancomycin mic values among recent clinical isolates, so called "mic creep". although still within the susceptible range, higher mics may be associated with increased chance of treatment failure. because of the conflicting reports and lack of published data from paediatric patients, we sought to assess possible mic change over time and to compare results generated by using different methodologies including etest, agar dilution, and broth microdilution (microscan) methods. methods: we studied mrsa isolates predominantly community acquired including all blood and normally sterile site isolates collected in our large children's hospital in / , , , and molecular bacteriology o genome sequence of a virulent, methicillin-sensitive staphylococcus aureus clinical isolate that encodes the panton-valentine leukocidin toxin l. faraj, l.a.s. snyder, n.j. loman, d.p. turner, m.j. pallen, d. ala'aldeen, r. james°(nottingham, birmingham, uk) objective: to determine the genome sequence of a virulent meticillinsensitive staphylococcus aureus (mssa) clinical isolate sanot . methods: roche sequencing determined the genome sequence of the clinical isolate at times coverage. newbler sequence assembly (roche) generated scaffolds that were annotated using gendb and compared with other s. aureus genome sequences. results: an -year-old asian girl presented with fever and a -week history of knee pain following a trivial fall. an mr scan revealed a large subperiosteal abscess around the upper tibia secondary to metaphyseal osteomyelitis. a pvl-positive, mssa was isolated from blood cultures and pus. the child deteriorated, required repeated debridement and developed septic shock. further investigation revealed aortic valve endocarditis with an aortic root abscess. whole genome sequencing revealed that sanot is the first sequence of an st s. aureus isolate to be determined. sanot is agr type iii and carries three coding regions that are not found in any other s. aureus genome sequences. amongst the unique genes present in these regions is a dihydrofolate reductase gene (dfrg) which is present in addition to the usual dfrb gene. downstream of the orfx gene, a . kb remnant of sccmec type ivc was found. this sequence has only previously been found in the mrsa genome sequence where it is located between the orfx and sccmec type ii sequences. mrsa is unique in sharing genome regions with s. aureus strain rf , a causative agent of contagious bovine mastitis. all but one of these genome regions are also present in sanot . conclusions: comparison of the genome sequence of sanot and the closely related mrsa ha-mrsa (emrsa- ) isolate reveals new insights in the evolution of both ca-mrsa and ha-mrsa isolates and the link to s. aureus rf . pvl-encoding mssa strains can be significant pathogens but are not currently under mandatory surveillance in uk. as the cost of whole genome sequencing falls further it will become feasible to use this technology to monitor the evolution of both mssa and mrsa in healthcare settings and reveal clinically relevant information that will help to improve patient outcomes. objectives: ca-mrsa often produce panton-valentine leukocidin (pvl), a leukocidin encoded by two co-transcribed genes located on lysogenised phages. five pvl-encoding phages have been described in s. aureus: phipvl, phi pvl, phislt, phisa mw and phisa . single nucleotide polymorphisms (snps) in the pvl genes tend to vary with lineage and may have structural and functional implications. we examined a selection of pvl-positive ca-mrsa reported in our hospital to determine whether sequence variation and the pvl-encoding phage vary with lineage. methods: twenty-two pvl-positive isolates were chosen to reflect mlst clonal complexes identified in our hospital: cc , , , , , and . isolates were characterised by antimicrobial resistance profile, sccmec and spa type, pulsed-field gel electrophoresis (pfge) profile and multilocus sequence typing (mlst); an oligonuleotide array (clondiag arraytube) was used to detect a range of toxin and antimicrobial resistance genes. primers were designed to amplify and sequence the luksf-pv genes. the pvl-encoding phage was characterised using a recently described pcr-based assay (ma et al. j clin microbiol ; : − ) . results: snps were identified at seven positions in the luksf-pv genes and the snp profile varied with lineage. three of the snps were coding mutations, which may have structural and functional implications. cc and cc isolates were both found to carry phisa mw. the pvlencoding phage was not definitively identified in the other lineages, although the cc isolates carried a phisa -like phage and the cc , cc and cc isolates carried elongated head-type phages. one of the cc isolates had an unexpected snp pattern compared with other cc isolates; this isolate also carried a novel or variant phage. conclusion: pvl gene sequence and the pvl-encoding phage vary with lineage in pvl-positive ca-mrsa isolates. this suggests that certain lineages are susceptible to infection or lysogeny with certain phage types. although ca-mrsa commonly carry pvl genes, some strains do not; it is possible that some pvl-negative types are resistant to infection with pvl-encoding phage, perhaps via restriction modification systems. crucially, our findings suggest the pvl genes have co-evolved with their phage and are not freely transmitted between different phages. further work is required to characterise the pvl-encoding phage in other isolates and to investigate whether the pvl sequence variants result in biological differences. objectives: community-associated mrsa (ca-mrsa) of many different mlst clonal complexes (ccs) can harbour lysogenised bacteriophage dna (prophage) encoding panton-valentine leukocidin (pvl). five pvl phages (phipvl, phislt, phisa mw, phi pvl, and phisa ) have been reported to date. we sought to determine the distribution of chromosomally integrated copies of these lysogenised pvl-phages amongst dominant clones of pvl mrsa in england and wales. methods: seventy isolates of previously characterised pvl-mrsa were analysed by pcrs developed by ma et. al, (jcm, ) , to identify and discriminate between the five known pvl phages. to maximise any underlying diversity, representatives of each cc were selected based upon their spa, staphylococcal cassette chromosome mec (sccmec), toxin gene and pulsed-field gel electrophoresis (pfge) profiles. these included isolates of internationally disseminated pvl-mrsa lineages ccs , and which resemble the usa , south west pacific (swp) and european clones, respectively. in addition we analysed pvl-mrsa from ccs , , , , and st . results: all seven cc isolates, which included representatives of the european clone, possessed an elongated-head-type phage and were positive by the pcr specific for the phisa mw phage. one of the cc isolates possessed a phi pvl phage, four swp representatives had elongated head type phages, whilst the remaining four cc isolates harboured an icosahedral-head-type phage. one cc was positive for both head shapes. the cc (including representatives of usa ), eight cc , six cc isolates and the st isolate were all positive for elongated-head-type phage. nine cc isolates were non-typeable for phage head shape and specific phage pcrs. three of four cc isolates, harboured a phisa -like phage of an unknown head type and the other cc isolate was non-typeable. all cc isolates possessed an icosahedral-head-type phage, were positive for the phipvl phage type and one possessed phi pvl type. we have determined the pvl phages present in a diverse panel of distinct pvl-mrsa clones and found considerable inter-lineage variation in the pvl prophage present. there was also evidence of intra lineage variation in some major ccs such as ccs , and . together with variation in mlst cc and sccmec, these data suggest pvl-mrsa have evolved on multiple occasions, sometimes within the same lineage. o transcriptional profiling of klebsiella pneumoniae genes controlled by the transcription factor, rama objectives: rama is an arac/xyls family transcriptional activator where over expression is associated with a multidrug resistance phenotype. in both multidrug resistant klebsiella and salmonella isolates, the rama gene has been associated with increase in expression of the acrab efflux pump. in salmonella it has been shown that a deletion of the rama locus prevents the emergence of multidrug resistant mutants. therefore in order to understand the role of this key regulator in the emergence and development of antibiotic resistance, transcriptomic analyses of its regulon were undertaken in k. pneumoniae. methods: rna was extracted from a combination of isogenic mutants and clinical isolates using the qiagen or ribopure kits. rna integrity was assessed using nanodrop and agilent nanochip systems. the rna was transcribed into double stranded cdna prior to labelling with cy . the cdna was hybridised to the nimblegen expression array platform designed from the k. pneumoniae mgh genome. results: approximately genes were found to be affected by rama expression, of which twenty (involved in metabolism, physiology, transcription, drug efflux, protection responses and the cell envelope) were confirmed by rt-pcr. the rama protein appears to affect drug efflux operons not previously shown to be associated with multidrug resistance and or affected by similar proteins such as mara. comparative transcriptome analyses of different k. pneumoniae clinical isolates overexpressing rama showed that variations exist in the levels of expression of the drug efflux genes. of note genes shown to be directly regulated by rama have a marbox-like sequence within the promoter sequences. conclusion: in this study, the transcriptome of the regulatory protein, rama, was determined in the pathogen k. pneumoniae. drug efflux proteins not previously associated with rama overexpression were found to be directly affected. the rama regulon overlaps with the mara and soxs regulons in e. coli and salmonella but is directly associated with regulating the expression of a subset of genes via a marbox sequence. interestingly, variations in the levels of the expression of the regulon genes were found in the different rama overexpressing strains. m. eshoo°, c. crowder, h. li, h. matthews, s. meng, s. sefers, r. sampath, c. stratton, d. ecker, y.w. tang (carlsbad, nashville, us) objectives: the potential for fatal outcome from tick-borne human infections such as ehrlichiosis emphasizes the need for rapid diagnosis. we developed and validated an ibis t assay (ibis biosciences, inc., carlsbad, ca) that can detect and identify a wide range of tick-borne pathogens from clinical samples. methods: a multi-locus assay was used that employs broadrange pcr primer pairs targeting all known bacterial tick-borne pathogen families. electrospray ionisation mass spectrometry of the pcr amplicons was used to determine their base composition. these base composition signatures were subsequently used to identify the organisms found in the samples. the assay was developed using field collected ticks and a wide range of clinical sample types and has been shown to be sensitive to the stochastic limits of pcr. results: whole blood ( ) , cerebrospinal fluid ( ) and plasma ( ) samples, which were originally submitted for ehrlichia species detection by a colorimetric microtiter plate pcr (pcr-eia), were collected consecutively from january to august , at vanderbilt university hospital. among the total specimens, pcr-eia detected ehrlichia species with a positive rate of . %. the ibis system detected ehrlichia in of the pcr-eia-positive samples and in of the pcr-eia-negative specimens, giving sensitivity and specificity of . % and . %, respectively. the ibis system further characterised the ehrlichia-dual positive specimens to the species level (e. cheffeensis, ; e. ewingii, ) with a % agreement to that identified by pcr-eia using additional species-specific probes. in addition we demonstrated the detection of borrelia burgdorferi from the blood and skin of a patient with lyme disease. conclusions: we demonstrate broad-range detection of tick-borne pathogens in a single assay using skin, whole blood, plasma, skin and csf. in addition to ehrlichia, the ibis system detected rickettsia rickettsii positive specimens, which were confirmed by serology and clinical findings. the ibis t system, which can be completed within five hours from specimen processing to result reporting, provides rapid and accurate detection and identification of a broad range of pathogens causing tick-borne human infections. r. sampath°, l. blyn, r. ranken, c. massire, t. hall, m. eshoo, r. lovari, h. matthews, d. toleno, r. housley, s. hofstadler, d. ecker (carlsbad, us) objective: to investigate the use of a novel platform-based approach for rapid characterisation of hai organisms. pathogens that cause healthcare-associated infections (hais) pose an ongoing and increasing challenge to hospitals, both in the clinical treatment and in the prevention of the cross-transmission of these problematic pathogens. here we describe the utility of a pcr electrospray ionization mass spectrometry (pcr/esi-ms) detection platform as an innovative, rapid approach for detection and complete characterisation of important hai pathogens. methods: we have developed pcr/esi-ms based methods to rapidly identify and characterise mrsa, vre, c. difficile (nap- strain), p. aeruginosa and a. baumannii. each target organism can be analyzed using an independent -well assay that can be run on the same platform and can provide species and strain id, virulence factors, antibiotic resistance and genotyping as appropriate. validation studies were performed using - retrospective, well-characterised clinical isolates for each organism. this was followed by a prospective study for one of the organisms, mrsa, that included screening of clinical specimens (nares swab) from patients who were admitted to a medical unit with a high prevalence of mrsa clinical infections. results: for each of the five hai organisms, pcr/esi-ms species identifications were compared to gold standard testing results from the clinical microbiology laboratory and showed % concordance. for s. aureus, p. aeruginosa and a. baumannii, molecular genotyping by pcr/esi-ms was compared to pulse field gel electrophoresis (pfge) clusters and showed > % concordance. characterisation of virulence and/or drug resistance was performed for mrsa, vre and c. difficile and showed − % correct detection compared to existing testing methods. analysis of clinical specimens for mrsa showed that of the swabs, ( %) contained mrsa, either singly or as a dual infection with cons, ( %) were mssa and ( %) contained meca+ coagulase negative staphylococcus (mr-cons). comparison to gold standard analysis showed % sensitivity for mrsa detection with . % specificity, % ppv and %npv. the pcr/esi-ms technology is a high throughput assay system useful for infection control and for epidemiological studies. it is capable of simultaneous identification of hai organisms while detecting presence of key phenotypic markers and genotypic strain characterisation. m. reijans°, j. ossel, j. keijdener, g. simons (maastricht, nl) objective: molecular diagnostics play an increasingly important role in the detection of infectious agents in cerebrospinal fluids. however, the growing list of targets and the relatively small sample volumes are challenges that demand an improved molecular diagnostic approach. the meningofinder is a multifinder assay allowing the simultaneous detection of viruses and internal control in reaction. until now, the analysis of multifinder assays was based on size-fractionation, identifying each multifinder probe due to its specific length. here we present an alternative approach allowing realtime detection of eight meningofinder probes in a single tube. the realtime detection enables a faster analysis, less handling and lowers the risk of contamination. method: the meningofinder assay is a multifinder assay which detects herpes simplex virus and (hsv − ), human parechovirus (hpev), cytomegalovirus (cmv), epstein-barr virus (ebv), enterovirus (ev) and varicella-zoster virus (vzv) plus an internal control in a single reaction. each meningofinder probe can be distinguished based upon the specific length of each probe by size-fractionation using gel or capillary electrophoresis. we developed an alternative detection method using fluorescently labelled probes which allow specific identification of multifinder probes in a realtime pcr machine. results: a large number of qcmd samples (n = ), several enterovirus types (n = ) and characterised clinical samples (n = ) were analyzed using the meningofinder. all meningofinder reactions were analyzed by capillary electrophoresis and by fluorescently labelled probes in a realtime pcr machine. the results of the meningofinder showed a very good correlation with the expected results (> %). furthermore, the results of both meningofinder analyses showed a high degree of correlation. the realtime detection of the meningofinder probes decreases the analysis time and post pcr handling dramatically. we developed a new assay for the realtime detection of meningofinder probes. the realtime analysis showed a very good correlation with the conventional capillary electrophoresis analysis. in addition, the realtime detection reduced contamination risk and patient results became available more quickly. the combination of multifinder technology combined with realtime detection shows great potential in fast and easy multiparameter screening of clinical samples for infectious pathogens. in-house naats were applied to nucleic acid extracts obtained by own in-house methodology in each centre. results: sensitivities for the detection of the respiratory viruses were % for commercial mx naat, % for in-house mw naat, and % for mono in-house naat. the viral load was low each time false-negative results were obtained. false positive results were obtained by all methods used, resulting in specificities ranging from %- %. for the atypical bacteria, the multiplex naats failed to detect low l. pneumophila positive samples and low m. pneumoniae positive sample resulting in sensitivities of % and % compared to % in the inhouse mono naats. the commercial mx naat also failed to detect strong positive samples. no false positive results were obtained for the atypical bacteria. revisiting phage therapy against problematic pathogens s how the past feeds the future: from d'herelle to modern phagotherapy the increasing antibiotic resistance problem boosts the interest in alternative treatments for infections. a prominent example for this is the so-called phagotherapy. it makes use of bacterial viruses − bacteriophages − as drugs against bacterial agents. these bacteriophages are isolated from nature, characterised and then tested against the bacterial strains that are targeted. in theory, this approach has several advantages. for instance, bacteriophages infect, as a rule, their bacterial prey very specifically. therefore, they do not harm the commensal bacteria of the patient. additionally, if a bacterial strain becomes resistant against a certain bacteriophage strain, evolution will provide for new and active bacteriophage strains. in practice, phagotherapy has been used for a long time. already one of the two discoverers of bacteriophages, félix d'herelle, was an ardent advocate of this method. in fact, he was the first to use bacteriophages against infections − against bacterial diarrhoea (shigella spp.). after that, phagotherapy has been used to quite some extent in europe, the us and other parts of the world until penicillin entered the market in the s. in some parts of the former soviet union and the eastern bloc, the method has been utilised until today. now, several companies and university researchers are developing bacteriophages for therapeutical purposes again. historical documents related to phagotherapy and oral history reveal a fascinating past. bacteriophages have been employed against a wide variety of bacterial diseases in a time in which there were virtually no other anti-infectives. for example, in india, millions of cholera patients were treated with bacteriophages in the s. anti-cholera phages were also poured into drinking wells as prophylactics. bacterial viruses have also been utilised by the german and soviet armies in the second world war. the history of phagotherapy makes for more than an exciting story, however. analysis of the old literature helps identify important factors for success and failure. this is especially relevant for a field which holds promise but which has had limited funds at its disposal in the past few years − and which, therefore, has been making rather slow progress. additionally, examination of the strategy used for phagotherapy in the soviet union and poland also contributes to a better application of this method today. the discovery of bacteriophages, particularly their ability to replicate and lyse pathogenic bacteria may have been among the most important milestones in the history of biomedical sciences. in the pre-antibiotic era of the early th century, phage therapy was becoming a powerful weapon against infectious diseases of bacterial aetiology. unfortunately, phage treatment and research was largely forgotten in the western world as antibiotics became widely available. nowadays, the rapid propagation of multi-drug resistant bacterial strains is leading to renewed interest in phage therapy. in contrast to its decline in the west, phage therapy remained a standard part of the healthcare systems in eastern europe and the ussr during the second half of the th century. phage preparations were used for diagnostic, therapeutic and prophylactic purposes to combat various bacterial infections. the eliava institute of bacteriophages, microbiology and virology (tbilisi, georgia) is perhaps the most famous institution in the world focused on the study of bacteriophages, particularly the isolation and selection of phages active against various bacterial pathogens. phages have been isolated against bacterial strains received from all over the former ussr and socialist east european countries; consequently, a huge collection of phages and pathogenic bacterial strains has been constructed at the institute. thousands of people were treated with individual phages and phage mixtures during the soviet era. the preparations developed in tbilisi have been studied through extensive preclinical and clinical trials. however, little of this information has ever been published and even when details are available, the trial reports do not meet internationally approved regulations and standards. bacteriophages have a number of advantages in comparison to antibiotics. phage therapy as an alternative approach for treatment of infections has become an evident and promising remedy. today, many people from various parts of the world express their willingness to take phage treatment against different infections, including those that are caused by antibiotic-resistant bacterial pathogens. the eliava institute has elaborated new, phage-based products and technological schemes for their production. strong collaboration with the medical community in the design of clinical trials according to international standards is absolutely critical to supporting the broader implementation of phage therapy. an australian male aged years died from an intracerebral haemorrhage ten days after he returned from a trip to rural yugoslavia. his kidneys and liver were donated to three female recipients aged years (kidney), years (kidney), and years (liver). four to five weeks after the organ donation, all three recipients died. all had febrile illnesses with altered mental status. subsequent testing of post-mortem tissues from the recipients identified a novel arenavirus, which was related to lymphocyctic choriomeningitis virus (lcmv). this viral detection process involved the use of high-throughput sequencing techniques to identify novel microbial rna sequences. confirmatory testing was performed using the techniques of reverse transcriptasepolymerase chain reaction, immunohistochemical analysis for arenavirus antigens, and immunofluorescent testing for igg and igm antibodies. the clinical features in these four patients as well as other similar problems with transplant-related illness from classic lcmv will be discussed, as well as details of the laboratory identification of this new virus, and implications for organ transplantation protocols in future. successful management of invasive fungal infections depends on timely and correct treatment. over the last decades a number of new tests have become available which have improved the diagnostic options. in contrast to the scenario for bacterial infections, acquired resistance in fungi is rare and thus species identification is a valuable tool guiding choice of treatment. therefore, microscopy & culture is still a corner stone in diagnosis, but culture and identification are time consuming (app. − and − days, respectively). the sensitivity and speed of microscopy have been improved by the use of fluorescent brighteners such as calcofluor white or blankophor. but only with the recent development of pna probes specific for a number of the candida spp. has species identification become possible directly from a positive blood culture before subculture on agar media. chromogenic agars allow a presumptive identification of several candida spp. and facilitate the recognition of yeast isolates in samples containing several yeasts or yeast and bacteria in combination. the use of such plates has been shown to lead to a better identification of mixed cultures in a recent nordic eqa scheme including more than laboratories. rapid species identification of the most important candida spp. is possible in the routine laboratory using easy commercially available kits. thus, a species identification of c. albicans, c. dubliniensis and c. krusei can be obtained within minutes using latex agglutination kits (bichro-dubli, krusei-color; fumouze diagnostics) and c. glabrata can be rapidly identified due to its high amounts of preformed intracellular trehalase enzyme (glabrata rtt; fumouze diagnostics). finally, pna probes and fluorescence microscopy can also be used for a same day identification of a range of the clinically relevant candida spp. (advandx). susceptibility testing is possible using etest and the results are comparable with those obtained by reference methodologies in head to head comparisons. however, recent data from eqa distributions suggest that detection of isolates with acquired resistance causes many laboratories difficulties. this illustrates that a critical number of isolates should be tested per technician per week and quality control strains should be included on a regular basis. in conclusion, a number of new diagnostic tests have become available over the last decade and the diagnostic laboratories are encouraged to take advantage of these new options. th eccmid, oral presentations since the introduction of newer antifungals with different in vitro spectra, the aetiology of invasive fungal infections (ifi) has become a major diagnostic issue as a prerequisite for a guided antifungal therapy. while molecular methods, such as pcr and sequencing for the diagnosis of ifi have been evaluated from specimens such as blood and bronchoalveolar lavage fluid for some years, they have been less studied for biopsies. characteristics inherent to these molecular methods, e.g. sensitivity, specificity and short turnaround time makes them promising as adjuncts to conventional diagnostic tests, e.g. culture and histopathology from organ biopsies. studies using tissue from animal models of mould infections suggest that pcr might be more sensitive than culture and allows for a better species identification than histopathology. however, most of these studies used assays detecting only a small range of agents or even single organisms. while this may increase the sensitivity of the assays and reduces the likelihood of contaminations it limits the usefulness in the clinical setting, given the broad range of potential fungal pathogens. studies using fresh clinical samples suggest that the detection and identification of a wide range of fungi is possible using broad range assays in combination with sequencing or by combining more specific pcr assays. further studies are needed to optimise dna extraction, define the best molecular targets and the best method for amplicon detection. the prevention of contaminations due to ubiquitous fungi and unspecific amplifications are a major problem, especially when using broad range assays. in contrast, fish probes may potentially be more specific than pcr due to the visualisation of fungal elements in tissue. in contrast to pcr, they appear to work well with formalin fixed specimens. species identification might be more challenging than by pcr and sequencing. direct comparisons between fish and pcr are needed to characterise the pros and cons of each method in determining the aetiology of ifi. molecular tissue diagnosis has the potential to evolve into a useful method to describe the aetiology of ifi even in culture negative samples. results might be obtained fast enough to guide the antifungal therapy in patients with ifi progressive to empiric antifungal therapy. in these patients, the risk associated with invasive tissue sampling might be outweighed by potential benefits of a guided antifungal therapy. the two groups of carbapenemases (serine carbapenemases and metallobeta-lactamases (mbls)) can be encoded by genes that can be carried on plasmids. the serine carbapenemases are distinctly either class a or oxa (class d); the latter being mainly associated with acinetobacter spp. the dominant mbl subgroups, vim and imp have genes that are reportedly carried on plasmids and chromosomes. recent evidence has shown that the majority of blavim- , even those initially reported, are indeed plasmid mediated and probably accounts for their rapid dissemination. blavim- genes have been recently shown to be carried on incn and incw plasmids. the "brazilian" mbl gene, blaspm- , is exclusively chromosomally encoded. the mbls sim- and aim- are both chromosomally encoded whereas gim- is encoded from a plasmid of approx. kb. the recently described blakmh- gene is also carried on a plasmid ( kb). hitherto, only two mbl-positive plasmid sequences are available thus far -those carrying blaimp- and blavim- . the former carries other resistance genes and are approx. kb (inchi ), whereas the latter is a small plasmid ( kb) and shows similarities with incp plasmids. oxa carbapenemase genes have been shown to be both plasmid and chromosomally mediated. thus far, the blaoxa- and blaoxa- / clusters can be both plasmid and chromosomal and have mainly been found in acinetobacter spp. the blaoxa- and blaoxa- clusters have been found in k. pneumoniae and acinetobacter spp., respectively, and both are plasmid mediated. blaoxa- and blaoxa- have been shown to be carried on kb and - kb plasmids, respectively. a blaoxa- plasmid has been recently sequenced and shown to carry two different replicases. the class a carbapenemase genes, blakpc, blaimi- and blages are all carried on plasmids. blakpc is found mainly in k. pneumoniae and carried on plasmids that vary in size − kb and mostly possessing the origin of replication incn. however, kpc- has recently described in a pseudomonas as being chromosomally mediated. blaimi- is exclusive to the usa and carried on a kb plasmid although blaimi- is chromosomal. the blages genes have been found in p. aeruginosa and enterobacteriaceae of which ges- , , and have been shown to be plasmid mediated although little else in known. this lecture will provide a synopsis, discuss the evolution of resistance due to plasmids and briefly predict what we may face in the c with respect to carbapenemase resistance. nosocomial infections caused by multidrug-resistant pathogens, especially gram-negative bacilli, have become a serious clinical concern in every healthcare setting worldwide. as well as carpapenemhydrolysing metallo-b-lactamases, ctx-m-type b-lactamases, and qunolone-resistance genetic determinants such as qnr, aac( )-ib-cr, and qepa, plasmid-mediated novel molecular mechanisms such as rmta, rmtb, rmtc, rmtd, arma, and npma responsible for pan-resistance to aminoglycosides have recently been identified in pseudomonas aeruginosa, acinetobacter spp., serratia marcescens, esherichia coli, klebsiella pneumoniae, proteus mirabilis etc. since , and these enzymes have indeed methylation activity of g or a at the a-site of the bacterial s rrna as found in aminoglycoside-producing actinomycetes. these plasmid-mediated s rrna methylases are speculated to be originated from some nonpathogenic environmental microbes that produce aminoglycosides or some similar compounds, so it is quite natural that several new enzymes would be further identified hereafter in both clinical and livestock farming environments. rmtb and arma have widely spread in asia, europe, america and australia via various pathogenic gram-negative bacilli, we should pay special attention to the further spread of such hazardous microbes. in my talk, i would like to give an outline of newly identified molecular mechanisms that confer pan-resistance to aminoglycosides in pathogenic microbes isolated from both human and veterinary environments. [ acquired resistance to quinolones mainly results from chromosomal mutations responsible for modification(s) of dna gyrase and topoisomerase iv, and for a decrease of drug accumulation into bacteria due to decreased permeability and/or overexpression of efflux systems. plasmid-mediated quinolone resistance (pmqr) was first reported in from the usa, and two other mechanisms have been identified to date. the first pmqr determinants, qnr proteins, belong to the family of pentapeptide repeat proteins. five determinants have been identified: qnra, qnrb, qnrc, qnrd, and qnrs with , , , , and different variants, respectively. they may act by binding directly to both dna gyrase and topoisomerase iv leading to protect them from quinolone inhibition. they confer resistance to nalidixic acid and reduced susceptibility to fluoroquinolones (fqs), but may facilitate recovery of mutants with higher level of resistance. the overall prevalence of qnra, qnrb, and qnrs determinants generally ranges from to %, and they have been identified worldwide mostly in esbl-producing enterobacterial isolates. the origin of the qnra and qnrs genes were identified as shewanella algae and vibrio splendidus, respectively. the second type of pmqr determinant, aac( )-ib-cr, is a variant of the aminoglycoside acetyltransferase aac( )-ib which confers resistance to kanamycin, tobramycin and amikacin. this variant possesses two substitutions (trp arg and asp tyr) that are sufficient to acetylation of ciprofloxacin and norfloxacin with a -to- -fold mic increase. the overall prevalence of aac( )-ib-cr may range from . to up to %, and it has been reported mainly in escherichia coli and klebsiella pneumoniae. the third type of pmqr determinant, qepa, has been identified in two e. coli clinical isolates from japan and belgium. the qepa gene encodes a -transmembrane-segment putative efflux pump belonging to the major facilitator superfamily. this protein confers decreased susceptibility to hydrophilic fqs (e.g. norfloxacin, ciprofloxacin and enrofloxacin) with an -to- -fold mic increase. the two epidemiological surveys for qepa may indicate its low prevalence (< %). the natural reservoir of qepa remains unknown but might be an actinomycetal species. discovering of three main mechanisms of pmqr within the last ten years is peculiar. it may reflect the emergence of novel mechanisms of resistance but also a deeper investigation of resistance mechanisms in clinical isolates. emerging infections: can we cope with them? a. kühn°, c. schulze, h. ranisch, p. kutzer, h. nattermann, r. grunow (berlin, frankfurt-oder, de) objective: little is known about the prevalence of francisella tularensis in humans and animals in germany. interestingly, the pathogen emerged recently when several marmosets (callithrix jacchus) died from tularaemia and a group of hunters became infected in the areas of western germany. to find out more about the distribution of the pathogen also in eastern germany we investigated the seroprevalence of tularaemia under foxes (vulpes vulpes) and raccoon dogs (nyctereutes procyonoides) in the area of brandenburg (around berlin). methods: sera of animals (n = and n = , respectively) from the years and were tested for f. tularensis − lps antibodies in an indirect elisa and suspicious samples were confirmed by western blot for lps ladder recognition using protein g − pod conjugate. furthermore we investigated the serum samples by a competitive elisa using a peroxidase-conjugated anti − lps monoclonal antibody. results: from the serum collection, we tested ( . %) foxes and raccoon dogs ( . %) positive for specific f. tularensis antibodies. the geographical distribution showed hot spots in the area of the investigated region. our results indicate for a higher seroprevalence in wildlife for tularaemia in eastern regions of germany than assumed. since the reported human cases for the last decade seem to be underestimated, the real prevalence of the pathogen is unknown. the high number of tularaemia antibody positive foxes and raccoon dogs indicates that this zoonose is present in wildlife in eastern germany. however, the impact of transmission of zoonotic pathogens from wildlife to domestic animals and humans is not yet well studied. in conclusion, the obtained data will contribute for creating of up-to-date strategy for more efficient control of the two rickettsial zoonoses. objective: helicobacter pylori is established as the primary cause of gastritis and peptic ulceration in humans. in a minority of patients with upper gastrointestinal symptoms long tightly coiled spiral bacteria, clearly distinct from h. pylori, and provisionally named as "h. heilmannii", can be observed in gastric biopsies. our objective was to isolate and identify the spiral organism, resembling "h. heilmannii" from the gastric mucosa of a finnish patient presenting with severe dyspeptic symptoms. methods: we used two different selective media for the isolation of the bacteria from gastric biopsy samples before and after treatment of the patient with a -day course with lansoprazole, tetracycline and metronidazole. the isolates were characterised by testing for urease and catalase activity, light and electron microscopy, and sequencing the partial s rrna and ureab genes. single enzyme aflp was used to analyse the genetic diversity among the isolates. results: growth of long spiral organisms was obtained from out of antrum and all corpus biopsies before and all three antrum biopsies after treatment of the patient. the partial s rrna gene sequence showed high sequence similarities with other gastric helicobacter species. the partial ureab gene showed high sequence similarity with h. bizzozeronii and was clearly distinct from other gastric helicobacter species. aflp indicated that the isolates belonged to the same clone however some minor genetic diversity was observed among the isolates. results: b. pseudomallei was primarily found in close proximity to streams and in grass-rich areas but was also correlated with environmentally disturbed soil such as caused by the presence of animals, farming or irrigation. prediction maps are currently being verified by sampling predicted b. pseudomallei "hot-" and "cold-spots". see in figure a prediction map for rural darwin with red areas indicating high probability for presence of b. pseudomallei. this study contributes to the elucidation of the environmental distribution of b. pseudomallei in endemic tropical australia and to the clarification of environmental factors influencing its occurrence. it also raises concerns that b. pseudomallei are spreading due to changes in land management. o concurrent multi-serotypic dengue infections in various body fluids w. kulwichit°, s. krajiw, d. chansinghakul, g. suwanpimolkul, o. prommalikit, p. suandork, j. pupaibool, k. arunyingmongkol, c. pancharoen, u. thisyakorn (bangkok, th) objectives: dengue virus infection is one of the rapidly-spreading emerging diseases worldwide. the virus is divided into distinct serotypes with limited cross-protective immunity; therefore, one can be reinfected with different serotypes. while each episode is usually caused by a single serotype, an individual can occasionally be infected by concurrent multiple ones. our group has previously detected dengue virus from urine and oral specimens of some patients. in this study, we sought to determine the characteristics of multi-serotype infections when analysing beyond the patients' blood compartments. methods: during during - and adult patients suspected of dengue infections were enrolled. plasma, peripheral blood mononuclear cells (pbmc), urine pellets, buccal brushes, and saliva were collected during and after the febrile episode. only specimens from patients with both positive dengue serology and pan-dengue-specific rt-pcr were included. serotype-specific rt-pcr was then performed on the aforementioned various specimens of each patient. results: patients met the above criteria. serotyping was successful in patients. den- was the most common serotype, accounting for half of the cases. of these ( . %) demonstrated multiserotypic infections when combining data from all specimen types in each individual. serotyping using single, conventional serum/plasma specimens, however, would detect only half of the cases. the phenomenon of concurrent multi-serotypic infections was present in all examined specimen types, including urine pellets, buccal brushes, and saliva. the most frequent combinations were den- + den- and den- + den- ( cases each). two patients were simultaneously infected by serotypes , , and and one by serotypes , , and . there was no demonstrable significant difference in clinical severity between single-and multi-serotypic infections. conclusion: in a dengue-hyperendemic country with simultaneous circulation of all four serotypes, the phenomenon of concurrent multiserotypic infections are more common than previously demonstrated by traditional serotyping on single serum/plasma specimens. this may be explained by the sensitivity limitation of the detection method or by biological behaviour of the virus. our findings have an implication for potentially more accurate epidemiologic studies in the future, and for further exploratory investigations regarding dengue virus in various secretions and excretions. o emerging concepts about the evolutionary history of hantaviruses h.j. kang, s.n. bennett, l. sumibcay, s. arai, a.g. hope, j.a. cook, j.w. song, r. yanagihara°(honolulu, albuquerque, us; tokyo, jp; seoul, kr) objective: recent discovery of genetically distinct hantaviruses in shrews (family soricidae), captured in widely separated geographic regions, challenges the conventional view that rodents are the principal and progenitor reservoir hosts of hantaviruses, and raises the possibility that other soricomorphs, notably moles (family talpidae), harbour hantaviruses. methods: using oligonucleotide primers based on conserved genomic regions of rodent-and soricid-borne hantaviruses, rna extracts from tissues of the japanese shrew mole (urotrichus talpoides), american shrew mole (neurotrichus gibbsii) and european common mole (talpa europaea) were analyzed for hantavirus sequences by rt-pcr. newfound s-, m-and l-segment sequences were aligned using clustal w and were analyzed phylogenetically by the maximum-likelihood and markov chain monte carlo tree-sampling methods, with the gtr+i+g model of evolution. results: novel hantavirus genomes, designated asama virus (asav), oxbow virus (oxbv) and nova virus (nvav), were detected in tissues of urotrichus talpoides, neurotrichus gibbsii and talpa europaea, respectively. sequence and phylogenetic analyses indicated that asav and oxbv were related to hantaviruses harboured by soricine shrews in eurasia and north america, respectively. by contrast, phylogenetic analyses of full-length s-and l-segment sequences showed that nvav formed a unique clade, clearly distinct and evolutionarily distant from all other hantaviruses. despite the high degree of sequence divergence at the nucleotide and amino acid levels, the secondary structures of the nucleocapsid proteins, as well as the l-segment motifs, of the moleassociated hantaviruses were well conserved. conclusions: while cross-species transmission has influenced the course of hantavirus evolution, such host-switching events alone do not satisfactorily explain the co-existence and distribution of genetically distinct hantaviruses among species in two taxonomic orders of small mammals spanning four continents. when viewed within the context of molecular phylogeny and zoogeography, the close association between distinct hantavirus clades and specific subfamilies of rodents, shrews and moles is likely the result of alternating and variable periodic codivergence at certain taxonomic levels through evolutionary time. thus, the primeval hantavirus might have arisen from an insect-borne virus, with ancestral soricomorphs, rather than rodents, serving as the original mammalian hosts. from south-eastern france m. kaba, b. davoust, j.l. marié, m. barthet, m. henry, c. tamalet, j.m. rolain, d. raoult, p. colson°(marseille, toulon, fr) objectives: autochthonous hepatitis e is currently considered as an emerging disease in industrialised countries and several studies suggest that hepatitis e is a zoonosis, especially in pigs, boars and deer. we aimed to study whether hepatitis e virus (hev) is commonly present in domestic pigs in southern france, and to determine the relationship between hev sequences detected from pigs and those described in human hepatitis e cases. methods: serum and stools samples were collected from three or six-month-old pigs from different regions of southern france. sixmonth-old pigs were from a slaughterhouse, and three-month-old pigs were from a pig farm. swine igg anti-hev antibodies testing was performed using a commercial elisa kit for clinical diagnosis with minor modifications. swine hev rna detection was conducted by realtime pcr and amplification/sequencing assays using in house protocols targeting the orf region of the hev genome. results: % of pigs were seropositive, and % of three-monthold pigs were hev rna-positive, whereas none of the six-monthold pigs were hev rna-positive. hev rna was significantly more frequently detected from stools than from serum ( % versus %; p < . ). phylogenetic analysis showed that swine hev sequences belong to genotype f or e and formed two clusters within which sequences showed high nucleotide homology (> %). these clusters were correlated with the geographical origin of pigs as well as with their repartition into pens and buildings in the pig farm where samples were collected. swine hev sequences from the present study were genetically close to hev sequences found from humans or swine in europe, although no strong phylogenetic link could be observed neither with these latter sequences nor with those from human hepatitis e cases diagnosed in the laboratory. conclusion: our data indicate that three-month-old farm pigs from southern france might represent a potential source of contamination to humans, and they underscore the great potential of hev to cause epizootic infections in populations of farm pigs. o clostridium difficile: changing epidemiology trends, - objectives: clostridium difficile infection (cdi) has become a growing concern world-wide with an increased reported incidence and an increase in the associated financial burden. our aim therefore was to review trends in cdi occurring from - inclusive. methods: all patients admitted to lothian university hospitals division (luhd) tested for c. difficile toxins a+b by eia were included. retrospective analysis of prospectively collected data was performed. the number of occupied bed days was provided by nhs-lothian statistics department. the most recent published costs associated with cdi were used to estimate potential costs to lothian nhs trust. results: , faecal samples were tested for c. difficile toxins from - inclusive; of these samples were positive. overall cdi was identified in . cases/ patient days and . cases/ inpatient hospital admissions. the incidence of identified cdi rose from . cases/ patient days in to . cases/ patient days in . incidence also increased with age from . cases/ patient days in the − years age group to . cases/ patient days in the − years age group. renal medicine and intensive care had the highest incidences of identified cdi with greater than cases/ patient days each followed by infectious diseases and gastrointestinal medicine whose rates were . and . cases/ patient days respectively. medicine of the elderly in comparison had an incidence of . cases/ patient days. of note % of all patients were transferred through a minimum of two specialties during the period in which they remained positive for c. difficile toxins. estimated costs over the study period for toxin testing alone were in the region of £ , and the minimal potential hospitalisation costs of patients with cdi was in the region of £ , , . conclusion: the incidence of patients identified with cdi has risen markedly and not surprisingly the incidence has also been noted to increase with age. medicine of the elderly however had a much lower incidence than several other specialties and therefore risk assessment of cdi development and containment should now also be targeted within other specialties. with % of identified cdi patients transferred through different specialties and the significant financial burden cdi imposes on healthcare institutions judicious application of infection control measures remains an important factor to prevent cdi spread. isolates of this strain were pvl negative, but positive for enterotoxin a (sea) and, in most cases, also for seb, sek and seq. a fifth strain was the "taiwan clone", st / -mrsa-v (wa mrsa- and - ) which also comprised two closely related sequence types. this strain carried a sccmec element of type v(t) or vii as well as pvl and, usually, seb, sek and seq. it was the most common cc strain in wa. the sixth strain differed from the "taiwan clone" in the presence of a sccmec type v element and in the absence of pvl. the differentiation of this clonal complex into various different strains indicates a rapid evolution and spread of sccmec elements, and the diagnostic microarray technology allows one to distinguish beyond mlst level and hence to accurately trace outbreaks and spread of these strains. a sample taker has daily contact with poultry and is excluded from analysis. b sample taker reported no contact with livestock elsewhere than in this study at that moment (spa-types of sample taker and farm are not corresponding). c sample taker tested mrsa-negative in following tests. d sample taker was not tested again. complete data sets (samples taken before, directly after and hours after a visit) were collected on visits by sample takers visiting farms. on farms mrsa was collected from pigs or stabledust ( %). these farms were visited times by different sample takers. one sample taker (# ) was positive for mrsa before visiting a farm, he was removed from the following analysis. fifteen of the ( %) visits to mrsa-positive farms resulted in acquisition of mrsa and / ( %) sample takers acquired mrsa at least once after visiting a positive farm. of these positive sample takers acquired mrsa twice and sample taker acquired mrsa three times after separate visits. of the acquisitions of mrsa, were negative after hours. the spa-types of mrsa isolates found on the farms and sample takers were grossly comparable. on the negative farms, none of the visits resulted in mrsa acquisition. for further information see the table. discussion: mrsa-cc was acquired by % of the sample takers after occupational exposure in this study. however, in of the cases the strain was not recovered the next day, therefore acquisition was of short duration, posing a limited treat to human health. some persons seemed to be more vulnerable to acquire mrsa during their work. the sample size of this study was too small to draw final conclusions concerning this inter-personal variation. this requires a more extensive study. [ objectives: community-associated mrsa is an increasing problem and an association with food animal contact has been made in some regions. this has led to concerns about the potential role of food in mrsa transmission. the objective of this study was to evaluate the prevalence of mrsa colonisation of retail pork in canada. methods: pork chops, ground pork and pork shoulders were purchased at retail outlets in four canadian provinces in conjunction with the canadian integrated program for antimicrobial resistance surveillance. both direct inoculation of meat into enrichment broth and rinsing of meat in broth were performed for pork chops and shoulders, followed by inoculation onto chromogenic agar. ground pork was tested only using the direct method. mrsa isolates were typed by pfge and spa typing. real time pcr was used to detect panton-valentine leukocidin genes. results: mrsa was isolated from / ( . %, % ci . − . %) of samples. there was a significant difference between provinces (p < . ) but no difference between different products, with mrsa isolated from / ( . %) pork chops, / ( . %) ground pork and / ( . %) pork shoulders (p = . ). / ( . %) samples were positive using direct culture while mrsa was isolated from / ( . %) of samples testing using the rinse method. nine samples were positive on direct culture but negative using the rinse method, while others were positive only with the rinse method and only were positive with both methods. seven samples (ground pork) that were positive on direct culture were not tested using the rinse method. main clones were present. the most common ( % of isolates) was a group of related spa types (t , t and new related type) were classified as canadian epidemic mrsa- by pfge, an st human epidemic clone that has been associated with horses. pfge-non-typable spa t were not surprisingly common, accounting for % of isolates. the rd main group was related spa types (t , t and new type) that were cmrsa- (usa ), an st clone that is common in humans in canada, that also accounted for % of isolates. the clinical relevance of mrsa contamination of pork is currently unclear. it is possible that contact with contaminated food could be a mode of mrsa transmission in the community, although further study of the prevalence of contamination, amount of mrsa in contaminated samples, sources of contamination and implications on human health are required. o prevalence of the novel trimethoprim resistance gene dfrk among german staphylococcal isolates of the bft-germvet monitoring study k. kadlec°, s. schwarz (neustadt-mariensee, de) objectives: very recently a novel trimethoprim resistance gene, dfrk, was identified on a tet(l)-harbouring plasmid in a porcine mrsa isolate from the bft-germvet monitoring study. this study included in total independent coagulase-positive and coagulase-variable staphylococci collected between and all over germany: isolates from infections of the urinary-genital tract of pigs, isolates from skin infections of pigs, isolates from respiratory tract infections of dogs/cats, and isolates from infections of skin/ear/mouth of dogs/cats. in this study, we investigated the prevalence and the plasmid location of the dfrk gene among these isolates. methods: pcr primers were designed and a pcr with subsequent restriction analysis of the pcr product was established to detect dfrk. isolates with positive results were tested for a plasmid location of dfrk by transfer experiments and dfrk-carrying plasmids were further analysed. the trimethoprim resistance gene dfrk was detected in another isolates. all isolates were from pigs: from skin infections and the remaining from a urinary-genital tract infection. six staphylococcus hyicus subsp. hyicus isolates, s. aureus isolates ( mrsa and mssa) and s. pseudintermedius. all these isolates harboured plasmids. in isolates ( s. hyicus, mrsa and the single s. pseudintermedius), the plasmid location of dfrk was confirmed by protoplast transformation with subsequent susceptibility testing and pcr analysis of the transformants. in all cases, the plasmids harbouring dfrk also carried a tet(l) tetracycline resistance gene. the results of a combined pcr assay with primers from tet(l) and dfrk confirmed that the dfrk gene was always located immediately downstream of the tet(l) gene. further analysis of these dfrk-and tet(l)-harbouring plasmids showed that they varied in size between and kb and that similar sized plasmids differed in their ecorv and hindiii restriction patterns. the novel trimethoprim resistance gene dfrk occurred in ( . %) of the porcine staphylococcal isolates from the bft-germvet study. in ( . %) of the isolates, it was located on structurally diverse plasmids, however, always in close proximity to a tet(l) gene. the linkage of the dfrk and tet(l) genes allows the maintenance and coselection of such plasmids under selective pressure by either tetracyclines or trimethoprim, both of which are widely used in veterinary medicine. (table) . the isolates were resistant to ciprofloxacin, clindamycin, erythromycin, gentamicin but susceptible to vancomycin. only one se was methicillin-susceptible and two isolates were quinupristin/dalfopristin non-susceptible. all strains were clonally related and clustered into three subtypes (a, a and a ). cfr gene was detected in a linezolid non-susceptible strain (mic, mg/l), which was recovered from a y/o male who underwent liver transplantation. plasmid analysis identified six plasmid bands ranging from c.a. . -to -kb in the cfrcarrying strain. hybridisation signals were observed from the -kb plasmid band as well as from a chromosomal band after i-ceui digestion. mutations at the s rrna, l or l were not detected. the cfr increased the linezolid mic value between -and -fold. this report highlights the ability of se to acquire linezolid resistances. the potential mobility of cfr combined with the clonal tendency for dissemination among staphylococcus spp., represent a serious threat to several potent gram-positive-active agents, including oxazolidinones. active surveillance combined with effective infection control and molecular studies seem prudent to minimise the spread of these resistance mechanisms. the objective is to get a glimpse of the potential impact of infectious diseases on music, as regards to the composer's or performing musician's own disease, living conditions or other relevant elements which might have affected the end result, the music we enjoy today. as music is an art of senses, full of drama, despair, realities of life − or just the opposite, blissful ignorance of those realities, full of romance, beauty, and delicacy − various forms of music was researched paying special attention to infections which potentially have played a significant role in the birth of that particular piece or performance. the entire research process was subjective, biased, and emotional, but done wholeheartedly. it aimed at to taking into account, not only the personal life of a composer or performing musician, but also the historical context in which the music was born. musical examples, served to the audience along with the essential background data, will show the extent to which infections have impacted music. regarding the aetiology of those infections, bacterial, viral and parasitic agents are well represented. in addition, many epochs in history have played their role. sometimes, the connections are surprising, even dramatic. if listened to with a tender ear, music quite often turns out to be affected also by infectious diseases. as physicians we should realise the strength with which some people are driven by this demonic, divine − but altogether beautiful force: music. the prevalence of antibiotic resistance has been increasing in asian countries in recent years. this problem has most likely arisen due to a combination of inadequate infection control practices particularly in hospital settings and the widespread misuse of antibiotics in hospital and community settings. factors that lead to antibiotic misuse include inappropriate antibiotic prescription due to a lack of clinical, microbiological and/or imaging data in many clinical settings in the asian region. a lack of separation of prescribing and dispensing by medical practitioners as practised in many countries in asia as well as the easy availability of over the counter medications also contribute to antibiotic misuse. optimal control of antibiotic use can only be achieved through a multipronged approach that includes better education of the public and medical practitioners on rational use of antibiotic, a review of the health system structure, as well as better control of over the counter sales of antibiotics. upgrading of microbiology and other laboratories and radiological facilities that will enhance the accuracy of clinical diagnosis is also urgently needed in most developing countries to keep pace with the complexities of managing patients in this new era to minimise the widespread practise of inappropriate antibiotic use. examination of the csf for microorganisms, wbc and differential counts, and concentrations of glucose and protein is the primary investigation to diagnose meningitis. however, this csf examination may not always be conclusive, and it can be difficult to distinguish bacterial from viral meningitis. therefore, improvement in diagnostic sensitivity and specificity of bacterial meningitis and development of rapid test for a bacterial aetiology are still needed. this presentation gives a review of the strength and weakness of several analyses and methods to reveal the microbiological agent (i.e. csf microscopy and culture, antigen or antibody detection, molecular methods to detect dna or rna) and the use of several mediators of the host immune response for diagnostic and prognostic purposes. bacterial meningitis is a medical emergency that requires a multidisciplinary approach. a diagnosis of bacterial meningitis is often considered, but the disease can be difficult to recognize. recommendations for antimicrobial therapy are changing as a result of the emergence of antimicrobial resistance. in this lecture, current concepts of the initial approach to the treatment of adults with bacterial meningitis will be summarised. the management of the critically ill patient with bacterial meningitis poses important dilemmas. controversial areas (i.e., prehospital admission antibiotics) will be reviewed and relevant literature will be discussed in the framework of current treatment guidelines, highlighting new developments in adjunctive dexamethasone therapy. acute bacterial meningitis (abm), especifically when caused by infection with streptococcus pneumoniae, still has an unacceptably poor prognosis with a mortality of − %. bacterial infection of the meninges causes one of the most powerful inflammatory reactions known in medicine. yet years ago, this inflammatory reaction was suggested to contribute substantially to brain damage. this concept underlies the use of anti-inflammatory agents as adjunctive therapy in abm. of all adjunctive treatments in abm, only corticosteroids have been properly evaluated in clinical trials. these trials recommend corticosteroids in patients with haemophilus influenzae type b and pneumococcal meningitis (pm). however, adjunctive corticosteroid therapy has several weaknesses such as a narrow treatment window and borderline effects on neurologic sequelae. thus, there is still the need for additional or alternate adjuvants in the therapy of abm. experimental studies using animal models (predominantly of pm) have provided insight into the pathogenic mechanisms underlying brain injury in abm. it is now clear that the autodestructive inflammatory reaction is initiated by the interaction of bacterial components with host pattern recognition receptors (prr) like toll-like receptors (tlr). prr signaling results in the activation of transcription factors like nf-kb which up-regulate the production of proinflammatory cytokines. cytokines like il- b are also potent triggers of nf-kb activation and therefore can exaggerate the inflammatory reaction (via positive feedback loops). as a consequence, great numbers of neutrophils are recruited to the meninges. activated neutrophils release many potentially cytotoxic agents including oxidants and matrix metalloproteinases that can cause collateral damage to brain tissue. additionally to the inflammatory response, direct bacterial cytotoxicty has been identified as a contributor to tissue damage in abm. thus, experimental studies point at four different targets of adjunctive therapy, namely interference with (i) the induction of inflammation (e.g., tlr blockade), (ii) the exaggeration of inflammation (e.g., il- antagonism), and (iii+iv) the generation of cytotoxic factors (either of host or bacterial origin, e.g., scavenging of oxidants). this presentation will give an overview of the pathophysiology of abm (with special emphasis on pm) and highlight promising targets for adjunctive therapy in abm, as deduced from experimental studies. a clinician's approach to managing difficult infections s acute post-surgical prosthetic joint infection optimal management of prosthetic joint infections (pji) remains undefined. important issues such us when the implant can be retained (conservative strategy), optimal duration of antimicrobial therapy (at) or the role of rifampin are yet matter of controversy. in spite of a number of reports, literature appears confusing. among the limitations of the literature we must emphasize: ) different criteria to classify pji; ) different criteria to select for conservative strategy (cs); ) no description of the initial population from which patients were selected for cs; ) very different at (from weeks to chronic suppressive therapy); ) low numbers of patients or short follow-up; ) absence of clinical trials. it is not so surprising that the rates of cs success have varied from to almost %. the most useful classification to approach pji was proposed by tsukayama ( ) . in his series out of patients with early pji managed by a cs (debridement, exchange of polyethylen and implant retention) were cured after weeks of at. the spanish group for the study of pji was constituted in within the spanish network for the study of infectious pathology (reipi), a public funded initiative. data from consecutive cases of early pji attended in hospitals were recorded in an online database. cases managed with cs could be analysed (mean followup of years). sixty-seven patients ( . %) were cured after a mean of days of at. in ( . %) the infection was not controlled (or relapsed) after a mean of days of at, and the implant had to be removed. in other patients ( . %) the implant was not removed, but suppressive at was given because of suspected ongoing infection. results were significantly worse in one hospital. no other factors resulted statistically significant, but there was a trend of worse results for mrsa produced infections (p = . ). time from the symptoms appearance to debridement was shorter in successfully treated cases (median, days) than in failures (median, days); p = . . good functional results were obtained in patients with successfully cs. in summary, a substantial proportion of early pji can be managed with cs strategy and a definite (non suppressive) at. it is difficult to identify patients at higher risk for failure, although mrsa aetiology and longer time until debridement seem to predict failures. different outcomes in some centres suggest that surgical technique could be an important factor for failure. more than million cardiac pacing systems are implanted worldwide and the estimated rate of infections after implantation of permanent endocardial leads is % to %, but varies between . to %. pacemaker infections correspond to different clinical situations including localised infection in the device pocket, pacemaker leads to systemic infection associated with bacteraemia and lead-associated endocarditis. this latter represents to % of all cases of pacemaker infections. the severity of pacemaker related infective endocarditis is sustained by a mortality range between to %. risk factors related to infections of implanted pacemakers are correlated with fever before h before implantation, temporary pacing before implantation and early re-interventions (haematoma, lead dislodgment). in contrast, an inverse correlation is observed between development of infection and antibiotic prophylaxis and implantation of a new system. data to guide therapy in patients with pacemaker infection are limited and the most appropriate management remains to be determined. according to different series, staphylococci accounted for to > % of the responsible organisms. coagulase-negative staphylococci (cns) are reported as predominant pathogens following by staphylocococcus aureus. the biofilm production, responsible for bacterial survival, and the emergence of methicillin-resistant in s. aureus and cns have complicated the management of pacemaker infections. this implies that empiric treatment of suspected pacemaker infection should coverage for staphylococci including methicillin-resistant strains. streptococci, corynebacterium spp, propionibacterium acnes, gram-negative bacilli and candida spp can cause occasional infections. the optimal therapy combines complete device extraction (percutaneous ablation or surgical removal during extracorporeal circulation) and prolonged course of antibiotics, in particular in case of multiresistant bacteria. leaving the device intact is associated with increased mortality and risk of relapsing or persistent infections. in absence of prospective studies, the duration of antibiotic treatment remains to be determined but month has been shown not to be associated with an increased incidence of relapse. shortest course of treatment ( weeks) has been proposed in case of vegetations strictly localised to leads without affecting cardiac valves. antibiotic therapy working alone should be reserved for highly selected patients. infection remains the most critical complication of ventriculoperitoneal shunt placement with an incidence of . − %. factors as the age of patient, aetiology of hydrocephalus, the type of shunt implanted, and the surgeon's experience are determined to be associated with increased risk of infection. children are more likely than adults to acquire shunt infection. the possible reasons are longer hospital stay, higher skin bacterial concentrations, immature immune systems, or more adherent strains of bacteria. staphylococci, as skin commensals, are the main causative organisms. nevertheless, in recent years a change in the epidemiology of microorganisms was observed with an increase of gram-negative bacteria. appropriate systemic antibiotics according to the antimicrobial susceptibility testing and surgical removal of the shunt with temporary external cerebrospinal fluid drainage and shunt replacement following the eradication of the infection are the cornerstone of the treatment of cerebrospinal fluid shunt infections. good compliance with infection control practices, inserion of the catheter under aseptic techniques and short-term perioperative antimicrobial prophylaxis in order to prevent the emergence of drug-resistant subpopulations are important steps in the prevention of shunt infections. o influenza in adults admitted to canadian hospitals: data from two seasons a. mcgeer, d. gravel, g. taylor°, c. weir, c. frenette, j. vayalumkal, a. wong, d. moore, s. michaud, b. amihod (toronto, ottawa, edmonton, montreal, saskatoon, sherbrooke, ca) objective: seasonal influenza (flu) remains a cause of substantial morbidity and mortality. antiviral treatment should be considered for all hospitalised patients with influenza. to better understand the epidemiology and burden of illness within the hospital sector in canada and the current use of antiviral therapy, we carried out a multihospital survey of virologically confirmed flu in hospitalised adults. methods: cnisp is a network of largely teaching hospitals across canada that collaborates to collect data on infections in hospitalised patients. during two consecutive years ( / and / ) hospitals within cnisp identified inpatients > years who had virologically confirmed flu. case patient charts were reviewed to capture demographic and clinical data and to determine whether flu was community (ca) or hospital acquired (ha). cases were reviewed at days to determine outcomes. deaths at days were reviewed to determine whether flu was a main or contributing cause. results: fifteen ( / ) and ( / ) hospitals were recruited from the cnisp network. virologically confirmed cases of flu were found, in / ( % flu a) and in / ( % flu a). mean patient age was years, % were male. there was documentation of patient vaccination that season in %. incidence of ca flu was / , admissions in / (range by hospital − ) and in / ( − ). admitting diagnoses in ca cases were: pneumonia or influenza %, exacerbation of copd %, sepsis or fever not otherwise specified %, cardiac diagnoses %, other diagnoses %. % of cases were ha, range by hospital . − . / , patient days. % of patients were managed with droplet and contact isolation practices, an n- mask was used in %. % of ca cases but % of ha cases received antiviral therapy p < . , almost entirely oseltamivir. % of cases were admitted to an icu; -day mortality was % with . % attributed to influenza. conclusion: there is considerable season-season and hospital-hospital variation in flu in patients in canadian hospitals. hospitalised patients ca flu present with a wide spectrum of clinical diagnoses; nearly a quarter of all cases were ha. few ca cases but most ha cases were treated with antiviral drugs. attributable day mortality was . %. v. papastamopoulos, e. kakalou°, t. panagiotopoulos, j. baraboutis, m. samarkos, a. skoutelis (athens, gr) objectives: our study sought to describe influenza vaccination coverage among adults in greece for the season / . methods: we conducted a random-sampling, telephone based household survey among adult individuals in greece. for this purpose a sample of adults representative of the basic demographic, social and geographical characteristics of the overall greek population according to the latest national survey, was used. two target groups were determined for analysis: persons > years of age and persons with chronic conditions such as respiratory and heart conditions (other than hypertension), diabetes mellitus and other conditions. results: the influenza vaccination rate for the season / among the adult population in greece was: % for the overall adult population ( . % for men, . % for women), . % for people > years of age, % for persons with chronic illness ( . % for persons with respiratory illness, . for persons with heart conditions, % for persons with diabetes mellitus). a high rate of % of the overall population reaching % among persons with chronic conditions report having had any type of contact with the national health system or a private physician within the last three years. among them only . % had been recommended to get vaccinated. among the ones recommended any vaccination, . % of persons with respiratory illness, % of persons with diabetes mellitus and . % of persons with heart conditions had been recommended to get the influenza vaccine. conclusions: available data show unacceptably low levels of influenza vaccination coverage among vulnerable groups such as the population over years of age and people living with chronic illness. influenza vaccination is the only preventive measure reducing influenza morbidity and mortality and its use has proven cost-effective among high risk groups. it is also the main vaccine recommended by physicians. however the overall rate of physicians recommendation of vaccination is very low. dynamic efforts are thus needed to design and implement strategies and policies that have demonstrated their rigorous effectiveness in enhancing influenza vaccination coverage rates. conclusions: nasopharyngeal sampling with flocked swabs is well tolerated and suitable to be used in an outpatient setting. implementation of real-time mono and multiplex naats results in a significant improvement of the rate in diagnosing lrti. hrv account for the majority of viral lrti in primary care followed by influenza and coronaviruses but also rsv and hmpv are prevalent in an adult population. in this study, polyomaviruses were detected of which were involved in a double infection. methods: observational analysis of a prospective cohort of nonseverely immunosuppressed adults with pp requiring hospitalisation ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . of them, were diagnosed by urinary antigen and/or were diagnosed by culture. overall, % of pneumococcal strains were available for serotyping (quellung) and % for pfge (smal) and or mlst. the diagnosis of septic shock was based on a systolic blood pressure < mmhg and peripheral hypoperfusion with clinical or bacteriologic evidence of uncontrolled infection. results: a total of ( %) patients with pp had septic shock at presentation. patients with shock were younger ( vs yrs; p = . ), were more frequently current smokers ( % vs %; p = . ), had received more commonly corticosteroid therapy ( % vs %; p = . ), and were more frequently classified into high-risk psi classes ( % vs %; p < . ) than those who did not have this complication. they were also less likely to have received prior influenza vaccine ( % vs %; p = . ) and had more frequently bacteraemia ( % vs %; p = . ). no significant differences were found in rates of penicillin-( % vs %) and erythromycin-resistance ( % vs %). serotype was more commonly associated with shock ( % vs %; p = . ), whereas serotype was rarely associated with this complication ( % vs %; p = . ). no significant differences were found regarding genotypes: st ( % vs %), netherlands-ser -st ( % vs %), netherlands-ser -st ( % vs %), spain-ser v-st ( % vs %). patients with shock required more frequently mechanical ventilation ( % vs %; p < . ), and had longer los ( vs days; p < . ). early ( % vs %; p < . ) and overall case-fatality rates ( % vs %; p < . ) were higher in patients with shock. conclusions: pp presenting with septic shock is still associated with a poor outcome. it occurs mainly in current smokers, patients receiving corticosteroids, and in those infections caused by serotype . prior influenza vaccination and pp caused by serotype are associated with a lower risk of shock. o high long-term mortality rate after initial recovery from severe community-acquired pneumonia background: despite the presence of antibiotics and vaccination strategies against pneumocci, community-acquired pneumonia (cap) is still a major cause for mortality in developed countries. however, it is unclear how an episode of cap influences long-term survival after initial recovery. therefore, we determined mortality up to years after discharge in patients hospitalised because of an episode of severe cap in a non-intensive care setting. methods: in hospitals in the netherlands, patients (pts) with severe cap (psi class iv and v without need for treatment in icu) were prospectively followed for days and mortality up to years after discharge was determined using the dutch municipal public records database. we used cox regression analysis to examine predictors for mortality. results: compared to strategy , strategy resulted in slightly higher costs (chf , vs. , ) but fewer infections (. vs. . ) during patients' mean length-of-stay, producing an incremental costeffectiveness ratio (icer) of chf , per mrsa infection avoided. strategy was dominated by strategies and (both more costly and less effective). sensitivity analyses suggest that prevalence of colonisation on admission is a stronger predictor of cost-effectiveness than the costs of infection or rapid screening, the probability of cross-transmission, or the incremental costs of isolation and contact precautions. increasing the relatively low on-admission prevalence at our centre by % lowers the icer to chf , per infection avoided. in contrast, increasing the cost of each infection, the cost of rapid screening, or the risk of cross-transmission by % only marginally affects the icer. conclusion: this analysis suggests that compared to risk factor identification and pre-emptive isolation, universal rapid screening upon surgical admission is not strongly cost-effective at our centre. however, local epidemiology plays an important role. in particular, settings with higher prevalence of colonisation on admission may find universal rapid screening more cost-effective. of note, no screening is undesirable, as costs and infections would be higher. results: admission and weekly screening coupled with patient isolation was found to dramatically reduce the number of mrsa acquisitions. the largest reductions were obtained with pcr technology, followed by chromogenic agar. the differences, however, were surprisingly small, and all screening technologies achieved reductions in mrsa acquisition of close to % compared with the no-intervention scenario. nonetheless, chromogenic and pcr-based systems were able to decrease the number of unisolated mrsa-bed-days by approximately and % respectively. conclusions: the small differences in the ability of the screening technologies to reduce mrsa acquisition reflect both a relatively low estimated isolation efficacy and the observed highly skewed distribution of icu-stays, and may provide some important insights into the reasons for recent disappointing trial results. in particular, the skewed length of stay distribution means that most mrsa-bed days are accounted for by relatively long-stay patients for whom rapid detection will make the least difference. key sources of uncertainty were found to be isolation effectiveness and attributable mortality due to mrsa infections, both of which are difficult to accurately estimate with currently available data. the model results allow us to quantify the expected value of reducing these key uncertainties, and help to provide a rational basis for setting future research priorities. objectives: we have shown that there is substantial colonisation of mrsa among nursing home residents and staff with our recently conducted point prevalence study in nursing homes which revealed an overall prevalence rate of % in residents and . % in staff. the aim of this study was, therefore, to test the effectiveness of an intervention in nursing homes which sought to improve standards of infection control as a means of reducing mrsa prevalence. methods: a cluster randomised controlled trial (crct) involving nursing homes, with each home representing the unit of analysis, was performed. the study ran for months with data collected at baseline, , and months. nasal swabs were taken at baseline from consenting residents and staff in all homes prior to randomisation with an audit of infection control procedures also undertaken. following collection of these baseline data, nursing homes were allocated to the intervention or control arm ( : ). intervention home staff were trained in infection control, specifically hand hygiene, catheter care, barrier approaches such as use of gloves, aprons and masks, and decontamination of equipment and the environment with usual practice continuing in control homes. after each data collection timepoint, feedback was given to the intervention homes in terms of their performance and further education and training provided as required. the primary outcome was the prevalence of mrsa in intervention homes compared to control sites. results: preliminary analysis of the data has revealed no significant change in the prevalence of mrsa in the intervention and control homes, taking account of the clustering, over the one-year intervention period [risk ratio . ; % confidence intervals (ci) . − . ]. however, there was an improvement in infection control audit scores in the intervention homes, with a mean score in control homes at months of . % compared with . % in the intervention sites; these scores were significantly different (paired t-test, p < . ). the results suggest that infection control education and training as implemented in this study was not sufficient to affect mrsa prevalence. therefore, a more detailed education and training package either alone or in combination with mrsa decolonisation of staff and residents, may be required to reduce mrsa prevalence within this unique environment. [ objectives: in a response to the rapid global increase in the nosocomial prevalence of multi-resistant micro-organisms, infection control measures, such as patient isolation, are increasingly used. it is unknown how these measures influence the quality of life (qol) of patients during short-term isolation, and this was determined in a prospective matched cohort study. methods: all adult patients needing isolation in a single-patient room between / and / in the umc utrecht were eligible and included − hours after start of isolation (after giving informed consent and being able to fulfil study requirements). for each index patient we identified two control patients, admitted to the same wards at the same time, yet not subjected to any isolation measure. anxiety and depression and qol were assessed using the hospital anxiety and depression scale (hads) and visual analogue scale (eq- d-vas) in all patients. opinions on and experiences with isolation were measured in isolated patients by means of a self-developed 'isolation evaluation questionnaire'. results: isolated patients and controls were included, with comparable baseline characteristics (age, sex, nationality, level of education, length of hospital stay and severity of underlying disease and co-morbidity (using the cumulative illness rating scale)). reasons for isolation were clostridium difficile-associated disease (n = , %), high risk for mrsa carriage (n = , %), or resistant gram-negative bacteria (n = , %). mean scores of questionnaires are presented in table . isin univariate analysis only duration of isolation of hours (compared to hours) was associated with a reduced quality of life (vas . compared to . , p . ). on a visual analogue score of opposite terms isolation measures were rated with means of . , . and . for safety, usefulness and quietness, respectively. conclusion: short-term isolation (up to hours) is not associated with anxiousness or depression, but with positive feelings about safety, usefulness and quietness. index patients (n = ), mean (sd) . ( . ) . ( . ) . ( . ) . ( . ) control patients (n = ), mean (sd) . ( . ) . ( . ) . ( . objectives: there is a lack of data about the impact of healthcare associated infection (hai) on the experience of individual patients. this information is essential to empower health organisations to understand, prioritise, develop and implement solutions that will minimise risks to patients. this study explored comparable narratives from patients who had experienced a staphylococcus aureus blood stream infection with patients who had not. we conducted qualitative semi-structured interviews with eighteen adults who had previously been an in-patient in an acute teaching hospital in scotland. nine patients had had a laboratory diagnosed staphylococcus aureus blood stream infection and nine had no blood stream infection. all patients were interviewed for − minutes. the interviewer asked patients about their thoughts around hai, what concerns they had or still do, what measures they took to safeguard themselves from hai and how their experience impacted on their confidence of the nhs. probing questions were then asked depending on the responses given to the initial questions. all interviews were recorded, transcribed and analysed thematically. results: analysis of transcribed interviews is ongoing. preliminary analysis showed that all patients had positive and negative comments about infection prevention and control practice in the hospital. specific concerns included poor communication, poor cleanliness, awareness of patient boarding, lack of facilities, staff shortages and multi-tasking. some patients who had experienced bacteraemia said they had not been informed about the infection. those who had been informed were not given clear information about treatment or subsequent results. most patients were not specifically told what they or their family should do to safeguard them from infection and little or no written information about hai was provided. most patients are worried about hai on future admissions. the concerns of patients were not fundamentally different if they did or did not experience blood stream infection. the patient's reported experiences show that they have a broad awareness of systems issues that may increase risk of infection. consequently we need to involve patients in the design and evaluation of systems change and information that will improve patient experience. improving the safety and reliability of the system will have direct benefits for all patients in the hospital, not just the ones at risk of hai. analysis of surgical specialties separately revealed a significant reduction of mortality in cardiothoracic surgery who had been treated with mup-chx ( . % ( / ) vs. . % ( / ), p = . , figure) . in other surgical specialties no significant difference was found. conclusion: peri-operative application of mup-chx in nasal carriers of s. aureus undergoing cardiothoracic surgery results in a threefold reduction of mortality after one year. o a lot done, more to do − a survey of teaching about healthcare-associated infections in uk and irish medical schools h. humphreys°, d. o'brien, j. richards, k. walton, g. phillips (dublin, ie; norwich, newcastle-upon-tyne, dundee, uk) objectives: patient safety and the prevention of healthcare-associated infections (hcai) are increasingly important health issues. medical doctors have traditionally been poor in complying with preventative measures to minimise hcai such as hand hygiene compliance. we surveyed medical schools in the uk and ireland to assess what is being taught and assessed in this area. methods: a questionnaire was drafted, piloted and then subsequently forwarded to the heads of medical schools as well as to known contact professionals with an interest in hcai in medical schools. the questionnaire surveyed topics covered in the curricula, the modalities used to assess knowledge and practice, the usefulness of various teaching methods and materials, e.g. lectures, and what education resources were available. results: replies were received from ( %) medical schools; two supplied data on their undergraduate and postgraduate courses. only ( %) covered hcai as a quality and safety issue but over % covered prevalence, recognised risk factors, transmission, and preventative measures. ( %) medical schools assessed competence in undertaking aseptic techniques and the disposal of sharps and mcqs were the most common ( %) means of assessment. case scenarios, resource materials and clinical skills stations were used in educating students in ( %), ( %) and ( %) medicals schools respectively. ( %) medical schools would be willing to share educational resources on hcai with other medical schools. conclusions: medical schools in the uk and ireland include hcai in their curricula but its importance as a safety and quality issue needs to be further emphasized. there is potential for agreeing a core curriculum on hcai and for sharing teaching resources such as videos and e-learning material. objectives: noroviruses are most common cause of outbreaks of gastroenteritis in uk national health service hospitals, leading to ward closure costing as much as £ million per annum. using a detailed data set on norovirus outbreaks from three hospital systems in the south west of england, we estimated ( ) the relative importance of introduction of norovirus from the community and within the hospital and ( ) the cost effectiveness of ward closure at different time points during an outbreak. methods: using regression models we examined the association between number of new outbreaks in a hospital and community levels of activity and number of outbreaks currently occurring in other wards within the hospital. we examined the effect of different ward types (admission, general and long stay units) and whether the ward was open or closed to new admissions on a given day. we then undertook as analysis of cost (-effectiveness) of unit closure by developing a dynamic transmission model taking into account that ward closure may reduce norovirus transmission within and between wards. the stochastic simulation model was based on the actual characteristics of an acute hospital and the norovirus transmission parameters quantified in the statistical analysis. we measured the costs and benefits of closing affected wards at , and days after the onset of symptoms in the first case. results: community level of norovirus infection had a significant effect on the occurrence of new outbreaks as did outbreaks in admission and general medical units. the cost of closing wards to new admissions varied between £ . million to £ . million depending on the assumed effectiveness of closure in curtailing transmission. cost of bed day loss − compared with staff illness -accounted for around % of the total cost of closure. although the total number of cases tends to fall with rapid ward closure (by around % compared with no closure), the actual cost of control is similar regardless of when the closure is performed. we have developed a modelling framework to assess the effectiveness and cost-effectiveness of strategies to control norovirus outbreaks in hospital settings. ward closure is effective at preventing cases but since closure itself is an expensive intervention, it may not always be cost-effective. . other prevalent ribotypes were ( %) and ( %). % of the isolates originated from hospitals located in healthboard areas. the remaining isolates of ribotype originated from hospitals across scotland. in vitro % of isolates were resistant to clindamycin with a mic range of − mg/l, mic of mg/l and mic of mg/l. furthermore % of the isolates were highly resistant to erythromycin (mic mg/l, mic mg/l), and to levofloxacin and moxifloxacin (mic mg/l, mic mg/l for both), while % of these isolates were resistant to cefotaxime (mic = mg/l, mic = mg/l). all isolates were susceptible to metronidazole, vancomycin, meropenem and piperacillin-tazobactam. high frequencies of clindamycin, erythromycin, levofloxacin, moxifloxacin and cefotaxime resistance were also found among isolates of ribotype ( − %) and ( - %). conclusion: until c. difficile ribotype was only reported infrequently in scotland. in , reports of ribotype became more frequent and clusters were detected in hospitals. the majority ( %) of ribotype isolates were resistant to clindamycin. three other european countries have previously reported clindamycin resistance in pcr ribotype , albeit with a higher mic of > mg/l. objectives: to analyze trends in mortality due to clostridium difficile enterocolitis and to describe the most affected groups in order to better understand current clostridium difficile changing epidemiology. methods: we reviewed mortality data from the flanders and brussels regions in belgium (about million inhabitants). we selected those records in which icd- code a . (enterocolitis due to clostridium difficile) appeared as underlying cause of death within the death certificate. age-and sex-specific mortality rates were calculated for the period - . direct standardisation was performed using the european standard population and % confidence intervals were calculated. stata ® and excel ® were used as statistical software. objectives: toxigenic clostridium difficile is an enteric pathogen typical in the hospital environment but also community-acquired cases have been reported. however, relatively few attempts have been made to clarify the role of soil or water as a source of c. difficile infection. in november-december , the drinking water distribution system in the town of nokia, finland was massively contaminated with treated sewage effluent resulting in a large gastroenteritis outbreak. the aim of the present study was to evaluate if contaminated water in this outbreak was also a potential source of c. difficile infection. a sample from the contaminated tap water and a treated sewage effluent sample were collected as soon as possible after the massive faecal contamination of the drinking water distribution system had occurred. c. difficile was isolated from heat-treated water samples by filtrating of ml, ml and ml volumes of water and placing the membranes on selective ccey agar plates, which were anaerobically incubated for d. stool samples from the patients fallen ill during the epidemic were examined for enteric pathogens, including c. difficile. all potential c. difficile colonies were subcultured on ccfa agar plates and toxin-positive isolates were identified by pcr. pcr ribotyping was performed according to the protocol of the anaerobe reference unit in cardiff, uk, using the cardiff-ecdc culture collection as a set of reference strains. after gel electrophoresis, the band patterns were analyzed using the bionumerics software. results: altogether c. difficile isolates were found in water samples. twelve isolates were toxin-positive; isolates were from contaminated tap water and isolates from treated sewage effluent, the latter being the contamination source. among the tap water and sewage effluent isolates, and distinct pcr ribotype profiles were identified, respectively. the human faecal c. difficile isolates detected were divided into distinct pcr ribotype profiles. none of the profiles were identical with that of the hypervirulent pcr ribotype . two isolates, one from tap water and another from a patient, had an indistinguishable pcr ribotype profile. conclusion: our observation implies that c. difficile contamination of a tap water distribution system had occurred. waterborne transmission of toxigenic c. difficile and subsequent c. difficile infection seems possible. objectives: an accurate and rapid method is needed for typing of toxigenic clostridium difficile. a commercial automated repetitive pcr system (rep-pcr; diversilab ® , biomérieux inc., st louis, usa) utilises amplification and subsequent automated electrophoretic separation of the repetitive extragenic palindromic sequences of c. difficile. our aim was to evaluate the performance of this rep-pcr method for genotyping of c. difficile isolates and to compare it to pcr ribotyping. in addition, the correlation between the rep-pcr and the virulence gene profiles of c. difficile strains was studied. methods: a total of toxin-positive c. difficile isolates were studied. we included consecutive isolates from two laboratories in finland, containing also strains of the hypervirulent c. difficile ribotype . in addition, selected c. difficile strains with > bp deletions in their tcdc genes were analyzed. the dna was extracted and the rep-pcr performed according to the manufacturer's instructions. the amplification products of rep-pcr were detected and analyzed using the diversilab system. further analysis was performed with the web-based software accompanying the system. the usefulness of the library construction option of the diverslab system for isolate comparison was tested. the virulence genes (tcda, tcdb, cdta, cdtb and tcdc) were analyzed by conventional pcr and the whole gene sequencing of tcdc was performed from isolates with deletions > bp. pcr ribotyping was performed using the protocol of the anaerobe reference unit in cardiff, uk. the correlation between the rep-pcr profile and the ribotype was excellent. all major ribotype groups were clustered in their own rep-pcr groups. interestingly, subgroups could be found with rep-pcr within two most prevalent ribotypes and . the automated rep-pcr proved to be reproducible; the results from separate dna isolations and pcr-runs/microfluid electrophoresis as well as the results performed by different individuals of laboratory personnel were comparable. the rep-pcr profiles and pcr ribotypes correlated also with the virulence gene profiles. conclusion: this automated rep-pcr represents an effective and reproducible method for the genetic characterisation of c. difficile strains in clinical laboratories with molecular biology facilities. the constructed c. difficile library allows comparing the relatedness of c. difficile strains and their fingerprints over time. objectives: clostridium difficile infection (cdi) is a serious diarrhoeal illness associated with high morbidity and mortality. currently available treatments (oral vancomycin or metronidazole) usually produce good resolution of diarrhoea but are associated with a % to % incidence of recurrence. opt- , the first in a new class of macrocyclic antibiotics, is bactericidal via unique inhibition of rna polymerase. this phase , non-inferiority clinical trial was conducted in more than sites in north america and compared the efficacy and safety of opt- and vancomycin in treating cdi. methods: eligible patients were adults with acute cdi symptoms and a positive stool toxin test. patients received oral opt- ( mg twice daily) or oral vancomycin ( mg times daily) for days. primary end point was clinical cure (resolution of symptoms and no further need for cdi therapy days after stopping study drug). secondary end point was cdi recurrence (diarrhoea and positive stool toxin test within weeks after treatment). global cure was defined as a clinical cure with no recurrence. results: patients were enrolled and % were evaluable. in the per protocol (pp) population (n = ), mean age was . ± . years and . % of patients were male. equivalent rates of clinical cure were observed with opt- ( %) and vancomycin ( %) in the pp analysis; similar outcomes were observed in a modified intent-to-treat (mitt) analysis. significantly fewer patients treated with opt- ( %) than vancomycin ( %) experienced recurrence in the pp analysis (p = . ) and in the mitt analysis ( % vs %; p = . ). significantly more opt- -treated patients achieved global cure ( %) than vancomycintreated patients in the pp analysis ( %; p = . ) and in the mitt analysis ( % vs %; p = . ). opt- was well tolerated with an adverse event profile similar to that of vancomycin. in this study -the largest comparative trial of a new antimicrobial agent versus vancomycin for the treatment of cdi -clinical cure rates after treatment with opt- or vancomycin were equivalent. however, opt- was associated with a significantly lower recurrence rate and a higher global cure rate than vancomycin. opt- is an oral, non-absorbed agent that has a convenient (twice daily) dosing schedule and low risk of adverse events. opt- represents a potential new treatment option for cdi that is associated with a lower recurrence rate than currently available treatments. results: sequence analysis (sa) revealed that locus a is absent in type and that some mismatches are present in the primer annealing sites for loci b, c and g. lowering the annealing temperature and increasing the magnesium chloride concentration for loci b, c and g resolved the low yield of pcr products. applying the mlva on type strains revealed that ( %) strains, encompassing isolates from human (n = ) and porcine (n = ) origin, are genetically related with a summed tandem repeat differences (strd) ). three clonal complexes (cc, defined by strd ) were recognized; one cc contained both human (n = ) and porcine (n = ) strains. the optimised mlva identified genetically related clusters and cc among the isolates from e and ni. ccs contain isolates from more than one hospital and indeed for several clusters isolates from both e and ni. isolates obtained from ni years earlier were part of one large cc. the optimised mlva can distinguish and/or group type strains from distinct settings. type strains from human and animal origin are genetically related. the clustering of some isolates from distinct settings is consistent with community sources for type . the last observations suggest zoonotic transmission. objectives: this paper updates our assessment of the contribution that community-associated clostridium difficile infection (cdi), as reported to the english mandatory surveillance scheme since , makes to both the acute and community sectors of the national health service (nhs) in england. methods: nhs acute trusts (hospital groups) in england are required to report all c. difficile toxin positive diarrhoeal specimens processed by their laboratories whether the patients were in hospital or the community at the time of onset of the illness or when the specimen was taken via a web enabled reporting system. positive specimens from the same patient within days are not reported. reported cases in patients under years of age were omitted from this analysis. enhanced surveillance data (including information on date of admission, patient location prior to testing, sex, age and patient category) on cdi have been collected through a web-enabled reporting system since april . risk factor information is completed on a voluntary basis. results: more than , cases of cdi in patients aged > years were reported, % of these cases were taken in non-acute settings of which % were taken by a general practitioner. a further % of specimens were taken on presentation or < days of admission into an acute trust. approximately % of all cases had at least one risk factor field completed, > , cases reported risk factor information on episode category; % of these cases were community associated and % were hospital acquired. the information reported suggests that only % of the community associated cases were from patients with continued infection or relapsed episodes of cdi, this is compared to % of the hospital acquired cases who had continued infection or relapsed episodes of cdi. conclusions: % of the c. difficile specimens reported by acute trusts were diagnosed in a community setting. published studies suggest that − % of these might be expected to have been acquired during a hospital stay within the previous month (i.e. were community onset hospital acquired cases). future work is required to investigate whether there are differences in the epidemiology, risk factors e.g. antibiotic exposure and outcome of patients with community onset disease. o clostridium difficile-associated disease: a newly notifiable disease in ireland m. skally, f. roche, d. o'flanagan, p. mckeown, f. fitzpatrick°( dublin, ie) new cases of clostridium difficile-associated disease (cdad) became notifiable in ireland on th may . the main objective of this new notification process was to provide a national overview of the epidemiology and burden of cdad. this paper review the first six months of preliminary data notified. methods: the interim case definitions for new and recurrent cdad cases proposed by the european society for clinical microbiology and infectious diseases (escmid) study group for c. difficile were employed. this report reviews the weekly events of cdad extracted from the computerised infectious disease reporting (cidr) system in january . census of population figures were used as denominator data in the calculation of incidence rates. results presented represent weeks of data submitted. results: there were new cdad cases notified on cidr between the th may and th december , representing a crude incidence rate (cir) of . cases/ population (estimated annual cir is . cases/ , ). all cases were laboratory confirmed. there was a higher occurrence of cases in females. the male:female ratio for the period was : . . in . % of cases the sex was unknown. . % of cases were in the greater than years age category. the preliminary data submitted on cidr indicate that . % of cases were hospital inpatients and . % of cases were either gp patients or outpatients. the origin of . % of samples is unknown. there was large variation between the public health regions (table ) . the incidence of cdad in ireland is prominent in older age groups and in healthcare settings. what is more remarkable is the regional variation of cases reported. this varies from . per , in the north east to . per , in the west. the seasonal trend is indistinguishable at present due to late and batch notifications from institutions. o clostridium difficile-associated diarrhoea in immunosuppressed patients with cancer objective: to assess the epidemiology, clinical features and outcome of clostridium difficile (cd) associated diarrhoea in immunosuppressed patients with cancer. methods: review of all episodes of cd associated diarrhoea documented in adults with cancer and haematopoietic stem cell recipients ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . microbiologic diagnosis included cd isolation from stool samples, direct detection of cd toxin, and testing for cytotoxin production by the isolated strain. we documented a significant increase of cd associated diarrhoea, from . / admissions in to . / admissions in (p < . ). there were episodes in patients. thirty-one patients were male ( %) with a mean age of years (± ). forty three ( %) patients had an haematological underlying disease and had solid tumour; ( %) had received previous chemotherapy, ( %) were stem cell transplant recipients ( presenting with gvhd) and ( %) were neutropenic (< ). in the previous month patients ( %) had received one or more antibiotics (cephalosporins . %, glycopeptides %, carbapenems . %, betalactam + betalactam inhibitors %, quinolones %). fever > ºc ( %) and abdominal pain ( %) were the most frequent manifestations, and the diarrhoea was hemorrhagic in % of the cases. most patients ( %) were treated with metronidazole (median days), and the antibiotic therapy was discontinued in %. in patients who had recovered from neutropenia, the diarrhoea resolved just by discontinuing the antibiotic therapy. no patient developed toxic megacolon or needed surgery. three patients ( . %) had relapses. overall mortality (< days) was % ( patients). the incidence of cd associated diarrhoea in cancer patients has increased significantly in recent years. it is related with important morbidity and mortality. better strategies to improve its prevention and treatment are needed. s linking research to the clinic: how laboratory findings relate to management of invasive candida infections the role of the research laboratory in the management of invasive candida infections goes beyond routinely available tests for identification of candida species and susceptibility testing of antifungal agents. cutting-edge molecular epidemiology technologies have been used to type isolates of candida species based on their dna sequences. multilocus sequence typing schemes have been designed for c. albicans, c. dubliniensis, c. glabrata, c. krusei and c. tropicalis. multi-locus sequence typing can be used to investigate possible hospital outbreaks of infection (finding widely different strain types within a unit indicates no outbreak, although the converse is not true). for c. albicans, typing multiple isolates from the same patient has shown that people tend to harbour as commensals a mixture of closely related but different strain types, which may provide for selection of the most appropriate type for invasion of a particular tissue or in response to antifungal treatment. strains in c. albicans clade , the largest group of related strain types, have a higher proportion of isolates resistant to flucytosine than other clades, and they all share a common resistance mechanism. research on mechanisms of resistance of candida species to many types of antifungal has progressed to the point that some investigators are looking to design dna chips that could be used both for identification and for susceptibility testing of a candida isolate. much research effort goes into detailed study of host-fungus crosstalk in experimental candida infections. animal models of infection have been greatly refined and the latest research shows how early release of chemokines that attract neutrophils into infected tissues contributes to the immunopathology of candida infection. this rapid, innate immune response also emphasizes the need for antifungal intervention at the earliest possible stage to provide the best chance for successful treatment of a disseminated candida infection − a finding now supported by clinical data as well as experimental models. translation of the latest research advances into practical diagnostic tests and new therapeutic approaches for candida infections always takes a long time − typically years − and not all research results find clinical applications. however, the level of effort invested in basic candida research ensures support for steady progress in diagnosis and management. the echinocandins are semi-synthetic lipopeptides that are increasingly used for the prevention and treatment of invasive fungal infections. understanding the pharmacokinetic and pharmacodynamic (pk/pd) characteristics of these compounds is critical for their optimal clinical use. the echinocandins have potent in vitro activity against candida spp., although c. parapsilosis is less susceptible than other candida species. the molecular mechanisms of resistance in candida species, which relate to amino acid substitutions in 'hot spots' within the fks gene, are becoming well characterised. susceptibility breakpoints for all three clinically available compounds have been determined recently by the clinical laboratory standards institute, with a 'susceptible-only' breakpoint of > mg/l suggested. the pk/pd of the echinocandins have been determined in experimental models of disseminated candidiasis, and of both disseminated and pulmonary invasive aspergillosis. these studies suggest that the echinocandins: ( ) display concentration-dependent antifungal killing (or effect); ( ) are extensively distributed into peripheral tissues, where they exhibit prolonged mean residence times at the site of infection; ( ) are fungicidal against candida spp. and induce dose-dependent morphological changes in aspergillus spp.; and ( ) result in a diminished propensity for angioinvasion by aspergillus spp. recent evidence also suggests that the echinocandins have important immunomodulatory properties, which may contribute significantly to their observed antifungal effect. pk/pd modelling and laboratory animal-to-human bridging techniques have been used to identify safe and effective dosages for the echinocandins for relatively uncommon clinical syndromes such as neonatal haematogenous candida meningoencephalitis. these techniques are an efficient method of identifying effective regimens for humans that can be expedited for study in clinical trials. pk/pd modelling techniques can and should be used to address outstanding clinical queries in relation to these compounds, including optimal dosages, decision-support analysis for the setting of in vitro antifungal susceptibility breakpoints and the clinical relevance of inherent or acquired reduced antifungal susceptibility. s invasive candidiasis: which antifungal treatment for which patient? management of patients with invasive candidiasis represents a complex issue owing to the heterogeneity of patients in whom these infections occur. established risk factors for invasive candidiasis, which include total parenteral nutrition, multiple organ failure and candida colonisation, are common to many types of patients that are treated within the critical care setting. furthermore, the severity of the underlying condition in these patients necessitates swift antifungal treatment to ensure optimal outcomes. an additional factor for consideration when treating candida infections is the changing epidemiology of candida species; potentially fluconazole-resistant species such as c. glabrata and c. krusei are becoming more common, particularly in patients with prior fluconazole exposure. a range of antifungal agents is available with in vitro activity against candida species. however, not all of these agents are suitable options for the clinical management of invasive candidiasis because of the overall complexity of both infection and underlying condition. for example, the position of the polyenes, particularly amphotericin b deoxycholate, is becoming less tenable as the risk of renal complications is increasingly regarded as unacceptable in patients that are likely to have or be at risk of multiple organ failure. furthermore, because of the increasing prevalence of fluconazole-resistant species, recent guidelines no longer recommend the use of azoles as first-line treatment for invasive candidiasis except in special cases, focusing instead on the echinocandin agents. there is now a wealth of clinical data available for the echinocandins. micafungin, for example, has been assessed in invasive candidiasis in clinical trials that included a wide variety of underlying conditions and patterns of infection, including neutropenic patients and those with deep infections such as peritonitis. furthermore, micafungin is the most extensively evaluated of the echinocandins in paediatric patients, having been tested both in children up to the age of years and in premature infants and neonates. optimal management of patients with invasive candidiasis depends on a strategy that takes into account the complex nature of the disease. judicious selection of antifungal treatment should be accompanied by consideration of non-drug-related factors that improve survival, such as careful assessment of intravenous catheters and their potential involvement in candida infections. patients with invasive candidiasis often have underlying conditions that are severe illnesses in themselves. these range from neutropenia during cancer chemotherapy to the multi-organ failure of intensive care unit patients. against this background of severe underlying illness, it can be difficult to appreciate the success or otherwise of treatment strategies for candida infections. in the last decade, major advances have been made in antifungal therapy with the introduction of . echinocandins; . extended-spectrum azoles; and . lipid formulations of amphotericin b. robust clinical studies for their successful use in candidaemia have been published. however, it is important to translate these studies into practical strategies for the care of individual patients. in this presentation, individual cases will be used to provide insights into the successes and failures of these antifungal classes for the management of invasive candidiasis. specific interest will be focused on the use of fluconazole versus the echinocandins. these micafungin-based cases will be supported by insights from the evidence-based literature combined with practical experiences at the bedside. the factors to be considered are: . spectrum of activity; . drug toxicity; . drug interactions; . drug resistance; . pharmacology; . diagnosis; . site of infection; . use of biomarkers/cultures in treatment strategies; and . costs. it is important to realise that large clinical trials exclude many patients with invasive candidiasis. therefore, with the use of individual cases, it is possible to provide further insights into the clinical use of these outstanding antifungal agents. patient management: the era of rapid diagnostic results (symposium organised by cepheid) s will community mrsa and clostridium difficile change infection control in hospitals? infections caused by methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococci, and clostridium difficile are inter-related in healthcare institutions. the emergence of epidemic mrsa and c. difficile strains has placed a greater burden on infection control systems in healthcare facilities, which often must increase surveillance and change disinfection strategies to halt the transmission of these pathogens in hospitals. ironically, the usa mrsa strain arose in the community but now is being transmitted frequently in healthcare settings, while the epidemic nap /bi/ c. difficile strain was originally a healthcare-associated pathogen, which now is causing considerable morbidity in community settings. to successfully slow the spread of these pathogens, infection control must work closely with both the laboratory and pharmacy services to ensure that these organisms are detected rapidly and that the selective pressure to maintain the organisms in the institution are reduced. clearly, bundles of interventions, rather than single approaches, are necessary to contain the spread of these organisms in hospitals. the continued influx of patients with communityacquired mrsa and c. difficile infections into healthcare institutions is a challenge for infection control practitioners that will clearly increase in the future. the food borne pathogen l. monocytogenes discovered by murray in is responsible for a severe infection with various clinical features (gastroenteritis, meningitis, meningoencephalitis and materno foetal infections) and a high mortality rate ( %). the disease is due to the ability of listeria to cross three host barriers during infection: the intestinal barrier, the placental barrier and the blood brain barrier. it is also due to listeria capacity to survive in macrophages and to enter into non phagocytic cells, such epithelial cells. recovery from infection and protection against reinfection are due to a t-cell response, explaining why listeria has since many years has become a model in immunology. nearly three decades of molecular biology and cell biology approaches coupled to genetic and post-genomic studies have promoted listeria among the best models in infection biology. in depth studies of the mechanism of entry into cells has help unraveling how listeria crosses the intestinal and placental barrier. unsuspected concepts in cell biology were discovered. post-genomic studies have recently allowed to unveil the listeria transcriptional landscape during switch from saprophytism to virulence. the talk will give an overview highlighting recent results in the frame work of well established data. the last several decades of research in medical mycology have offered great insights into fungal cell biology, epidemiology, phylogenetics and the cells and molecules involved in the pathogenesis of fungal disease. a legitimate question is to ask to what extent our extensive advances in comprehension of the biology of fungal pathogens have contributed to improvements in diagnosis and treatment. to what extent do patients benefit from translation of basic research into tools for clinical management? and the equally valid question: to what extent does biological science benefit from study of fungi that are opportunistic pathogens? the speaker will examine some of these questions from the perspective of long experience in the field and the curmudgeonly attitude that develops with age. objectives: the incidence of invasive meningococcal disease (imd) has been reported in the czech republic since . in response to the emergence of a new hypervirulent clonal complex, cc , nationwide enhanced surveillance of invasive meningococcal disease was implemented by the national reference laboratory for meningococcal infections (nrl) in . the case definition is consistent with the ecdc guidelines. culture and pcr are used for confirmation of cases. notification is compulsory and is performed by local epidemiologists. strains of neisseria meningitidis isolated from imd cases are referred by the field laboratories to the nrl to be characterised by serogrouping, pora and feta sequencing (http://neisseria.org/nm/typing/) and multilocus sequence typing (mlst) (http://pubmlst.org/neisseria/). in the nrl, the epidemiological database is matched against that of strains to avoid duplicate reporting in the final enhanced surveillance database. results: despite the stable trend in imd incidence ( . / ) since , the case fatality rate was high ( . %) in . the disease was caused mainly by serogroup b meningococci ( . %) in , followed by serogroups c ( . %) and y ( . %). the most frequent clonal complexes were cc , cc / and cc (typical for serogroup b) and cc (typical for serogroup c). the highest age-specific morbidity rates were observed in the lowest age groups, i.e. − months and − years ( . / and . / , respectively), and were associated with high prevalence of serogroup b. the case fatality rate was the highest in infants under year of age ( . %). the incidence of imd caused by serogroup c is currently low and there is no indication for mass vaccination with menc conjugate vaccine. menb vaccine is needed for infants, but the sero/subtype coverage by the currently developed porin-based vaccines is low for czech meningococcal isolates (maximum . % for nine-valent meningococcal pora vaccine). methods:the vaccination programme incorporates dedicated vaccine clinic with a multi-disciplinary team including a nurse, data manager, a pharmacist specifically appointed to the unit. additional interventions to improve vaccine uptake and outcome have included use of sms texting to announce availability of influenza annually and improve adherence to completion of hepatitis b vaccination, educational programmes changes in guidelines e.g. varicella vaccination and creation of a vaccine passport. we reviewed vaccination clinic activity in the cohort of , hiv positive patients since introduction of a dedicated vaccine service. results:there has been a large increase in the uptake of vaccinations since introduction of this service. the varicella vaccination uptake increased from ( ) to ( ) due to targeted vaccine programme.(see graphic, legend reads left to right) conclusion: strategies implemented increased the uptake of recommended vaccinations in our hiv population. these included appointment of a dedicated health professional team, use of it supports, education of staff and patients and development of a vaccine passport. we developed the vaccine passport to help with patient education and awareness and it will serve as a record of vaccine administration for physicians off site. in the latter year, post guideline change, we have targeted our varicella non immune population. the next intervention planned is to assess all late entrants to our healthcare system to determine need for catch up vaccines, including mmr. results: column purified recombinant protein sspb was found to be a good antigen for both groups of animals used for immunisation. antibodies against the recombinant sspb tested by opsonophagocytosis were found to enhance phagocytosis of gbs strains belonging to different serotypes at the average . times relatively to control. affect against gas strains was less pronounced ( . times) but still statistically significant. antibodies were also capable to interfere with adherence of gbs strains carrying sspb relatively to the strain without the protein. adherence of the strain with sspb towards different cell lines was dramatically higher which proves the function of the protein as adhesin. in passive protection test carried out with mice challenged with virulent gbs or gas strains introduced intranasaly were eliminated from the lungs of the animals times faster in case of the usage of anti sspb serum relatively the control. in the experiments with active protection sspb immunised animals were found be significantly better protected against gbs and gas infection. (table ) . similar results were obtained in the analysis of factors associated with -day mortality. conclusion: these data suggest that outcomes of both community-onset and nosocomial bloodstream infections due to s. aureus may be improved by an expert consultation service. the factors most critical for better outcomes and modifiable in time by id specialist consultation remain to be determined and may be explored as process of care quality indicators. objective: worldwide, the present tuberculosis epidemic is characterised by an alarming emergence in drug resistance. given the limited therapeutic options in mdr (and especially xdr) tuberculosis, there is a need to define the resistance levels and mechanisms present in clinical isolates categorised as drug resistant on the basis of critical concentration testing, so as to facilitate rapid therapeutic decisions. methods: we determined quantitative resistance levels of drug resistant isolates of mycobacterium tuberculosis sampled in switzerland over the past years. resistance-conferring genetic alterations were identified by probe assays and pcr-mediated gene sequencing. results: rifampicin resistant isolates unanimously showed a high-level resistant phenotype (> mg/l) associated with mutations in rpob. in contrast, a significant fraction of clinical tb isolates categorised as isoniazid resistant on the basis of critical concentration testing showed a low-level resistant phenotype (mostly mutations in inha); heterogeneous phenotypic resistance levels were associated with mutations in katg. one third of streptomycin resistant clinical isolates had a low-level resistance phenotype (< mg/l). ethambutol resistance occurred mostly in mdr strains and was linked to alterations in embb, but resistance never exceeded mg/l. our data indicate that some first line agents may be considered as therapeutic treatment option despite in vitro resistance at the critical concentration. diagnostic mycobacteriology would benefit from standardised measures of quantitative drug susceptibility testing in particular for those drugs were significant variations in phenotypic resistance levels are found in clinical isolates, e.g. isoniazid, ethambutol and streptomycin. introduction recent advances in the diagnostics of varicella zoster virus (vzv) infections have changed the perception of this virus as a cns pathogen. a real-time pcr method amplifying a nt segment of the vzv gb region gave . log improved sensitivity over conventional pcr and was employed for routine diagnosis of vzv dna in samples of cerebrospinal fluid (csf). in addition, a new elisa method for detection of antibodies in the csf to glycoprotein e was developed, using a mammalian cell expression system for optimal glycosylation of the antigen. these methods were utilised for studies of vzv-induced cns infections. in a retrospective study, almost all patients had a reactivated vzv infection, but only % showed skin lesions. the following diagnoses were made: acute aseptic meningitis (aam), n = ; encephalitis, n= ; meningoencephalitis, n = ; cranial nerve affections, n = ; encephalopathy, n = ; and cerebrovascular disease, n = . in patients in whom vzv dna levels were determined, significantly higher viral loads were found in those with aam and encephalitis compared to patients with cranial nerve affection (including ramsay hunt syndrome). of the % (n = ) who had a follow-up, % (n = ) had neurological complications after months. sixty-two percent had a ct/mri scan of the brain performed and % of these had pathological findings. vzv encephalitis showed a more broad disease spectrum as compared with herpes simplex encephalitis (hse), as will be presented. detection of intrathecal synthesis of vzv ge antibodies was positive in the vzv encephalitis patients, as well as in some of the hse patients, arguing for a previous suggested role for vzv as a co-pathogen at least in some cases of the latter disease. vzv vasculitis was a more common finding ( % of all cases) than expected from the literature of case reports. mr findings showed that middle and posterior cerebral arteries were targeted. surprisingly, despite substantial vzv dna loads in the csf of these patients, investigated serum samples were pcr negative. thus, vzv might be suggested to be neuronally transported to the arterial walls rather than haematogenously spread. conclusions: vzv is a serious and underestimated cause of cns infection. a substantial number of the patients presented with serious neurological symptoms and sequela, and pathological findings on ct/mri of the brain were abundant, especially in patients with encephalitis and vasculitis. pk/pd controversies for the clinician s pk/pd and azoles the triazoles have revolutionised the treatment of invasive and allergic fungal diseases. fluconazole, itraconazole, voriconazole and posaconazole are available for clinical use. isavuconazole and ravuconazole are in development. the triazoles have broad spectrum antifungal activity. the pharmacokinetics and pharmacodynamics (pk-pd) of the triazoles have been extensively investigated in murine models of disseminated candidiasis. the pd parameter that optimally links drug exposure with the observed antifungal effect is the ratio of the area under the concentration-time curve (auc) to mic (auc:mic). there is increasing information on the magnitude of the auc:mic that is required for optimal antifungal effect. pk-pd principles have been used to define in vitro susceptibility breakpoints. the triazoles are fungistatic against candida spp. their mode of action against aspergillus spp. is less well defined, although they clearly exhibit dose-dependant decrement in fungal burden in laboratory animal models of invasive pulmonary aspergillosis. the triazoles accumulate in tissues and this is important for an understanding of their antifungal effect. in humans, the triazoles are characterised by complicated pharmacokinetic properties. both itraconazole and voriconazole exhibit nonlinear pharmacokinetics. the triazoles all exhibit clinically relevant exposureresponse relationships. recent work from our laboratory suggests that itraconazole exhibits clinically relevant concentration-toxicity relationships. higher concentrations of voriconazole are associated with a progressively higher probability of hepatotoxicity, photopsia and central nervous system toxicity. because of the significant pharmacokinetic variability and clinically relevant drug exposure-response relationships, therapeutic drug monitoring (tdm) is frequently used. a strong argument can be made for the routine monitoring of itraconazole and voriconazole. there may also be grounds to consider monitoring posaconazole levels. tdm should be considered for all patients receiving triazoles who have refractory disease. furthermore, tdm should be considered when compliance, drug interactions and variable pharmacokinetics result in uncertainty about resultant drug exposures. an understanding of the pk-pd relationships of the triazoles has been instrumental in optimising their clinical efficacy. innate immunity s the inflammasomes: danger sensing complexes triggering innate immunity the nod-like receptors (nlr) are a family of intracellular sensors of microbial motifs and 'danger signals' that have emerged as being crucial components of the innate immune responses and inflammation. several nlrs (nalps and ipaf) form a caspase- -activating multiprotein complex, termed inflammasome, that processes proinflammatory cytokines including il- beta. amongst the various inflammasomes, the nalp inflammasome is particularly qualified to sense a plethora of diverse molecules, ranging from bacterial muramyldipeptide to monosodium urate crystals. the important role of the nalp inflammasome is emphasized by the identification of mutations in the nalp gene that are associated with a susceptibility to inflammatory disorders. these and other issues related to the inflammasome will be presented. it is now years since charles janeway hypothesized the existence of clonally derived pattern recognition receptors and pointed to the importance of these in initial responses to bacterial and viral infections. janeway's hypothesis has been validated by the discovery of three groups of prrs. first, are the toll-like receptors which detect microbial lipids and non-self nucleic acids at the cell surface an in intracellular compartments. in addition cytoplasmic sensors of bacteria (nods) and of viral nucleic acids (rigs) have also been characterised. as well as being critical for responses to infections, these prrs also underlie a large burden of autoimmune and inflammatory disease in the human population and are thus important targets for therapy. in my talk i will describe the molecular mechanisms by which these conserved pathogen associated moecules are recognized by the tlrs with particular reference to lipo polysaccharide and single stranded viral rnas. i will also present new results which show how receptor activation is coupled to downstream signal transduction and in particular the role played by oligomeric signaling platforms assembled form adaptors and other signaling molecules involved in the pathway. i will discuss the potential for structural analysis to be used in the rational design of new drugs. this session proposes a critical review of the most salient recently published papers in the field with a special focus on control of multi drug-resistant organisms, prevention of infections in the intensive care unit, surgery etc. and highlights the need for validity/scope assessment. it emphasizes also the importance to prioritise information published in the abundant literature available so as to be able to summarise and understand the potential changes in clinical practice, and identify unresolved issues and areas of possible future clinical research. tourism is europe's face to the world. it is also a major source of revenue, employment and productivity. each year over million arrivals are recorded into the continent, and of those, approximately million are from latin america. returning travelers are even more numerous and more often associated with disease transmission into europe. within countries of the european continent, imported cases of environmental and zoonotic illnesses such as cholera, dengue, malaria, viral haemorrhagic fevers and west nile virus infections are a rare but established fact. diseases imported from latin america with the potential for autochthonous transmission (chikungunya, malaria, yellow fever) and or high infectivity (viral haemorrhagic fevers) will be described in detail and the possibility of european outbreaks from latin american countries will be discussed. cutaneous leishmaniasis (cl) is a worldwide disease, endemic in countries, that has shown an increasing incidence over the last two decades. so far, pentavalent antimony compounds have been considered the treatment of choice, with rates of curing close to %. however, the high efficacy of these drugs is counteracted by their adverse events. recently, in vitro and in vivo studies have shown that no plays a key role in the eradication of the leishmania parasite objective: to determine whether a no donor patch (developed by electrospinning technique) is as effective as meglumine antimoniate in the treatment of cl while causing less adverse events methods: a double-blind, randomised, placebo-controlled clinical trial was conducted with patients diagnosed with cl in santander, colombia, south-america. the patients were randomly assigned to two groups. during days group received simultaneously meglumine antimoniate and placebo of nitric oxide patches while group received active nitric oxide patches and placebo of meglumine antimoniate. biochemical determinations (aspartate aminotransferase, alanine aminotransferase, creatinine and pancreatic amilase) were measured at he beginning and at the end of the treatment. a follow up was realised , and days after the beginning of the treatment results: the study included ( . %) women and ( . %) men. the average age in group was . ± . years; while in group it was . ± . years. clinical and demographic data were similar in the two groups. after the follow up period, the complete clinical healing of group was . % versus . % for group (p= . ). treatment with no patches generated both, a lower frequency of non-serious adverse events (fever, anorexia, myalgia, arthralgia, headache), and a reduced variation in biochemistry determinations (asat the treatment with no patches resulted in a lower percentage of complete clinical response compared with meglumine antimoniate. despite its inferior effectiveness, the safety, the lower frequency of adverse events, the facility of administration (topical) and the low cost of the patches justifies its evaluation in further poblational studies, especially in populations as the colombian ones, where the serious adverse events due to glucantime have increased dramatically. objectives: trichinellosis is a zoonotic disease which has never been reported in taiwan and is rarely linked to consumption of reptiles. we investigated the first documented outbreak of trichinellosis in taiwan consisting of patients who became acutely ill after eating at the same restaurant in may . we conducted a retrospective cohort study by interviewing the patients and persons who ate together with them. a case was defined as illness in an attendee who had fever (> . ºc) or myalgia weeks after the festivals and was seropositive to trichinella antigen using an enzymelinked immunoassay and immunohistochemical staining. environmental study of the soft-shelled turtle farm was performed. results: of the attendees, persons met the case definition (attack rate = %). the most common presenting symptoms were myalgia ( %), fever ( %), and periorbital swelling ( %). all patients sought medical care; five were hospitalised. of the patients who underwent blood test, all had moderate eosinophilia. all patients' serum samples were strongly reactive to trichinella excretory-secretory antigen. the only food item significantly associated with illness was the raw softshelled turtle meat (relative risk undefined; p = . ). traced back to the farm, histological examination of soft-shelled turtles was negative for trichinella species. the most likely cause of this outbreak was consumption of raw soft-shelled turtle served in the festivals. this investigation indicates taiwan is not free of trichinellosis. prevention and control programs of trichinellosis should be established. the public should be aware of the risk of acquiring trichinellosis from consumption of raw soft-shelled turtle. objective: to develop and evaluate a modified, rapid giemsa staining procedure for detection of malaria parasites in blood smears. disadvantage of the rapid commercially available staining methods is that they require highly experienced technicians for interpretation of results because the interpretation can be difficult. for this reason, many laboratories use the giemsa stain. shorter giemsa staining times have been reported previously, however, to our knowledge, the effect of and minute staining in different giemsa dilutions have not been evaluated. the stock solution of giemsa stain (merck, darmstadt, germany) was used in different dilutions ( : and : ) and incubated for different lengths of time ( min and min). the staining effect was compared to our standard giemsa stain ( : , min). sensitivity was determined by examining smears of p. falciparum from fresh and edta blood. the level of parasitaemia was followed in two patients admitted to our hospital with p. falciparum parasitaemia's of . % and . % (see table; patient a and b) by examination of blood smears taken at different time points after initiation of therapy. these samples were used to evaluate the different giemsa dilutions and staining times. smears were read by three independent observers (a clinical microbiologist, a laboratory technician specialised in parasitology, and a resident in clinical microbiology). in the table results of the three staining methods on blood from two patients from ghana with high parasitaemia's on admission and during follow-up are shown. all smears were equally easy to read and yielded parasite counts within internationally accepted ranges of variation (see united kingdom national external quality assessment service). conclusion: staining blood smears for detection of plasmodium falciparum parasites with a : dilution of giemsa stain for five minutes provides easy to read slides and results comparable to those obtained with the standard giemsa staining. advantage of the rapid method is the shorter turnaround time, disadvantage is the larger amount of stain used. objectives: diarrhoeal diseases are common in developed and developing countries and are major causes of morbidity and mortality worldwide. the need to differentially diagnose protozoan parasites versus other gastrointestinal (gi) aetiologies is well recognized. the most common gi protozoan parasites infecting humans worldwide are considered to be entamoeba histolytica, giardia lamblia, blastocystis hominis, dientamoeba fragilis and cryptosporidium spp. laboratory detection of these parasites is relying on microscopic analysis of stool samples and water concentrates, as well as enzyme immunoassay (eia) tests. utilising the microscopic examination usually results in underdetection of gi parasites, while usage of eia is often not cost-effective. methods: savyon diagnostics is currently engaged with developing an approach aiming to address the unmet needs and the current limitations in this field. this approach includes major aspects: ( ) the ability to detect a panel of all the above organisms in one test kit, ( ) the possibility to perform the diagnosis in two steps − first, simultaneous detection of these organisms without distinguishing between the different species for screening of large number of specimens, and second, distinctive detection of the specific aetiology in the positively-found specimens, and ( ) the ability to apply eia diagnosis in formalin-preserved specimens for all the mentioned parasites. results: polyclonal antibodies were produced in-house based on native antigen extracts, recombinant antigens and synthetic peptides. the resulted inventory of antibodies enabled finding the optimal combination that provided the desired performance parameters for separate detection of each of the parasites in fresh, frozen or formalin preserved faeces specimens. the analytical limit of detection and the performance in characterised clinical specimens were comparable to microscopy or to reference eia, when available. the results show unique detection of e. histolytica in formalin-preserved specimens, which is comparable to detection in fresh specimens. furthermore, we demonstrate simultaneous detection of the parasites without compromising performance characteristics in fresh or preserved specimens. the presented work is a paradigm of an innovative approach, expected to advance the diagnosis of protozoan parasites in gi patients, thus, enabling appropriate and cost-effective diagnosis and treatment. objectives: systemic administration of certain facultative anaerob bacteria to mice bearing solid tumours leads to accumulation in tumours compared to normal target organs, like spleen and liver, and to retardation of tumour growth. salmonella enterica serovar typhimurium (s. typhimurium) as well as escherichia coli nissle (ecn) are such bacteria. preliminary experiments showed that such bacteria that exhibit the ability to form biofilms in vitro might also do so in tumours. in the present study this was systematically investigated. methods: biofilm formation of bacteria were detected on low-salt biofilm plates. additionally, salmonella-or e. coli-infected ct tumours of balb/c mice that were left untreated or were treated with anti-gr to deplete neutrophilic granulocytes were removed two days post infection, fixed and prepared for electron microscope analysis. the expression of different genes which are probably involved in the biofilm formation were tested via real-time pcr. results: when examined after colonising tumours s. typhimurium sl and sl as well as ecn are almost exclusively found extracellular although they are able to invade the ct cells in vitro. interestingly, like in vitro all three bacteria form biofilms to various extend when residing in the tumours. this was followed in more detail for s. typhimurium sl . biofilms were not formed by sl when neutrophils had been removed by antibodies. in addition, when arda a central switch for biofilm formation in the salmonellla had been deleted no biofilms could be found. importantly, now bacteria could be found intracellularly most likely in neutrophilic granulocytes. conclusion: the formation of biofilms by facultative anaerobic bacteria when residing in solid tumours is a novel and surprising finding. when neutrophils were removed, no biofilms are formed, while uptake into neutrophils is allowed when the ability of the bacteria to form biofilms was blocked. hence, it appears that the bacteria use biofilm formation as a defence system against the immune system of the host. objectives: rama is an arac/xyls family transcriptional activator found in klebsiella pneumoniae, salmonella spp. and enterobacter spp., the overexpression of which is associated with an mdr phenotype. recently a tetr-like gene that lies upstream of rama, known as ramr, has been identified as a repressor of rama. k. pneumoniae kp is a diazotrophic endophyte strain which has been reported to exhibit notable resistance to antibiotics. despite its mdr phenotype kp has been shown to exhibit attenuated pathogenicity in mouse models in comparison to clinical k. pneumoniae strains. the aims of this study were to: determine the levels of rama expression and establish its role in kp 's mdr phenotype; determine the effect of ramr complementation on rama expression and antibiotic susceptibility. methods: genome and sequence analysis performed in k. pneumoniae strain kp demonstrated a bp deletion within the ramr gene. cloning and complementation with full size wild type ramr was performed in kp (hereby known as kp /ramr). rt-pcr was used to assess levels of gene expression which were subsequently quantified using bio-rad quantity one software. mic testing was performed against chloramphenicol (cm), norfloxacin (nor) and tetracycline (tet) according to bsac guidelines. biofilm formation was measured using a modified protocol of o'toole and kolter. results: kp containing the mutated ramr gene ( bp deletion) was shown to overexpress rama and the putative outer membrane protein roma. complementation of the ramr gene resulted in the repression of both rama and roma transcription by − fold. interestingly, the ramr complemented strain demonstrated increased biofilm formation (up to -fold increase) over a hour period in both lb and m medium after static growth at ºc. mics of the tested antibiotics were reduced up to -fold in kp /ramr compared to the ramr mutated kp . conclusions: this result demonstrates that ramr acts as a repressor of both rama and putative outer membrane protein roma thereby increasing its susceptibility to antibiotics. however the restoration of a functional ramr in kp also increases biofilm formation significantly, suggesting that ramr plays a role in the regulation of biofilm formation genes and possibly bacterial virulence. rifampicin showed the highest activity on biofilm matrix and bacteria in sa and pa biofilms. results also indicated that biofilm viable mass was more susceptible to treatment than the biofilm matrix, which is mainly responsible for biofilm persistence. further research should specifically focus on compounds destroying matrix and which can be used as an adjunct to antibiotic therapy. [ objectives: staphylococcus epidermidis is a common cause of foreignbody infections (fbi) because of its ability to form biofilms. biofilms are very resistant to antibiotics. active and passive immunisation against biofilm-associated bacterial antigens may be an alternative. we studied the effect of immunisation against the lpxtg protein sesc in s. epidermidis biofilms in vitro and in vivo. we previously reported that sesc is present in all s. epidermidis strains tested. sesc is mainly expressed during the early and late fbi and at a higher level in sessile cells than in planktonic cells. methods: we used rabbit polyclonal anti-sesc-iggs ( mg/ml) to study biofilm inhibition in vitro and in vivo in our rat model ( mg igg per rat) on -day old biofilms. we also vaccinated rats twice with sesc according to standard protocols. serum samples taken at day and weeks after the st and nd immunisation were tested by elisa and showed an increase in anti-sesc antibody levels. s. epidermidis strains b and are biofilm forming strains and have been described before. for in vitro experiments, s. epidermidis b or were mixed with anti-sesc-iggs and incubated for hours at ºc. subsequently cells were added to each well. after h at ºc biofilms were washed and stained with crystal violet and od was measured. for in vivo experiments, catheter fragments were pre-incubated with s. epidermidis b and implanted subcutaneously in each rat. after explantation, the average number of cfu was determined after hrs. results: our data show that rabbit anti-sesc-iggs inhibit in vitro biofilm formation by s. epidermidis strains b and by % and %, respectively (n = ). in the in vivo rat model, rabbit anti-sesc-iggs reduced the bacteria in a -day old biofilm -fold (n = ). active immunisation with recombinant sesc led to a -fold reduction of cfu compared to control rats in day-old biofilms (n = ). after days, the reduction in biofilm-associated bacteria in the immunised rats was -fold (n = ) (fig .) . conclusion: sesc represents a promising target for prevention of s. epidermidis biofilm formation. the higher effect of passive immunisation compared with active immunisation is probably due to the subcutaneous injection of anti-sesc-iggs at the place of catheter insertion. objectives: staphylococcus epidermidis has emerged as a pathogen associated with infections of implanted medical devices impeding their long-term use. characteristics of s. epidermidis that allow persistence of infection are the ability of bacteria to adhere to surfaces in multilayered cell clusters, followed by the production of a mucoid substance more commonly known as slime, encoded by the ica operon. the adherent bacteria and slime are collectively known as biofilm. the coupled effects of specific chemical terminal surface groups and flow conditions on slime production and biofilm formation by s. epidermidis were investigated in correlation to the expression of two genes of the ica operon. methods: reference control strains (atcc , slime-positive and atcc , slime-negative), and two clinical strains isolated from different hospitalised patients, (one ica-positive/slime-positive and one ica-positive/slime-negative) were tested. bacteria grown in bhi medium were suspended in physiological saline at a concentration of~ × cells/ml. hydroxyl (oh)-terminated (hydrophilic) and methyl (ch )terminated (hydrophobic) glass surfaces were used as substrates in a parallel plate flow chamber. bacterial adhesion was examined under two flow rates: ml/min and ml/min for two and four hours. total rna from both planktonic (p) and adherent (a) bacteria, after detachment with trypsin, was isolated by the trizol method. reverse transcription followed by relative real-time pcr (rrt-pcr) towards a bp part of s rrna gene, allowed the detection of expression levels of icaa and icad. adherent bacteria were investigated with scanning electron and confocal laser microscopes. results: higher expression levels of both icaa and icad genes onto glass and especially methyl-terminated glass surfaces were calculated by rrt-pcr, under higher flow rate in two hours by the reference and the clinical slime-positive strains. these results correlate well with adherent bacterial cell counts and images taken by both microscopes. the icapositive slime-negative clinical strain showed lower expression levels of ica genes, less adherent ability and pia production on glass surfaces, as observed by microscopes. higher flow rate enhances the expression level of both ica genes, with a peak in two hours. hydrophobic biomaterial surfaces seem to play a crucial role to initial adherence, increasing ica gene expression and pia synthesis. consenting men and women with dfi (predefined by clinical signs and symptoms) caused by mrsa were potentially eligible including those associated with bacteraemia. patients with initial osteomyelitis were excluded. patients could receive l mg bid either iv or po. primary end point were cure or improvement rates (c+i) and microbiologic eradication (me) at days after the beginning of l. secondary end points were c+i on days and after the beginning of treatment and hospital discharge day, need of amputation, duration of therapy and mortality rates. all the adverse events were collected. results: patients were enrolled. relation men:women was . .the age of patients was . ± years and the average period from the diagnosis of diabetes was . ± . years. associated bacteraemia was present in . % of patients included. primary end points: c+i days after the beginning of l was achieved in . % of patients and me was obtained in . % of patients. secondary end points: c+i on day , hospital discharge day and day after the beginning of treatment and were; %; . % and . % respectively. only patients needed a minor amputation. the primary and secondary end points in the subgroup of bacteraemic episodes were not statistically different of those previously described. the mean duration of therapy was . ± . days. global mortality was . %. only one episode of polineuropathy was reported. neither thrombocytopenia nor lactic acidosis was found. conclusions: l achieved excellent c+i even at first evaluation visit in documented dfi caused by mrsa. l also showed high me rates. although patients received prolonged periods of treatment, l was a safe drug. objectives: azithromycin microspheres formulation (azm) was developed to enable a higher dosage of g to be administered as a single oral dose without decreasing the safety profile. this study compared azm with moxifloxacin (mox) aimed at confirming the efficacy and safety of azm in acute exacerbations of chronic bronchitis (aecb). methods: this prospective, multicentre, randomised, double-blind, double dummy study compared azm g single dose with mox mg once daily for days, enrolled aecb patients years old and above, with anthonisen type exacerbations, and with at least exacerbations of aecb in the past months. subjects were to have a history of smoking of at least pack-years and documented forced expiratory volume in second (fev ) less than % of predicted. they were followed up for up to months. results: a total of patients were treated ( in each of the treatment groups) the distribution of the age, and mean fev were similar for the treatment groups. pathogens were isolated from . % of the patients ( . % of patients on azm and . % of patients on mox). the clinical success (signs and symptoms related to the acute infection had returned to the subject's normal baseline level, or clinical improvement was such that no additional antibiotics were deemed necessary) rate for the per protocol population at test of cure (toc) at day − was . % for azm and . % for mox group ( % ci − . , . ). bacterial eradication rate (bacteriologic pre protocol population) at toc was . % for azm group and . % for mox group ( % ci − . , . ). although the study population had history of at least exacerbation in the past months, less than half of the subjects experienced a recurrence during the follow-up, and there was no statistically significant treatment difference in time taken to first occurrence of aecb. both treatments were well tolerated. the incidence of treatment related adverse events was low, being reported by % of subjects receiving azm and % of subjects receiving mox. most aes were mild or moderate in severity. the most common aes were gastrointestinal disorders, being reported by % of subjects receiving azm and % of subjects receiving mox. conclusions: a single oral dose of azm was as effective as a -day course of mox in the treatment of aecb and was well tolerated. objectives: optimal duration of gentamicin containing regimen for therapy of human brucellosis is not clearly determined. methods: this randomised clinical study was conducted to compare the efficacy of gentamicin mg/day for days plus doxycycline mg twice daily for eight weeks (gd group) versus streptomycin gr im for weeks plus the same dose of doxycycline for days (sd group). all cases were followed for one year after cessation of therapy. efficacy of both regimens (failure of therapy or relapse) were compared. results: seventy-nine patients with the mean age of ± . years and cases with the mean age of . ± . years were treated with regimen of gd or sd, respectively. the clinical manifestations in these two treated groups were similar. failure of therapy was seen in one patient in gd group and in cases in sd group ( objectives: to study the efficacy of telavancin (tlv), an investigational bactericidal lipoglycopeptide, for the treatment of complicated skin and soft tissue infections (cssti) caused by presumed or confirmed grampositive organisms. methods: atlas and atlas were methodologically identical, double-blind, randomised, multinational, phase studies. adult men and women presenting with cssti including major abscess were randomised : to tlv mg/kg intravenous (iv) q h or vancomycin (van) g iv q h for to days. test-of-cure (toc) visit was conducted to days after end of study treatment. the all-treated population (at) included patients with confirmed diagnosis of cssti who received dose of study medication. this analysis examined the baseline characteristics and cure rates at toc for patients with major abscess in the combined atlas at population. results: in the pooled at population of atlas, patients presented with major abscess. more than % of these patients required hospitalisation. the baseline lesion surface area exceeded cm in % of the cases, while % of the patients presented with lesions exceeding cm (table ) . elevated white blood cell counts were found in more than % of the cases (table ) . nearly all patients required surgical drainage, with approximately / performed prior to the first dose of study medication. very few patients required a surgical procedure more than days after the start of study medication. clinical cure rates at toc are presented in table . overall, adverse events in the at population were similar between the treatment groups with regard to type and severity. conclusion: telavancin administered once daily was non-inferior to vancomycin for the treatment of major abscess. objectives: b. fragilis and related species, members of the normal bowel flora, are the most widely isolated anaerobic bacteria from different infections. to follow the development and spread of the resistance among these strains is difficult, as antibiotic susceptibility testing of clinically relevant anaerobes in different routine laboratories in europe is less and less frequently carried out due to the fact, that clinicians treat many presumed anaerobic infections empirically. to follow the changes in the antibiotic resistance of bacteroides strains three europe-wide studies were organised during the past twenty years. the evaluation of the results of these studies may show changes in the resistance to different antianaerobic drugs. only clinical isolates and no normal flora members of bacteroides strains belonging to different species were collected from different countries throughout europe during these studies. agar dilution method was used for the antibiotic susceptibility determination. actual breakpoints accepted by nccls (clsi) and eucast were used. molecular genetic investigations were carried out to detect resistance mechanisms. since the first study the chromosomally mediated beta-lactamase production and tetracyclin resistance is the most prevalent among bacteroides strains in europe. clindamycin resistance in bacteroides is mediated by a macrolide-lincomycin-streptogramin (mls) mechanism and its frequency differs in different countries in europe. resistance to beta-lactam-beta-lactamase inhibitor combinations was studied using amoxicillin-clavulanic acid and/or piperacillintazobactam. increase in resistance was observed to both combinations throughout the years. the same is true for cefoxitine and in the third study several hetero-resistant isolates were found. the occurrence and spread of resistance to imipenem and metronidazole among bacteroides strains merit special clinical importance. the presence of the cfia gene is much more prevalent than the expression of the imipenem resistance; however the spread of the cfia gene among species other than b. fragilis is still very rare. the molecular genetic methods looking for the resistance genes among strains with elevated mics against these antibiotics prove that resistance breakpoints should be reconsidered. the resistance to moxifloxacin shows great differences in different countries. the lowest resistance rate was observed in the case of tigecyclin. many factors may affect the response to treatment such as site of infection, surgical procedures, severity of the illness, patient status, presence of other pathogens (mixed infection), pk/pd parameters of the antibacterial drugs. thus, correlation between treatment failure and antibiotic resistance among anaerobes remains difficult to assess. the main discrepancies came from intra-abdominal infections and a worrisome disjunction between surgeon and microbiologist opinions emerged in the 's. but, patients in whom primary therapy failed had more resistant strains compared with patients in whom therapy succeeded. in contrast many failures may be due to the lack of isolation of anaerobes from clinical samples! during anaerobic bacteraemia, salonen et al. demonstrated that mortality increased dramatically from % for initially effective treatment to % when an ineffective treatment was started. facing new mechanisms of resistance and global increase resistance to many antibiotics among anaerobes may lead nowadays to a different answer. clindamycin vs. penicillin studies for the treatment of lung infections pointed out the failure due to b-lactamase production among gram-negative anaerobes. we found many reports of failure after clindamycin treatment in osteomyelitis, septic arthritis, brain abscess in presence of clindamycin-resistant anaerobes (bacteroides fragilis group and prevotella), probably because when resistance occurs, clindamycin mic's are high. similarly, the lack of coverage of an undetected resistant anaerobe allows the selection of an anaerobic strain resistant to the treatment chosen against the associated aerobes such as imipenemresistant eghertella lenta or metronidazole-resistant strains of prevotella or bacteroides fragilis. the later failures may give opportunity to set up a new metronidazole breakpoint for resistance (mic > mg/l). the main problem is related to the difficulty to detect some heterogeneous resistant strains, that needs prolonged incubation period on agar medium. this kind of situation is probably the most suitable to correlate the bacterial antibiotic resistance with the failure of the antibiotic treatment. methicillin-resistant s. aureus isolates causing community-acquired infections (ca-mrsa) in children is a major problem in several areas around the world. ca-mrsa are associated with both skin and soft tissue infections and invasive infections. recurrent soft tissue infections and infections within the family caused by ca-mrsa isolates are common. ca-mrsa s. aureus isolates containing gene coding for pvl have been associated with serious staphylococcal pneumonia as well as osteomyelitis complicated by subperiosteal abscesses or venous thromboses. in addition to vancomycin, ca-mrsa generally are susceptible to clindamycin and trimethoprimsulfamethoxazole. treatment of superficial skin and soft tissue infections involves surgical drainage of abscesses followed by an oral agent such as tmp-smx or clindamycin. minocycline or doxycycline is a consideration for children > years old. empiric vancomycin is typically administered for more serious and invasive infections such as osteomyelitis, septic arthritis, serious head and neck infections or suspected staphylococcal pneumonia. clindamycin is efficacious in treating invasive ca-mrsa infections caused by susceptible organisms. linezolid or daptomycin is another option in selected circumstances. mri is the optimal imaging modality for assessing children with ca-mrsa osteomyelitis. aggressive surgical drainage of subperiosteal abscesses or sites of pyomyositis is recommended. venous thombosis is increasingly recognized as a complication of ca-mrsa osteomyelitis. anti-coagulation until the thrombus has resolved is recommended. the optimal approach to prevention of recurrent ca-mrsa infections is unclear but a strategy that includes emphasizing personal hygiene, plus/minus antimicrobial soaps, mupirocin to the nose or "bleach baths" is frequently suggested. s understanding the pathogenesis of group a streptococcal disease: the bedside-to-bench approach invasive group a streptococcal (gas) infection presents itself in a range of guises, most notoriously necrotising fasciitis and the streptococcal toxic shock syndrome. as a human pathogen, gas pathogenesis research should ideally be shaped by clinical questions arising from either epidemiological or case-based investigation of human disease. in the mid s, large epidemiological studies pointed to a central role for specific t cell-stimulating superantigens in the aetiology of streptococcal toxic shock. this sparked a series of clinical and laboratory investigations that demonstrated production of superantigens during infection which were indeed capable of triggering massive t cell activation in patients but were unlikely, alone, to account for all the features observed in toxic shock. genomic, clinical and laboratory-based investigations have identified novel and highly potent superantigens that appear to directly contribute to sepsis pathogenesis and, together, may constitute targets for adjunctive treatments in invasive disease. epidemiological, clinical, and laboratory studies have highlighted a role for blunt trauma in the aetiology of at least a quarter of cases of gas necrotising fasciitis. one of the most striking findings on examination of tissues from patients suffering with necrotising fasciitis is the failure of neutrophils to migrate to the focus of infection. investigation of patients with invasive gas infection led to the discovery that gas produces an enzyme that can cleave and inactivate human chemokines and study of patients with bacteraemia has highlighted a likely role for the causal enzyme spycep in disease pathogenesis; this bacterial surface enzyme has also shown promise as a potential vaccine antigen. notwithstanding a potential role for individual virulence factors in disease causation, clinical studies have demonstrated that gas bacteria may persist at the site of infection despite high concentrations of bactericidal antibiotics, and this has been borne out by experimental studies; the reasons behind such persistence are unclear but may include internalisation of gas by immune cells, formation of biofilm, and antibiotic penetration of necrotic tissues. the persistence of viable bacteria in such cases is not widely recognized and deserves focused consideration in the research laboratory. genome-wide analysis of microbial pathogens and molecular pathogenesis processes has become an area of considerable activity in the last years. these studies have been made possible by several advances, including completion of the human genome sequence, publication of genome sequences for many human pathogens, development of microarray technology and high-throughput proteomics, and maturation of bioinformatics. despite these advances, relatively little effort has been expended in the bacterial pathogenesis arena to develop and use integrated research platforms in a systems biology approach to enhance our understanding of disease processes. we have exploited an integrated genome-wide research platform to gain new knowledge about how the human bacterial pathogen group a streptococcus causes disease. results of these studies have provided many new avenues for basic pathogenesis research and translational research focused on development of an efficacious human vaccine and novel therapeutics. new data stemming from use of a systems biology approach to provide new data about group a streptococcus pathogenesis will be presented. streptococcal toxic shock syndrome and necrotising fasciitis caused by group a streptococcus are rapidly progressive invasive diseases that are associated with significant morbidity and mortality, ranging from − % despite prompt antibiotic therapy and surgical debridement. s. pyogenes is known to primarily cause disease by activating and modulating host immune responses. the exotoxins with superantigenic activities have been demonstrated to be crucial triggers of excessive inflammatory responses and consequently systemic toxicity, organ dysfunction, tissue necrosis and shock. another important virulence determinant is the m-protein, which is classically known for its antiphagocytic properties, and lately, was shown to trigger pro-inflammatory responses as well as induction of vascular leakage and shock. this likely represents important mechanisms contributing to the rapid development of shock and systemic toxicity in patients with severe invasive group a streptococcal infections. the understanding of these infections as hyperinflammatory diseases highlighted the potential of immunotherapy to improve outcome. one such strategy includes the administration of intravenous polyspecific immunoglobulin (ivig) as adjunctive therapy. the mechanistic actions of ivig in this setting are believed to include opsonisation of the bacteria, neutralisation of the superantigens and suppression of the pro-inflammatory responses. there is growing evidence to support the use of ivig in patients with streptococcal toxic shock syndrome. these studies include one observational cohort study based on canadian patients identified through active surveillance of invasive group a streptococcal infections, and one european multicentre placebo-controlled trial. however, the question remains whether ivig is efficacious also for the severe streptococcal deep tissue infections. an observational study of seven patients with severe streptococcal deep tissue infections suggested that the use of high-dose ivig in patients with severe gas soft tissue infections may allow an initial non-operative or minimally invasive approach, which can limit the need to perform immediate wide debridements and amputations in unstable patients. the fact that seven patients with severe group a streptococcal infections survived with this approach definitely warrants further studies to be conducted on the use of ivig in these severe infections. hepatitis o prevalence and outcome of pregnancy in chronic hepatitis c virus infection i. julkunen°, a. sariola, m. sillanpää, k. melen, p. koskela, p. finne, a.l. järvenpää, s. riikonen, h.m. surcel (helsinki, oulu, fi) objectives: in the western countries the incidence of hepatitis c virus (hcv) infection has steadily been increasing especially among young adults. it is thus likely that an increasing prevalence of hcv infection is also found in pregnant women. methods: to assess the frequency of hcv infection in the metropolitan area of helsinki selected anti-hcv antibody testing was carried out for pregnant women during the years - . in addition, hcv prevalence was analysed in serum specimens collected from pregnant maters during the years of - . results: altogether mothers were identified among mothers. the frequency of anti-hcv positivity rose from . % in to . − . in - . in early 's only % of mothers knew about their seropositivity, whereas by the end of the follow-up period almost % of mothers knew about their hcv infection already before the pregnancy. intravenous drug abuse was the major risk factor ( % of cases) for contracting the disease. in % of the mothers chronic hcv infection was well under control and in this population the mean serum alanine aminotransferase (alt) values decreased towards the end of the pregnancy. however, % of anti-hcv ab positive mothers developed intrahepatic cholestasis (odds ratio . ) as characterised by itching and elevated serum bile acid levels. the correspondig value in the control pregnancies was only . %. anti-hcv ab positive mothers were younger, delivered earlier and gave birth to babies with smaller birth weight as compared to control deliveries. to have a more comprehensive view of the problem of hcv infection during pregnancy randomly selected serum specimens from the finnish maternity cohort were tested. - serum specimens were tested in selected cohorts ( , , , and ) . in the nationwide prevalence was . % and it steadily role to . % in . in the metropolitan area of helsinki the prevalence was higher being . % and . in and , respectively. conclusion: our study indicates that there is an increasing problem of hcv infection in pregnant women in finland. although most women cope well with their disease during pregnancy there is a subpopulation of mothers who develop cholestasis and their liver status should thus be followed-up carefully. testing of all mothers for serum anti-hcv antibodies is recommended. objectives: the viral genome of hepatitis c virus constitutes a . kb single-stranded positive-sense rna which encodes altogether viral proteins. in order to study the humoral immune responses against different hcv proteins in patients suffering from chronic hcv infection, we produced three structural (c, e and e ) and six nonstructural proteins (ns , ns , ns a, ns b, ns a and ns b) in sf insect cells by using the baculovirus expression system. the recombinant hcv proteins were purified and used in western blot analysis to determine antibody responses against individual hcv protein in hcv rna and antibody positive human sera that were obtained from patients suffering from genotype , , or infection. results: these sera were also analysed with inno-lia score test for hcv antibodies against core, ns , ns ab and ns a, and the results were similar to our western blot method. based on our western blot analyses we found that the major viral antigens were the core, ns b, ns and ns a proteins and they were recognized in %, %, % and % of patient sera, respectively. there were no major genotype specific differences in antibody responses to individual hcv proteins. a common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized ns protein and only three sera (from genotype ) recognised ns b. the data shows significant variation in the specificity in humoral immunity in chronic hcv patients. anti-hcv antibody pattern also remains very stable within one individual. alt and ast levels were tested in all subjects. the presence of hbv-dna was determined quantitatively in plasma samples of hd patients with anti-hbc alone (hbsag negative, anti-hbs negative and anti-hbc positive) by real-time pcr using the artus hbv rg pcr kit on the rotor-gene real-time thermal cycler. results: of patients enrolled in this study, subjects ( . %, % ci, . %- . %) had anti-hbc alone. hbv-dna was detectable in of hd patients ( %, % ci, %- %) with anti-hbc alone. plasma hbv-dna load was less than iu/ml in all of these patients. our study showed that detection of anti-hbc alone could reflect unrecognized occult hbv infection in hd patients. the majority of these infections are associated with low viral loads. were included in the study. all the subjects had never been exposed to antiretroviral therapy. genotypic resistance testing was performed at the time of diagnosis with a sequence-based assay (trugene hiv- genotyping test) targeted at the protease region (codons to ) and rt region (codon to ) of the hiv-l genome. results: of patients ( . %) harboured a virus with at least one mutation associated with phenotypic resistance; / with mutations associated with resistance to nucleoside reverse-transcriptase inhibitors (nrtis), / to non-nucleoside reverse-transcriptase inhibitors (nnrtis) and / to protease inhibitors (pi). resistance to nrtis was associated with the key mutation m v, while resistance to nnrtis was associated with y c and k n mutations. among mutations to pi, major resistance mutations l m and d n were found in three patients, whereas there was a high prevalence of accessory pi resistance mutations at positions , , and . conclusion: our data estimate the prevalence of primary resistance and mutations patterns among naive hiv patients, underlining the importance of genotypic resistance testing in hiv patients before starting treatment, especially when nnrtis would be included in the initial antiretroviral therapy. objectives: few data are available on the genetic mechanisms of protease inhibitor (pi) resistance in non-b hiv- , and pi resistanceassociated mutations (rams) are commonly observed in pi-naive patients with subtype a/e infection. this study aimed to compare pi-rams between pi-naive and -experienced patients. methods: genotypic resistance testing was conducted among a cohort of hiv- infected patients who had virologic failure. patients were categorised into groups: pi-naive and pi-experienced. we focused on pi-rams previously described by ias-usa . results: we studied patients (mean age, . years; % male). median cd cell count and hiv- rna at virologic failure were cells/cu.mm. and copies/ml, respectively. % of patients were infected with subtype a/e; the others had subtype b ( %), ab ( %), and c ( %). there were patients in pi-naive group and patients in pi-experienced group. the clinical characteristics between groups were similar (p > . ) except for the duration of antiretroviral therapy which was shorter in pi-naive group ( . vs. . months, p = . ). percentage of patients who had primary pi-rams was % in pinaive and % in pi-experienced groups (p = . ). the most common primary pi-rams in the latter group were v a ( %) and i v ( %). percentage of patients with secondary pi-rams in the corresponding groups was % and %, respectively (p = . ). median number of secondary pi-rams was also similar between groups (p = . ). the most common secondary pi-rams in both groups were m i ( %), h k ( %), l m ( %), i v ( %), l p ( %), l i we also defined a "silent score" (ss) and a "resistance score" (rs) as the number of synonymous mutations and of resistance mutations (in the second sequence in comparison with the first one) divided by number of days between the two tests, respectively. ( ); pts with drms in non-b-st (%) were ( . ), ( ), ( ) and ( . ). a significant increase of non-b-st (p = . ) and a significant decrease in drms (p < . ) were observed. crf _ag was the prevalent non-b st ( %). . % of non-b st pts were italians. among b-st, drms predicted a reduced susceptibility to one drug class in , , and cases in the different periods; to two drug classes in , , and ; to three classes in , , and . in non-b-st, a reduced susceptibility to one drug class was found in , , and cases; to two drug classes in , , and ; to three drug classes in , , and , respectively. among pts with one or two classes of resistance, a decrease of percentage of protease inhibitors related drms, and a persistence of non nucleoside rt inhibitors involving drms, mainly n and a, were observed. methods: from hiv+ persons with a history of, or an acute episode of opc, oral fungal burden was evaluated bi-weekly and buccal mucosa tissue was collected bimonthly for a period of one year. tissue was evaluated for the presence of cd + t cells and e-cadherin by immunohistochemistry or flow cytometry. objectives: to define the secular trends in the epidemiology of candidaemia in queensland, australia (population, . million) over a -year period. methods: all episodes of candidaemia within queensland public hospitals from - were identified from laboratory information systems. data on species identification, antifungal susceptibility, demographics, and hospital ward of diagnosis, and denominator data (hospital admissions, accrued patient-days (pt-days) and fluconazole usage) were collected. results: over the -year period, unique episodes ( % case ascertainment) were identified from healthcare facilities ( tertiary, paediatric, secondary and smaller hospitals). the median patient age was . years. the overall incidence-density was . / ptdays, highest in paediatric ( . / pt-days) and tertiary hospitals ( . / pt-days). over the years, the incidence-density increased . -fold in tertiary hospitals and . -fold in secondary hospitals (both p < . for trend), but not in paediatric or smaller hospitals. the incidence-density in icus ( . / pt-days) was -fold higher than in non-icu wards, but did not significantly increase over the study period. the relative proportion of episodes occurring in adult general medical/surgical (ie non-oncology/non-icu) wards significantly increased (p < . ), accounting for % of episodes at the end of the -year period, whereas that occurring in paediatric and adult oncology wards decreased (p < . and p = . respectively). overall, c. albicans accounted for %, c. parapsilosis % and c. glabrata %. although the incidence-density of all species increased over the study period, the relative proportion caused by c. albicans decreased (p = . ) and c. parapsilosis increased (p = . ). despite significantly increased fluconazole usage (from . to . ddd/ pt-days, p < . ), the relative proportion caused by c. glabrata/c. krusei did not change (p = . ). the overall incidence of candidaemia has increased almost % in queensland public hospitals over the last years. the relative proportion of episodes occurring among general medical/surgical patients and caused by c. parapsilosis has increased. candidaemia is an increasing problem the epidemiology of which continues to evolve. it is increasingly affecting patients outside traditional risk groups. conclusions: this surveillance study and pharmaco-economic modelling has proved immensely beneficial in setting up inhouse processing, improved tat, reduced costs of outsourcing and subsequent use of expensive antifungals. reduction in mortality has been noted but is not statistically significant. c. albicans was the commonest isolate; fluconazole resistance is minimal and associated mortality is lower than reported from europe. many pts received systemic prophylaxis ( %); itraconazole and fluconazole were used in and pts respectively. no differences emerged between empirical vs pre-emptive therapy and none of the drugs resulted to significantly influence outcome. in % of pts initial empirical/pre-emptive drug remained unchanged after ia diagnosis, while in % clinicians shifted to a combined treatment. conclusion: this study allows as to analyzed multiple factors as potentially influencing outcome. we confirmed that aml phase and neutropenia influence ia outcome. present data confirm the perception that during last years the application of a correct and timely diagnostic work-up and the availability of more efficacious and less toxic drugs (i.e. voriconazole, liposomal amphotericin b, caspofungin) have modified the course of ia. however none of the new drugs emerged as the most efficacious in our series. even combined treatment did not confer any advantage in survival analysis. (< % each). the first line therapy was monotherapy with voriconazole ( %), caspofungin ( %), lipid formulations of amb ( %) or used antifungal drugs combination ( %). the mortality rate at day was % when first line therapy included voriconazole compared to % when it did not (p < . ). conclusion: comprehensive collections of cases based on systematic reporting and description of cases using a dedicated network of hospitals in selected regions and stringent definition criteria applied by trained clinicians and microbiologists are useful to describe ia, to assess its burden and secular trends, and to identify potential changes in diagnostic and therapeutic procedures. this network will expand to other regions in the near future, and data will help assessing the impact of new management strategies such as prophylaxis with posaconazole, the impact of modification of new diagnostic criteria as recently proposed (clin infect dis, ), and identifying new populations at risk for ia. nosocomial aspergillosis represents a serious threat for severely immunocompromised patients and outbreaks have been attributed to airborne sources. the role of hospital-independent fungal spread sources e.g. the private homes or business suites are not known. we investigated the relationship between fungal exposure prior hospitalisation and the ensuing onset of invasive mould infections (imi) in patients at risk. patients admitted to the department of haematology and oncology or to the department of transplant surgery of the innsbruck medical university received a structured questionnaire regarding their fungal exposure prior hospitalisation. questions inquired heavy fungal exposures up to five days prior hospitalisation. patients were enrolled in this study and % were smokers, % suffered from an airborne allergy, % lived in old buildings, % were ruralists, % and % were exposed to any outdoor or indoor fungus sources. poor housing conditions and other fungus exposures were associated with the onset of community-acquired imi only in patients with acute myelogenous leukaemia (p < . ). aml patients being more at risk for imi when smoking cigarettes (p < . ), living on the country site (p < . ), having two or more fungus exposures (p < . ) and suffering from allergy to dust, pollen and/or moulds (p < . ). a similar trend was for lung transplant recipients receiving extensive immunosuppressive agents to treat allograft rejection. overall, % of imi were community-acquired cases. hospital-independent fungal sources highlight risk-factors for imi in severe immunocompromised patients and the rate of communityacquired imi does increase. an analysis of an individual patient's risk factors for fungal infection and the type of fungus to which they are most susceptible, indicates the preventative strategies that are likely to be successful. to the icu-mhs with aspergillus spp detected in significant amounts in clinical samples. the underlying conditions of the patients were heart transplantation (n = ), major heart surgery (n = ), and other (n = ). eight ( . %) patients developed proven/probable ia ( with lung infection, with mediastinitis, with disseminated ia, and with prostate involvement). the mortality of patients with ia was . %. the icu-mhs is divided into areas, one of which is equipped with hepa filters. only case of ia occurred in the protected area. we measured the fungal conidia levels in the air of each of the areas ( samples analyzed) monthly. a total of strains of a. fumigatus ( clinical strains from patients and environmental strains) were genotyped using microsatellites (de valk et al, jcm ) . the mean airborne conidia levels ( months) before and after the outbreak were, respectively, . ( − ) cfu/m and . ( − ) cfu/m . no cases of ia occurred during these periods. however, all cases of ia were linked to peaks of abnormally high airborne conidia levels ( , , and cfu/m ). a. fumigatus was involved in cases of ia; patient was infected by non-fumigatus aspergillus (not further genotyped). in patients ( mediastinitis, pulmonary ia and colonisation), we demonstrated similar genotypes in the air and in clinical samples. patient was located in the protected area and had a unique genotype. patient had two different clusters of genotypes: one cluster was similar to that of patient and the other was also found in patient and in the air. the genotype present in patients and was also detected in the air during a -month period. conclusions: epidemiologic and molecular typing suggests that there is a causal relationship between aspergillus causing ia and those present in the air. our finding also supports the need for hepa filtration in icu-mhs. j. guinea is contracted by fis (cm / ). sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were calculated in reference to proven and probable cases of ia. reasons for performing bronchoscopy on patients were also recorded. the protocol received approval by the local ethic committee. results: from the samples studied, ( . %) were classified as proven, ( . %) as probable, and ( . %) as possible cases of aspergillosis. twelve samples ( . %) represented colonisation, and bal samples were obtained during routine surveillance. pulmonary aspergillosis was the main clinical presentation of ia ( . %). using roc analysis, the best cut-off for galactomannan testing in bal was defined as . (sensitivity . %, specificity . %, ppv % and npv . %). median bal gm index for the group of patients with proven/probable aspergillosis and for 'negative cases' were . and . , respectively (p < . ). overall mortality was % (n = ). the odds for death for patients diagnosed with ia were . , in comparison to patients who did not have this infection ( % ci . − . ). conclusion: gm testing in the bal added to the diagnosis of ia in lung transplant recipients. in order to avoid false-positive results, a higher test cut-off should be applied to bal samples, in comparison to sera. increasing the cut-off to . resulted in a very high npv, with an associated sensitivity of > %. objectives: ) determine the performance characteristics of the galactomannan (gm) assay in broncho-alveolar lavage (bal) in haematology-oncology patients; ) evaluate the prognostic value of the gm assay in this particular population. methods: the platelia gm eia assay (bio-rad) was performed on all bal specimens obtained from haematology-oncology patients at our institution between march and april , in addition to routine laboratory stains and cultures. all results were reported to physicians. we conducted chart reviews to classify cases as proven, probable, possible or without invasive pulmonary aspergillosis (ipa) according to the revised definitions of invasive fungal disease from the eortc/msg consensus group. for performance characteristics, proven and probable cases were considered as ipa; possible cases were considered as without ipa. the result of bal gm was not considered as a criterium to classify cases in order to avoid incorporation bias. in patients with > positive (gm index > . ) specimen, only the first one was considered for the analysis. mortality was calculated at days following the first bal procurement. data were analyzed with stata . . results: there were bal samples from patients, including haematopoietic stem cell transplant (hsct) recipients. we found proven, probable and possible cases of ipa (total of ipa cases; . %). gm on bal was positive in ( . %) specimens. the sensitivity and specificity of the gm assay in bal were % and . % respectively. positive predictive and negative predictive values were . % and %, respectively. false-positive results were found in patients without ipa and in with possible ipa. an index value . was significantly associated with a -day mortality risk ( / patients with a positive gm died within days after bal compared to / with a negative gm (or = . , %ci . − . ; p = . ). this association was even stronger when restricted to hsc recipients (or = . , %ci . − . ; p = . ). the clinical utility of gm assay in bal mainly lies in its negative predictive value, identifying patients at low risk of ipa. this test also carries a prognostic value in predicting patients at higher risk of mortality. (see table below) . not significant differences have been found among pneumocystis colonisation and copd status evaluated by fev- %. as well as no significant differences respect to age, sex or lymphocytes and leucocytes blood count were found. background: infliximab, a monoclonal antibody targeting tumour necrosis factor alpha (tnf-a), is indicated for the treatment of rheumatoid arthritis (ra) and other autoimmune diseases. however, its use has been associated with opportunistic infections, including pneumocystis jirovecii pneumonia (pcp). moreover, p. jirovecii has been observed colonising to humans with several disorders. objectives: to obtain information about p. jirovecii colonisation among patients with rheumatologic disease treated with infliximab. this information could be useful for assessing new strategies in the prevention of pcp in patients at risk. methods: consecutive patients treated with infliximab for rheumatic disorders were included in the study. oropharyngeal washes (ow) samples were collected for p. jirovecii detection. clinical and demographic data were collected (sex, age, rheumatologic diagnosis, duration of infliximab use, concomitant use of other drugs for rheumatologic treatment, use of any other anti-tnf-a agent, use of anti-pc drugs in the last six months, smoking, and diagnosis of chronic pulmonary respiratory disease). p. jirovecii colonisation was identify in ow samples by pcr at mtlsu-rrna gene, with primers paz -x and paz -y. we adapted a method previously described to a real-time pcr setting, using a lightcycler . (roche, germany). individuals in whom the presence of p. jirovecii was detected at two independent assay in the absence of respiratory symptoms or radiological findings suggestive of pcp were considered to be colonised. results: clinical and demographic data for patients treated with infliximab are presented in table objectives: most research with human bocavirus, a recently found respiratory pathogen, has been done by molecular biology (polymerase chain reaction, pcr). the results have been ambiguous because the virus has often been found in co-infection with other viruses, and also in clinically healthy subjects. it has been proposed that, for bocavirus, antigen detection could better indicate the aetiology than qualitative nucleic acid detection. we have developed a rapid antigen detection test for the virus. the one-step test for bocavirus vp antigen is based on a separation-free two-photon excitation fluorometry (arcdia tpx assay technique). the assay protocol is simple; the swab sample is dissolved in sample buffer, and the solution is dispensed ( ml) onto a -well microtitre plate (containing the reagents in dry form) for incubation and automated quantitative measurement. the immunoassay applies microspheres as solid-phase carriers of purified bocavirus-specific polyclonal antibodies. the virus antigens concentrate onto the solid-phase which is probed in real-time with fluorescently labelled antibody reagents. strong positive samples are reportable in minutes, while low positive and negative samples are reported in hours. the performance of the method was studied with recombinant human bocavirus-like particles (vp ), and purified respiratory pathogens (group a streptococci, streptococcus pneumoniae, and influenza a and b, respiratory syncytial, metapneumo, adeno, and parainfluenza − viruses). results: analytical detection sensitivity of the method (lowest limit of detection, -control + sds) was ng/ml, dynamic concentration range was three orders of magnitude, and intra-assay imprecision was − %. cross-reactions with the other respiratory pathogens were not found. the new method enables rapid detection of bocavirus antigens. the new test is very easy to perform in comparison to standard elisas. the analytical sensitivity of the method is expected to allow analysis of clinical samples. the sensitivity of the antigen detection test could be significantly increased by the use of monoclonal antibodies ( - fold). our future objectives include increasing the detection sensitivity, and analysis of clinical samples in order to study the correlation of antigen detection and the clinical aetiology. life-year for patients who survived. all analyses were performed using treeage software ( ). results: the overall mortality rates for empiric vancomycin (v) and semi-synthetic-penicillin (ssp) was % and %, respectively, as apposed to % for those receiving the rapid mrsa pcr testing. these mortality rates were similar in both the eu and us subsets. furthermore, the number needed to test in order to save one life was and for empiric v and ssp, respectively. using sensitivity analysis the prevalence of mrsa was varied from % to % and yielded an absolute mortality difference favouring the pcr testing group of % and %, respectively as compared to empiric v and % and % compared to empiric ssp. in eu the c/e for empiric v and ssp treated patients was € and € , respectively as compared to € for rapid pcr testing. in the us the c/e for empiric v was $ , as compared to $ for rapid pcr testing. using sensitivity analysis the prevalence of mrsa was varied from % to % and yielded favourable c/e in both the eu and us for rapid pcr testing regardless of the empiric treatment regimen. conclusion: rapid mrsa pcr testing using the xpert mrsa/sa blood culture pcr assay appears to improve mortality rates and is cost effective in the eu and us across a wide range of mrsa prevalence rates. background: rapid detection of gastro-intestinal carriage of glycopeptide-resistant enterococci (gre) from screening cultures is crucial for an efficient control of their spread. we assessed media − chromogenic, chromid, (biomérieux), and chromagar (chromagar microbiology), and selective, vre selective (oxoid) and eccv (bd) − for their ability to detect gre using well-characterised isolates and stool samples from hospitalised patients at high risk of gre colonisation. methods: twenty-five isolates consisting of gre. faecalis/faecium carrying various van genes and non-vre at concentrations of - cfu/ml and cfu/ml, respectively, and stool samples were randomised and spiral plated on all media and scored by blinded investigators for characteristic colonies after hrs incubation. standard confirmatory tests were done on putative gre colony or on characteristically coloured colony each for e. faecalis/faecium from the selective and chromogenic media, respectively. detection of van genes, and ddl or soda based speciation was done on pcr-sequencing. mean sensitivity (sen) and specificity (spec), and confidence intervals (cis) were estimated for each medium by a logistic regression model using a penalised likelihood approach based on the reader response for the stool samples and isolates, and additionally on confirmation test results for the stool samples, both at the aggregated (gre detected) and penalised level (correct species-colony colour correlation). results: chromagar showed the highest sen based on reader response at the aggregated and penalised level for both stool samples and isolates (table) . using confirmation test results at the aggregated level, sen for eccv was highest while the two chromogenic media showed a decrease in sen by at least % in comparison to the values obtained based on reader response. sens for the chromogenic media were even lower (< %) based on confirmation test results at the penalised level. eccv and chromid showed the highest specs with both reader response (stool samples) and confirmation test results at the aggregated level, and chromid also at the penalised level, with narrow cis indicating a high precision of this parameter estimate. for isolates, specs were highest for chromagar at both levels. conclusions: chromagar showed the best overall performance considering both sen and spec estimates. eccv performed well as a selective medium for gre detection from stool samples. objectives: metallo-beta-lactamases (mbls) expressed from pseudomonas are able to confer resistance to all beta-lactams with the exception of aztreonam. however, enterobacteriaceae possessing mbls exhibit moderate cephalosporin and low carbapenem mics and thus are often underestimated. herein, we describe data from new etest prototypes specifically designed to detect this problematic resistance mechanism. methods: mbl-positive (vim or imp derivatives) enterobacteriaceae clinical isolates from countries and randomly selected enterobacteriaceae negative controls (including the atcc type strains) were tested against the different etest mbl prototypes. beta-lactam substrates used were imipenem (ip), meropenem (mp), ceftazidime (tz) and cefotaxime (ct) with or without the inhibitors dipicolinic acid (dpa) and edta. the etest standard procedure for gram negative aerobes was used and a reduction of beta-lactam mic by equal to or greater than dilutions by edta or dpa was interpreted as positive for mbl. presence of esbls was tested using the etest ct/ctl, tz/tzl and cefepime (pm)/pml strips. ampc production was detected using the etest cefoxitin (fx)/fxi and cefotetan (cn)/cni strips. of the select specimens that were negative for gbs, grew turquoise-blue colonies, but the majority that required further work to rule out gbs grew after hours. two strains of gbs that were missed grew as white colonies on select, and even at h, did not exhibit the characteristic turquoise-blue colour. conclusion: ssb enrichment followed by select subculture was extremely sensitive ( . %) and superior to cna/ssb for detection of gbs from genital specimens. however, non-gbs organisms can produce turquoise-blue colonies on select and further work must be performed to rule out the presence of gbs. objectives: screening for chlamydia trachomatis (ct) specific antibodies is valuable in investigating recurrent cause of miscarriage, pelvic inflammatory disease and tubal damage following repeated episodes of pelvic inflammatory disease. immunofluorescence (if) is considered the gold standard for detection of ct antibodies. the present study aims to compare the performance of other commercial tests for the detection of serum igg antibodies specific for ct: two ct igg pelisa both using major outer membrane protein (momp; ["momp-medac", ct-igg-pelisa; medac, wedel, germany and "momp-ruwag", ct pelisa; ruwag, bettlach, switzerland), one ct hsp- igg pelisa ("hsp -medac", chsp -igg-pelisa; medac, wedel, germany), and a new automated epifluorescence immunoassay ("inodiag", "must chlamydiae; inodiag, signes, france). methods: a total of patients with (n = ) and without (n = ) miscarriages were tested by all serological tests described above. sensitivity and specificity were calculated using if as gold standard. a second standard, defining true positive or negative samples as sera respectively positive and negative in all others tests, was also used (see table) . objectives: participation in diagnostic microbiology internal and external quality control (qc) processes is good laboratory practice, an essential component of a quality management system and compulsory in some european countries. currently, there is no qc scheme for diagnostic oral microbiology. the aim of this study was to collate information on current qc needs, and processes undertaken in diagnostic oral microbiology laboratories. method: an on-line questionnaire was devised to ascertain interest in participating in an oral microbiology qc scheme and sent to oral microbiology diagnostic laboratories. the laboratories were identified from participants attending the european oral microbiology workshop in helsinki, . following this, a pilot round of qc samples was distributed to all interested laboratories. results: we identified individuals that worked in diagnostic oral microbiology laboratories and received ( %) positive responses. of these laboratories (representing european countries) % did not participate in either internal or external qc. each laboratory processed on average a total of samples annually. % of participants were in favour of a european-wide oral microbiology qc scheme. the preferred frequency for receiving external qc specimen was once in − months. the most preferred specimen types were periodontal pocket and oral pus specimens (both %), followed by oral mucosal swabs and caries activity tests. all participating laboratories were willing to share and harmonise their specimen processing and interpretation standard operating procedures. the pilot round specimen was a periodontal pocket sample. six laboratories reported their findings in the specified time. the predominant pathogens (aggregatibacter actinomycetemcomitans, porphyromonas gingivalis) were identified by of laboratories. in addition to conventional culture, one laboratory used pcr. laboratories performed antibacterial sensitivity testing primarily by disc diffusion. conclusions: this is the first attempt to a standardised europeanwide approach to diagnostic oral microbiology. the findings from this feasibility study have indicated that a qc scheme for oral microbiology is of interest and have raised a number a pointers for subsequent rounds of specimens. further work to improve the quality, to standardise the methodology and the interpretation of diagnostic oral microbiology at the european level is on-going. objectives: since severe sepsis with acute organ dysfunction can be fatal within hours, it is customary to start empirical broad-spectrum antimicrobial therapy in all patients hospitalised for a suspicion of systemic inflammatory response syndrome. however, increased use of broad-spectrum antimicrobials over the years has contributed to the emergence of drug resistant strains of bacteria. especially, drug resistance among gram-positive bacteria, the leading cause of sepsis, is now a serious problem. the objective of this preliminary study was to develop a method for distinguishing between gram− and gram+ bacterial infection. methods: in this prospective study, leukocyte and neutrophil counts, crp, esr, and quantitative flow cytometric analysis of neutrophil complement receptors (cr /cd ) and (cr /cd b), were obtained from hospitalised febrile patients, of which had bacterial and viral infection. the patient data were compared to healthy controls. results: it was noticed that in gram− infection (n = ) the average amount of cd b on neutrophils was significantly higher than in gram+ infection (n = ). on the contrary, serum crp level was significantly higher in gram+ than in gram− infection. other measured parameters did not differ significantly between gram+ and gram− infections. we derived a crp/cd b ratio dividing the serum crp value by amount of cd b on neutrophils. in thirteen ( %) out of patients with gram+ sepsis had crp/cd b ratio cutoff value of . (figure ). of these patients, ( %) were diagnosed with streptococcus pneumoniae, with staphylococcus aureus, with enterococcus faecalis, and with both streptococcus intermedius and streptococcus oralis. corresponding percentages in patients with local gram+ infection, gram− infection, clinical pneumonia, other clinical infection, and viral infection were %, %, %, %, and %, respectively. conclusion: the detection of gram+ sepsis is possible after combination of neutrophil cd b data and serum crp level. crp/cd b ratio viral infections of the central nervous system s displayed % sensitivity and % specificity for detection of gram+ sepsis. the proposed crp/cd b ratio test could, for its part, assist physicians to decide appropriate antibiotic treatment in patients with severe bacterial infection. a bacterial biofilm is a structured consortium of bacteria cells surrounded by a self-produced polymer matrix. biofilms may be monospecies or polyspecies biofilms. biofilm growing bacteria give rise to chronic infections, which persist in spite of therapy and in spite of the host's immune-and inflammatory responses. biofilm infections are characterised by persisting pathology and immune response (in contrast to colonisation). bacterial biofilms use both biofilm specific (b) and conventional (planktonic) resistance mechanisms (p) when they are exposed to antibiotics. the following resistance mechanisms have been described in bacterial biofilms: . stationary phase physiology (b), low oxygen tension (b) and slow growth (b) especially inside biofilms whereas the surface of biofilms is more similar to planktonic growth. . penetration barriers (b), binding to the polymer matrix (b). . mutations, hypermutators (b, p). . chromosomal betalactamase is upregulated (b, p). . antibiotic tolerance/adaptive resistance (b). . efflux pumps (b, p). . alginate production (b). . high cell density and quorum sensing (b, p). . pbp − sos response ? (b). the knowledge of these resistance mechanisms can, however, be used to design new therapeutica approaches especially as regards quorum sensing inhibitors. we consider two factors that contribute to treatment failure in the absence of inherited resistance, the density of the population being treated and the physiological state of the bacteria. we also explore how these factors might contribute to the evolution of inherited resistance during the course of treatment. we conclude with a computer-and chemostat-assisted consideration of the potential clinical implication of these density and physiology effects and make suggestions for treatment protocols to deal with them. using in vitro cultures of staphylococcus aureus atcc or the clinical isolate ps and antibiotics of six different classes we determined the functional relationship between the inoculum density and the efficacy of the antibiotics. as measured by the rates and extent of kill and/or the minimum inhibitory concentration (mic), the efficacy of all of these antibiotics declined with increases in the density of bacteria, albeit to different extents. for daptomycin and vancomycin, much of this density effect can be attributed to bacteria-associated declines in the effective concentration of the antibiotic in the medium. for gentamicin, vancomycin, ciprofloxacin and oxacillin, our bioassays failed to reveal significant reductions in their effective concentration in the medium. the effects of the physiological state of s. aureus on the efficacy of these antibiotics were examined for bacteria from cultures in "stationary phase" for different times and from chemostats run at different generation times. these experiments are currently under way but by the time of the symposium we will have the full (and true) story. it is, however, clear that the efficacy of all of these antibiotics declines with the time in stationary phase (its "age"). and, even slowly dividing cultures from chemostats are more susceptible to antibiotic-mediated killing that early stationary phase batch cultures. the efficacy in killing non-growing bacteria varies among the bactericidal antibiotics examined. to ascertain the potential clinical implications of these density and physiological effects, we use both computer and in vitro simulations of antibiotic treatment. the results of these simulations provide compelling support for the proposition that antibiotic treatment regimes, including those designed to prevent the ascent of resistance, should take into account the anticipated density and physiological state of the target population of susceptible bacteria. there have been an increasing number of neurotrophic viral infections playing an important role in the world over the last decade. the list includes west nile virus, nipah and hendra virus (both paramyxoviruses), as well as chikungunya virus which suddenly emerged. furthermore, the relation between jc virus in progressive multifocal leukoencephalopathy (pml) in patients with multiple sclerosis treated with a new immunosuppressive drug, has triggered our attention. the development and implementation of molecular based amplification method has assisted us to detect these viruses more efficiently. these technologies have been used now routinely in a large number of laboratories to enable the detection of more commonly known neurotrophic viruses, like hsv, vzv and the neurotrophic picornaviruses like enterovirus and parechovirus. the pitfalls of these molecular methods have been generally solved by implementing regular quality control testing schemes, like organised by qcmd (quality control of molecular diagnostics) and the introduction of internal controls during the whole diagnostic process. finally, with the ability to quantify the amount of nucleic acid present in csf, more information on the pathogenesis of these viral infections, as well as significant tool to monitor the antiviral effect of treatment options for these viruses, has become available. to as a rare disease in europe restricted to some endemic foci. however, current data suggest that the incidence of ae has significantly increased, and the disease is spreading to the north, west, and east. ae has become an emerging disease in the baltic countries. thus, human infections with e. multilocularis have arrived in the "centre" of europe. ae is a lifethreatening disease, and is characterised by a tumour-like lesion in the liver. the larva can infiltrate the surrounding tissues and metastasize to distant organs. in an attempt to classify the large variety of anatomical findings in ae, the pnm-classification system was developed and serves as a benchmark for standardised evaluation of diagnostic and therapeutic measures. modern imaging techniques, such as ultrasound, ct or mri and pet/ct contributed not only to a much better description of the lesions, but also to a judgment upon the activity of the metacestode. the differential diagnosis of ae varies from haemangioma-like lesion of the liver or cancer. the diagnostic skills are limited, and are the reason for frequent misdiagnosis in geographic areas where ae is rather unknown. continuous treatment with benzimidazoles is the backbone of a lifelong management of ae. however, radical resection is the procedure of choice and should always be strived for. ae is still a rare disease in europe, but where it occurs, it is often diagnosed too late. patients are misdiagnosed for months and years, before receiving the correct treatment. at that late stage the disease has progressed, and radical cure of the liver lesion(s) is not anymore possible. recent reports provided hints for an accelerated larval growth of echinococcus spp. in the immunodeficient host. a careful monitoring of patients receiving immune-modifying drugs is warranted. the modern clinical management and long-term parasitostatic treatment with benzimidazoles are highly effective. thus, a higher alertness for the "tumours from the centre" would increase the prognosis of this hepatic disease resembling liver cancer. the percutaneous treatment of liver hydatid cysts were considered to be contraindicated due to two main potential risks: anaphylactic shock and abdominal dissemination of the disease. since the first case percutaneously treated was published, several series of successful percutaneous treatment of the liver and the other abdominal organs, peritoneum, thorax, soft tissue and orbital cavity hydatid cysts have appeared in the literature. percutaneous treatment of hydatid liver disease is an effective and safe procedure with its unique advantages (e.g., shorter hospital stay, low complication rate). today, the percutaneous approach has an important role in treatment of hydatid cysts not only in the liver but also in the other organs and tissue. therefore it must be first treatment option whenever it is indicated. in europe, dirofilaria immitis and dirofilaria repens are responsible of autochthonous filariases in dogs. adults of d. immitis kills the dogs with an heart location and d. repens is often found in subcutaneous nodules in dogs and cats. the microfilariae are present in the blood of these animals. dirofilariasis is due to the transmission of microfilariae by some mosquito bites (aedes, culex, anopheles, mansonia, psorophora and taeniorhynchus). usually non pathogenic to humans, these parasites are particularly present around the mediterranean basin. d. immitis is very rare in humans in europe, sometimes found in a pulmonary nodule and the heart location is not described. d. repens is more frequent and emerging in humans. usually, only one larva develops, producing an immature adult worm inside a subcutaneous nodule. ultrasound examination may suggest the parasitic origin of the lesion before an extraction and a parasitological diagnosis of the worm. more often, a fortuitous diagnosis is made on histological examination. very rarely, an adult worm may mature and produce systemic diffusion of microfilariae. dirofilariasis due to d. repens can present problems in diagnosis and treatment. an ocular and subconjunctival location of the worm and a subcutaneous nodule enclosing an immature adult are the commonest clinical forms. exceptional pulmonary locations are described. the subcutaneous locations described are: skull, cheek, breast, inguinal area, buttocks, arms and legs. cases of testicular location with painful symptoms have been observed. blood hypereosinophilia was exceptionally observed in human. it is treated surgically, by excision, without chemotherapy. while the majority of esbls, isolated in clinically-relevant gram negative bacteria (gnb) (mostly enterobacteriaceae, p. aeruginosa, a. baumannii) are tem-, shv-or ctx-m-types, a few others have been reported (sfo, bes, bel, tla, ges, bel, per, veb-types, and some oxa-esbls). laboratory detection of esbl-producers is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. selective culture media (macconkey and drigalski agar supplemented with cefotaxime and/or ceftazidime) have been proposed for detection of gnb resistant to expanded-spectrum cephalosporins (esc). media using chromogenic based substrates and selective antibiotics have been developed recently for the detection and presumptive identification of esbl-producing enterobacteriaceae directly from clinical specimens. detection of esbls based only on susceptibility testing is not easy due to the variety of b-lactamases and their variable expression of blactam resistance. commercially available esbl detection methods yield at most % accurate esbl identification, since some esbl-producers may appear susceptible to some escs. therefore, any organism showing reduced susceptibility to esc should be investigated using esbl confirmatory tests. these tests should be able to discriminate between esbl-producers and those with other mechanisms conferring esc resistance. these phenotypic tests (double-disk synergy test, esbl etest, and the combination disk method) are based on clavulanate inhibition and esc susceptibility testing. they often need slight changes by either reducing the distance between the disks of esc and clavulanate, the use of cefepime (not hydrolysed by ampcs), the use of cloxacillincontaining plates (that inhibits ampc), or by double inhibition by edta and clavulanate (masking metallo-enzymes). enzymatic tests have also been proposed for identification of esbl-producers. several pcr-based techniques (end-point or real time) have been developed on clinical samples or on colonies. several esbl genes have been detected using pcr coupled to either pyrosequencing, inverse hybridisation, to dhplc, or to fluorescent probes. these techniques even though more specific require technical knowledge, special equipment, are costly and detect only known genes, regardless of their expression. detection of esbl-producer remains a challenge for the microbiology laboratory and one shall be aware that esbl screening media are now available. resistance to antimicrobial agents has become common in many bacterial species, particularly those that cause human infections. the rapid detection of resistant organisms directly in clinical samples by real-time pcr coupled with molecular beacons, or of potentially resistant bacteria and yeast in blood culture bottles by peptide nucleic acid-fluorescence in situ hybridisation (pna-fish) is already having a positive impact on antimicrobial therapy. the direct detection of mycobacterium tuberculosis in sputum in approximately hours with concomitant detection of mutations in rpob indicating rifampin resistance (as a surrogate for multidrug resistance) in the near future will likely improve the outcomes for tuberculosis patients in many developing and developed countries. several molecular technologies, including microarrays, bacterial tag encoded flx amplicon pyrosequencing (btefap), and ultra deep sequencing, have not yet transitioned to clinical laboratories but will likely provide even greater information about antimicrobial resistance not in just a single species, but in a whole community of microorganisms. complex wounds, like diabetic foot ulcers, containing multiple resistance genotypes are amenable to analysis by btefap. the implementation of these technologies in the clinical laboratory will be expensive but the potential to dramatically improve therapeutic outcomes especially for life-threatening diseases is unprecedented. objective: to determine the appropriateness of antimicrobial therapy (amt) in dutch hospitals. method: data were obtained from a prevalence survey performed within the dutch surveillance network for nosocomial infections (prezies). amt administrated on the day of the survey was registered. antiviral and antifungal drugs, tuberculostatics, cements containing amt and prophylaxis administrated in the operation-theatre were excluded. the appropriateness of amt was assessed according to a standardised algorithm based on the local antimicrobial prescription guidelines. per patient a classification in appropriate use, inappropriate use and insufficient information was made. figure: relative risk of ia use of amt against largest hospital (hospital c). results: a total of , patients were included of which , ( %, range per centre (rpc): − %) received amt. in the latter group, amt was considered appropriate in % (rpc: − %), inappropriate in % (rpc: − %) and was not judged because of insufficient information in % (rpc: − %). there was considerable variation in inappropriate use among the participating centres (figure). in univariate analysis older age, the use of quinolones, being on the urology ward and presence of a suprapubical catheter were associated significantly with inappropriate use. admission on the icu and presence of an intravascular catheter were associated significantly with appropriate use. in a multivariate analyses the presence of suprapubical catheter, being on the urology ward and the use of quinolones were determinants for inappropriate use. this study showed large differences in overall use and appropriateness of use of amt between hospitals. based on these results it is possible to define targets for intervention to improve the prudent use of amt. the high fraction of patients with insufficient information in several centres may have influenced the analyses and should be addressed in future studies. m. struelens°, s. metz-gercek, r. mechtler, f. buyle, a. lechner, h. mittermayer, f. allerberger, w. kern objectives: the eu-project antibiotic strategy international (abs) qi team developed process qis for auditing the performance of key treatment and prophylactic practices. an international network of pilot hospitals tested these tools for feasibility, reliability and sensitivity to improvement. methods: qis included: . surgical prophylaxis (indication, drug choice, timing and duration of administration); . management of community-acquired pneumonia (cap) (blood culture and legionella antigen tests and drug choice for empirical treatment); . management of s. aureus bacteraemia (echocardiography, iv catheter removal and duration of therapy); and . iv-po switch for bio-available antibiotics. a minimum of consecutive cases per centre and qi were retrospectively reviewed from clinical, laboratory and administrative records and assessed for data availability, inter-observer reliability, data collection workload and performance score. results: a total of patients were evaluated in acute care hospitals from countries, with a range of to cases and to centres per indicator. seven centres had already implemented antibiotic quality improvement and audit programmes. availability of data was > % of cases and ranged between % (catheter removal in s. aureus bacteraemia) and % (diagnostic tests for cap). / indicators were found to be reliable with kappa . (good to excellent agreement). the workload per case ranged from a median time of (cap) to min (iv-po switch). the intention to treat qi scores showed high levels of adherence to the surgical prophylaxis qi bundle, with median values of to % for hip prosthesis and to % for colo-rectal surgery. for cap management, diagnostic testing appeared sub-optimal (< % compliance with idsa guidelines). for s. aureus bacteraemia management, indicator results ranged from to %. for use of bio available antibiotics, a median of % iv administrations were avoidable. there were marked differences of scores between centres for all qis. conclusions: the abs qis are reliable and broadly applicable tools for auditing antibiotic treatment and prophylactic practices. inter-hospital variation in adherence to recommended practice indicates substantial potential for improvement with different local priorities. these qis can be recommended for assessing the effect of quality of care interventions at either local or multi-centre level. d.j. noimark°, e. charani, s. smith, b. cooper, i. balakrishnan, s.p. stone (london, uk) introduction: reduction of clostridium difficile infection (cdi), which often follows use of third generation cephalosporins, is a national priority. over a three year period, antibiotic policies were reviewed and changed in an elderly medicine department according to local sensitivities of common pathogens and levels of cdi. a laminated pocket-sized card describing antibiotic policies was given to all doctors in the department on induction with instructions not to depart from these without microbiologists' approval. this prospective controlled interrupted time series examines whether this intervention increased compliance with antibiotic policy and decreased cdi incidence. methods: the department's "narrow-spectrum, no cephalosporin" antibiotic policy was changed on st august to replace trimethoprim with cephradine ( st generation cephalopsporin) as empiric treatment for urinary tract infection, reflecting local escheriscia coli sensitivities. in october , all cephalosporins and quinolones were removed from the policy as cdi levels had increased. notional day antibiotic usage was calculated from prospective pharmacy generated data with aspirin, calcium, bisphosphonate & laxative prescription use as a non-antibiotic control, and analysed by segmented regression with a robust variance estimator. cdi rates were prospectively collected separately & analysed by a poisson regression model. results: an immediate response to change in antibiotic guidelines was observed (figure) . from august -sep there was a highly significant increase in cephalosporins ( - % of which was cephradine alone) (p < . ), a significant fall in trimethoprim (p < . ) and a significant increasing trend in cdi ( no tools existed to assess the readiness of public hospitals to receive this technology, and therefore guide resource allocation to facilitate implementation. aim: to assess the readiness of victorian public hospitals to introduce electronic antimicrobial stewardship. method: literature on readiness for change, organisational culture and information technology acceptance were reviewed. group interviews with project teams at site initiation meetings, one on one interviews with project officers at subsequent meetings, and observation where appropriate were all used to determine potential barriers and enablers. this information was recorded using a 'readiness assessment tool' and analysed to identify a number of key domains. to triangulate the data, questionnaires were distributed to project officers asking them to assess their sites' readiness to implement the system. results: a novel 'readiness assessment tool' was developed. it covered the domains of technical readiness, skills readiness, process readiness, administrative support readiness, resource readiness and hospital organisational characteristics. assessments at several hospitals highlighted a variety of issues at different sites and allowed early efforts to address these. a formative readiness assessment can be used to identify systematic problems that might facilitate or hinder uptake of electronic antimicrobial stewardship and to inform the adopters of potential resources required. [ ] buising, k, thursky, k, robertson, m, black, j, street, a, richards, m & brown, g ( ) . electronic antibiotic stewardship-reduced consumption of broad-spectrum antibiotics using a computerised antimicrobial approval system in a hospital setting. j antimicrob chemother. w.v. kern°, m. steib-bauert, a. pritzkow, g. peyerl-hoffmann, h. von baum, u. frank, m. dettenkofer, c. schneider, k. de with, h. bertz (freiburg, ulm, de) objectives: fluoroquinolone prophylaxis (fqpx) may reduce morbidity and mortality in cancer patients (pts) with neutropenia, but the development of fluoroquinolone resistance (fqr) in escherichia coli and other target organisms limits its usefulness. we evaluated changes in the incidence density of gram-negative bloodstream infection (gnb) and in the in vitro fqr rates after the introduction of fqpx (with levofloxacin) as a standard of care for pts with high risk neutropenia in a university hospital. methods: we collected individual data for pts admitted during baseline and during the first months following the intervention to assess clinical outcomes. individual pt data were compared with aggregate data ( -month periods). aggregate data analysis (unit-wide antibiotic consumption, gnb and numbers of in vitro fqr bloodstream isolates) was continued for a total of eight -month periods for both the haematology-oncology service and for general internal medicine. the new policy was introduced in the second half of the year when unit-wide baseline fqr of e. coli and of coagulase-negative staphylococcal (cons) bloodstream isolates had been % and % in the haematology-oncology unit, and % and % in general internal medicine, respectively. the individual pt data analysis revealed that pts not given fqpx had a much higher incidence of gnb than those given fqpx ( - ) . the monthly use of iv and oral quin was calculated based on data from the pharmacy department. statistical analyses were performed using segmented linear regression analysis. bayesian model averaging was used to account for model uncertainty. results: before the interventions the use of quin (both iv and total) was stable. the best fitting models indicated that the first intervention was associated with a stepwise reduction in iv use of prescribed daily doses (pdd) ( % ci: , (p < . )). there was also an indication of smaller reduction in iv use associated with intervention , but only the intervention effect was robust to model uncertainty. the overall use of quin was also significantly reduced (figure) with a large stepwise reduction of pdd ( % ci: , ) associated with intervention . this study showed that the hospital-wide use of quin can be significantly improved (and decreased) by an active policy consisting of multiple interventions. marwick°, j. broomhall, c. mccowan, s. gonzalez-mcquire, k. akhras, s. merchant, p. davey (dundee, high wycombe, uk; raritan, us) aim and objectives: to describe the antibiotic treatment and outcomes stratified by severity in a representative sample of adult patients aged or older who were treated in hospital for skin and soft tissue infections. inadequate. we also judged that % of patients received unnecessarily broad spectrum therapy. conclusions: ssti is common and is associated with significant mortality. however, choice of empirical therapy is not evidence based, with significant under treatment of high risk patients. ab were mostly ( / ) prescribed by gps and delivered by public (n = ) or hospital pharmacies (n = ). surveillance of ab use in nhs was organised in only ms. in countries a nh specific pharmaceutical formulary was available. prescription profiles by prescriber were available in countries. other quality improvement initiatives in nhs such as regular training of prescribers, promoting microbiological sampling, collection of antimicrobial resistance profiles or pharmacist advice on ab prescription were scarce. guidelines for ab treatment of most frequent infections were available in many countries but were focussing on ambulatory care and did not consider the specific nh situation. only in country the presence of an infection control practitioner was compulsory and partnership with hospital infection control teams was legally imposed in ms. conclusion: important structural, functional and regulatory nh differences exist between eu countries. specific tools to improve infection prevention and ab therapy in nhs should take into account these differences. a european nh network was created in the framework of the esac nh subproject, which will organise point prevalence surveys on ab use in . c. escherichia coli in south-western finland j. jalava°, o. meurman, h. marttila, a. hakanen, m. lindgren, k. rantakokko-jalava (turku, fi) objectives: extended-spectrum betalactamases (esbls), especially enzymes of the ctx-m group, are spreading rapidly in europe. enterobacteriaceae with reduced susceptibility to third generation cephalosporins and a positive esbl confirmatory test are also increasing in southwest finland. the purpose of this work was to study the resistance genetics of these esbl-positive enterobacteriaceae. methods: the study comprises a total of clinical enterobacteriaceae strains isolated from both inpatient and outpatient specimens. all enterobacteriaceae strains that were esbl confirmatory test positive between january and december were included in this study ( escherichia coli, klebsiella pneumoniae, one isolate per patient). of these strains, ( %) were urine isolates. resistance determinations were done using disk diffusion method (clsi) or vitek and esbl confirmations by the double disk method using cefotaxime and ceftatzidime with and without clavulanate. thus far, strains (those collected by end of june ) have been analysed for the presence of the most important esbl genes (tem, shv and ctx-m) using pcr and pyrosequencing as described before (haanpera et al. aac, : ; ) . results: in only esbl-positive strains were found. all of them harboured a ctx-m type esbl gene. since then, the number esblproducing enterobacteriaceae strains has increased significantly being tenfold in compared to year (figure) . a high majority, ( %) of the strains analysed thus far had a ctx-m-type esbl gene. most of those ( %) belonged to the ctx-m- group according to the pyrosequencing results. ctx-m- group was the next common, with % of the ctx-m genes belonging to this group. only two strains with ctx-m group enzyme were found. conclusions: enterobacteriaceae strains which produce esbl are increasing rapidly in southwest finland. this is especially true with e. coli strains isolated from urine. towards the end of the study period, the esbl enzymes were almost exclusively ctx-m, ctx-m- group being the most common. further research is needed to characterise genetic elements that carry these esbl genes. esbl strains and the proportion of ctx-m genes in - . ( ) ( ) ( ) ( ) ( ) ( ) ( ) in france (n = ), spain (n = ), portugal (n = ), uk (n = ), kuwait (n = ), canada (n = ) and china (n = ), including hong kong (n = ) were studied. clonality was established by pfge and phylogenetic groups of ec and kp were determined as reported. susceptibility testing (clsi), blactx-m- transferability and location (i-ceu-i/s nuclease) were investigated. plasmid analysis included determination of inc group (pcr-replicon typing, hybridisation, sequencing) and comparison of rflp patterns. association of blactx-m- with isecp , isecp -is or iscr was established by pcr and sequencing. we identified pfge types among isolates: / ec, / kp and / cf. distribution among phylogroups were as follows: i) ec: a (n = ), b (n = ), b (n = ) and d (n = ), and ii) kp: kpi (n = ) and kpii (n = ). resistance to tetracycline ( %), nalidixic ( %), streptomycin ( %), sulfonamides ( %), ciprofloxacin ( %) and trimetroprim ( %) was common. were spreading horizontally in our hospitals and, here, we characterised the plasmids responsible in the major k. pneumoniae strains identified during the survey. methods: plasmids from representative k. pneumoniae strains with ctx-m- enzyme were extracted by alkaline lysis and compared by apai, psti and ecori restriction analysis. they were transferred into e. coli dh a by electroporation. transformants were selected on cefotaxime-containing agar and were screened by pcr for beta-lactamase genes, the aminoglycoside resistance genes aac( )-ib and aac -iib, and the plasmid-mediated quinolone resistance genes qnra/b/s. results: twelve isolates were characterised, representing major strains (a-d, and f) found in the most-affected hospitals. restriction analysis divided their plasmids into several groups. representatives of strain a (n = ) had essentially the same plasmid (group ), as did the two representatives of strain d (group a). one strain f isolate had a plasmid (group b) very similar to plasmid a from strain d, indicating possible horizontal transfer. plasmids of group were retrieved from representatives of strains b and c, again indicating probable transfer. plasmids from three other strains differed substantially from each other and from plasmids , a, b and . nevertheless, on all plasmids, blactx-m genes were linked to an upstream isecp element, known to be involved in their mobilisation. all encoded multi-resistance: all but one group and one ungrouped plasmid carried aac( )-ib; blaoxa- and aac( )-iia were detected on all except group plasmids; blatem was found on group , b, one group and two ungrouped plasmids. blashv and qnra/b/s genes were not detected. the considerable diversity of plasmids encoding ctx-m- enzyme in major slovenian k. pneumoniae strains suggested only limited transfer, even when multiple strains were present in the same hospital. evidence of plasmid transfer was between strains b and c, and possibly between strains d and f, although these plasmids were not strictly identical. analysis of resistance genes encoded by the plasmids revealed diversity, with groupings coinciding largely with those based on restriction profiles. a. ingold, g. borthagaray, a.k. merkier, d. centrón, h. bello, c.m. márquez°(montevideo, uy; buenos aires, ar; concepción, cl) objectives: to examine the genetic context of class integron harbouring blactx-m- in fifteen nosocomial k. pneumoniae isolates from south america in order to enhance the understanding of the antibiotic resistance spread among the region. methods: dna was extracted with the use of axypreptm bacterial genomic dna miniprep kit. the analysis of the cassette array was carried out with the use of primers hs /hs targeting adjacent conserved regions. the examination of the surroundings were performed using two pcr primer pairs, hs /hs and hs /hs , to amplify the initial(iri) and the terminal(irt), inverted repeat boundary, respectively. the primer pair hs /hs was used whenever a negative result was obtained with hs /hs . all pcr products were purified and sequenced and the data was analyzed with ncbi blast tool. the sequence obtained with primers hs /hs revealed the presence of three different transposons backbones at the iri end. the tn -like module and the tn -like module were present in isolates, the tn -like module was present in isolates. no amplicons were obtained with the use of primers hs /hs that amplify a tn -like insertion. two uruguayan isolates with a tn boundary at the iri end were tested with hs /hs that target a tn -like backbone and one generated a product consistent with a tn -like mer region. uruguayan isolates carried a single aada cassette ( / ) and the other one contained a dfra -aada array, while the four argentinian isolates carried the combination aaca -aada -orfd. chilean isolates arrays are in process. conclusions: among the extended-spectrum beta-lactamases, the cefotaximases constitute a rapidly growing cluster of enzymes that have disseminated geographically. there is a high frequency of isolation of ctx-m- producing k. pneumoniae associated with a class integron in the region. despite being common the presence of iscr linked to blactx-m- in k. pneumoniae isolates, this study provides new and relevant information in the sequence context at the iri. here we report about the cassette array diversity and the diversity of elements in which the class integron are embedded. different integron/transposons carrying the blactx-m- gene seem to be circulating and different regional patterns could be emerging, this study highlights the ability of different genetic elements to act cooperatively to spread and rearrange antibiotic resistance. l. vinué, a. garcía-fernández, d. fortini, p. poeta, m.a. moreno, c. torres, a. carattoli°(logroño, es; rome, it; vila real, pt; madrid, es) objectives: ctx-m enzymes are frequently detected in europe. in particular, ctx-m- and ctx-m- -producing strains have been recovered from both humans and farm animals in spain, italy, greece, and portugal, suggesting the existence of community reservoirs for these enzymes. the aim of this study was to compare escherichia coli strains and plasmids harbouring blactx-m- and blactx-m- genes isolated from human and animals. methods: four e. coli ctx-m- and eight ctx-m- epidemiologically unrelated producers from sick or healthy animals (pig, dog, cow and chickens) and from humans (urine, blood and faecal samples) were analysed by xbai-pfge, plasmid transferability, pcr-based replicon typing, plasmid restriction analysis and southern blot hybridisation. all isolates were from spain but the dog isolate was from portugal. the genetic context of the blactx-m genes was previously investigated for all the strains. results: three ctx-m- strains (one from healthy chicken and two from hospitalised patients) showed the same pfge pattern. a chromosomal localisation of the blactx-m- gene was suspected in these strains. the five remaining ctx-m- producers showed the blactx-m- gene on plasmids belonging to the incn ( strains) or untypable groups ( strain). two incn plasmids showed identical pvuiirestriction patterns: one was identified in a strain from a healthy chicken and one was from a hospitalised human patient; these two strains were isolated in and , respectively and showed different pfge patterns. ctx-m- producers (three from animal strains and one a healthy human) did not show clonality by pfge and the blactx-m- gene was always located on plasmids, three belonging to the incn and one to the inci groups. two of the incn plasmids carrying the blactx-m- gene showed highly related restriction patterns: one was from a healthy dog and one from a healthy human. conclusion: this study demonstrated the presence of clonal e. coli ctx-m- producers in animal and human sources and also detected epidemic incn plasmids disseminating among unrelated isolates from humans and animals, clearly suggesting a potential animal reservoir for the blactx-m- / genes. o characterisation of bladim- , a novel integron-located metallo-beta-lactamase gene from a pseudomonas stutzeri clinical isolate in the netherlands l. poirel°, j. rodriguez-martinez, n. al naiemi, y. debets-ossenkopp, p. nordmann (k.-bicetre, fr; amsterdam, nl) objectives: characterisation of the mechanism involved in the uncommon resistance to carbapenems observed from a pseudomonas stutzeri isolate recovered from a patient hospitalised in the netherlands with a chronic tibia osteomyelitis. that strain was resistant to ticarcillin, piperacillin-tazobactam, imipenem and meropenem, of intermediate susceptibility to ceftazidime and cefepime, and susceptible to aztreonam. methods: screening for metallo-beta-lactamase (mbl) production was performed using the e-test method with a strip combining imipenem and edta. shotgun cloning was performed with xbai-digested dna of p. stutzeri and pbk-cmv cloning vector. selection was performed on amoxicillin and kanamycin-containing plates. results: e. coli top (pdim- ) recombinant strains were obtained, displaying resistance to penicillins and ceftazidime, reduced susceptibility to cefepime, imipenem and meropenem, and full susceptibility to aztreonam. sequence analysis identified a novel ambler class b betalactamase dim- for "dutch imipenemase" (pi . ) weakly related to all other mbls. dim- shared % amino acid identity with the most closely related mbl gim- , and and % identity with the imp and vim subgroups, respectively. dim- hydrolyzes very efficiently imipenem and meropenem, expanded-spectrum cephalosporins, but spares aztreonam. the bladim- gene was as a form of a gene cassette located at the first position in a class integron, but the be of that gene cassette was truncated giving rise to a fusion with an aadb gene cassette encoding an aminoglycoside adenylyltransferase. the third and last gene cassette corresponded to the qach cassette encoding resistance to disinfectants. conclusion: a novel mbl gene was identified in p. stutzeri further underlining (i) the diversity of acquired mbl genes, especially among non-fermenters, (ii) that pseudomonas sp. may be a reservoir of these genes and (iii) the possibility of spread of important resistance determinants in northern part of europe. isolates in greece p. giakkoupi, o. pappa, m. polemis, a. bakosi, a. vatopoulos°( athens, gr) objectives: metallo-beta-lactamases of the vim family are the main mechanism of carbapenem resistance in p. aeruginosa in greece. in this preliminary report we attempted to survey the subtypes of vim betalactamase currently prevailing in p. aeruginosa clinical isolates in greek hospitals, the genetic relatedness of the respective isolates, as well as the genetic environment of the blavim gene. methods: fifteen mbl producing and epidemiologically unrelated p. aeruginosa clinical isolates were collected in september from fifteen different hospitals around greece. mbl production was initially identified by an edta synergy test. identification of blavim gene, as well as mapping of the blavim cassette carrying integrons were performed by pcr and sequencing of the products. the o serotypes of the isolates were determined by a slide agglutination test using p. aeruginosa antisera (biorad). molecular typing was performed by pulse-field gel electrophoresis of spei-restricted genomic dna. results: blavim- gene was detected in nine isolates, blavim- in five and blavim- in only one isolate. the blavim- cassette of all nine isolates was located on the bp variable region of a class i integron, preceded by aaca gene cassette. blavim- cassette of all five isolates was the first cassette of the bp variable region of a class i integron, followed by the aaca and blapse- gene cassettes. blavim- was the unique cassette of a class i integron. vim- producers belonged to o , o and o serotypes, whereas four isolates were non-typeable. vim- producers belonged to the same three serotypes, whereas only one was non-typeable. the vim- producer belonged to o serotype. the nine vim- producing p. aeruginosa isolates revealed a great degree of variability in pfge molecular typing, belonging to seven types. contrary, the five vim- producing p. aeruginosa isolates displayed higher genetic similarity and fell into one major type with % homology, which also included the vim- producing isolate. there was no correlation between the results of serotyping and molecular typing. conclusions: mbl production in p. aeruginosa in greece seems to be mainly due to specific class i integrons harbouring either blavim- or blavim- genes. genetic variability was higher among bacteria carrying vim- beta-lactamase, a fact indicating wider intraclonar spread of the respective integron. j.m. rodriguez-martinez, l. poirel°, p. nordmann (k.-bicetre, fr) objectives: extended-spectrum beta-lactamases of ampc-type (esacs) contributing to reduced susceptibility to imipenem have been recently reported from enterobacteriaceae. the aim of the study was to evaluate the putative role of natural ampc-type beta-lactamases of p. aeruginosa in a similar resistance profile. methods: thirty-two non-repetitive p. aeruginosa clinical isolates recovered in our hospital in were included. they were selected on the basis of criteria of intermediate susceptibility or resistance to ceftazidime and intermediate susceptibility or resistance to imipenem. mics were determined by agar dilution and e-test techniques. the level of expression of the ampc beta-lactamases was evaluated by measuring specific activities. pcr, sequencing, and cloning allowed to characterise the different bla(ampc) genes. identified esacs were purified and their km and kcat values for beta-lactams determined by spectrophotometry. results: using cloxacillin-containing (an ampc beta-lactamase inhibitor) plates, the susceptibility to ceftazidime was restored for out of isolates, suggesting overproduction of the ampc. in addition, in presence of cloxacillin, reduced mic values were also observed with ceftazidime, cefepime and imipenem for out of those isolates. cloning and sequencing identified distinct ampc b-lactamase variants among the isolates. recombinant plasmids expressing the ampcs were transformed into reference p. aeruginosa strain and reduced susceptibility to cefepime and imipenem was observed only with recombinant p. aeruginosa strains expressing ampc beta-lactamases that had an arginine residue at position . the catalytic efficiencies (kcat/km) of the ampc variants possessing this arginine residue were increased against oxyiminocephalosporins and imipenem. in addition, in-vitro assays demonstrated that those ampc variants constituted a favourable background for selection of additional degree of carbapenem resistance. conclusions: some ampcs of p. aeruginosa possessing extended activity torward carbapenems may contribute to carbapenem resistance. background: most oxa-type esbls are oxa- , oxa- or oxa- derivatives. they display a very low homology, the percentage of which is between % and %. oxa-type esbls are divided into five groups according to the different homology by frederic bert, etc. group includes oxa- , oxa- , oxa- and its derivants;group includes oxa- , oxa- , oxa- and oxa- ;group includes oxa- , oxa- , oxa- and oxa- ; group is named after oxa- ; group only includes a single enzyme called lcr- . oxa-type esbls has been reported widespread in the world since the first report in , such as turkey, france, england and so on. but there is few report about it in china. objective: to investigate the prevalence and genotype distribution of oxa-type extended-spectrum beta-lactamases (esbls) in clinical pseudomonas aeruginosa strains isolated from xiangya hospital of central south university in changsha city, hunan province, china. methods: ninety-seven non-repetitive clinical isolates of p. aeruginosa were collected between october and january from the hospital. they were screened for oxa-type esbls production by polymerase chain reaction pcr with five pairs of primes specific for blaoxa genes, respectively. then amplification of oxa-type esbls production was performed by pcr with specific primers. the purified and amplified products were sequenced to confirm the genotype of the oxa-type esbls. results: the sequences of the three oxa-type esbls pcr products were then compared in genbank database and there were no the completely same ribonucleotide and amino acid sequence with them. they were two novel oxa-type esbls, named as blaoxa- and blaoxa- , which have been registered in genbank database under accession numbers eu and eu , respectively. conclusions: there have occurred infections caused by p. aeruginosa producing oxa-type esbls in xiangya hospital of central south university. two novel oxa-type esbls in p. aeruginosa strains have been discovered in our study, which are named blaoxa- and blaoxa- , respectively. pneumonia is one of the most common nosocomial infections and is associated with high mortality. in the last years, gram-positive bacterial pathogens have risen in prevalence as a cause of hospitalacquired pneumonia (hap), including that occurring during mechanical ventilation (ventilator-associated pneumonia; vap). in particular, staphylococcus aureus is a major cause of hap, including vap. the rise of multidrug-resistant infections is a source of concern, with methicillinresistant s. aureus (mrsa) accounting for > % of s. aureus isolates in some european hospitals. this symposium will take the format of a question-and-answer roundtable session in which experts will answer questions and initiate discussion surrounding emerging concerns and appropriate therapeutic strategies in nosocomial pneumonia, including that caused by multidrug-resistant gram-positive pathogens. recently, shifts in the susceptibility of s. aureus to established therapeutic agents for nosocomial pneumonia have added to the challenge of selecting appropriate empiric therapy. in patients with suspected multidrug-resistant infections or those who are mechanically ventilated, prompt initiation of therapy, often before the pathogen has been confirmed, is critical. vancomycin is the gold-standard treatment for multidrug-resistant infections and resistance has been remarkably slow to emerge. however, clinical reports in europe of 'mic creep' and the emergence of vancomycin-intermediate s. aureus (visa), hvisa and linezolid-resistant mrsa have presented new clinical dilemmas. elevated vancomycin mics are linked to treatment failure and increased mortality. hence, while vancomycin remains a useful therapeutic tool, treatment decisions present an increasing challenge, especially in groups of patients in whom rapid eradication of infection with appropriate agents is critical. telavancin is a novel lipoglycopeptide under investigation for treatment of nosocomial pneumonia. a number of key features suggest telavancin as a potentially attractive option for nosocomial pneumonia. telavancin has a unique dual mechanism of action that disrupts both bacterial cell wall biosynthesis and cell membrane integrity. the agent is rapidly bactericidal against a broad range of clinically relevant grampositive bacteria, including mrsa. two pivotal phase iii studies have demonstrated telavancin efficacy equivalent to vancomycin in hap, including vap, including in seriously ill patient subgroups and in that caused by mrsa. hantaviruses are enveloped rna viruses, each carried primarily by rodents or insectivores of specific host species. they have coevolved with the hosts in which they cause almost asymptomatic and persistent infections. in humans some hantaviruses cause disease: haemorrhagic fever with renal syndrome (hfrs) in eurasia. in europe puumala (puuv) from bank voles and saaremaa (saav) from field mice cause mild hfrs and dobrava (dobv) from yellow-necked mice severe hfrs. in asia hfrs is caused mainly by hantaan and seoul viruses. in americas some viruses cause hantavirus cardiopulmonary syndrome: sin nombre, andes and other viruses carried by sigmodontine rodents, not found in eurasia. in addition, in europe the common vole carries tula and rats seoul virus. however, they have not been definitely associated with disease in europe, although both can infect humans. we discuss the epidemiology, molecular genetics, detection of infection in carrier hosts and humans (including rt-pcr and -min serological tests), functions of hantaviral proteins, risk factors for humans to catch hantavirus infection (including smoking) and disease (including risk and protective hla haplotypes), role and mapping of epitopes of cytotoxic t-cells, mechanisms of hantavirus-induced apoptosis, newly discovered clinical features (including hypophyseal haemorrhages in puuv infection), and long-term consequences and pathogenesis of hfrs (endothelial permeability, thrombocytopenia, tnf-alpha and il- ). puuv occurs widely in europe except in the far north and mediterranean regions, saav in northern, eastern and central europe and dobv mainly in the balkans. the epidemiological patterns differ: in western and central europe hfrs epidemics follow mast years with increased oak and beech seed production promoting rodent breeding. in the north, hantavirus infections and hfrs epidemics occur in − year cycles, driven by prey-predator interactions. the infections and hfrs are on the increase in europe, partly because of better diagnostics and partly perhaps due to environmental changes. in several european countries hantavirus infections are notifiable and in some countries (e.g. belgium, finland, france, germany, scandinavian countries, slovenia) their epidemiology is relatively well studied. in large areas of europe, however, hantavirus infections and hfrs have not been studied systematically and they are still heavily under-diagnosed. mrsa screening − will we ever agree? s mrsa: universal screening! the successful control of any outbreak or epidemic relies on detection of those harbouring the pathogen (infected and colonised persons) combined with eliminating spread to new individuals. the approach to containment and reduction of the global mrsa pandemic is now being discussed. a challenge for this infection is that most persons harbouring mrsa do not exhibit signs of disease and thus in order to detect all potential spreaders of this organism some surveillance must be done. the required level of detection (surveillance through screening) is not known and likely varies with the prevalence of colonisation and disease. for a given mrsa prevalence, the factor that seems most crucial in reducing spread is the percentage of potential isolation days captured. the operational processes that highly influence this are ) the sensitivity of screening detection (including sites tested and laboratory methods used), ) the speed at which results of newly detected positive patients are reported from the laboratory (assuming pre-emptive isolation is not employed), and ) the selection of patient populations who are to undergo screening. laboratory testing has a major impact on detecting mrsa colonised patients with real-time pcr having a sensitivity of % and a possible hour reporting time compared to direct chromogenic agar cultures with a sensitivity of % and > hour reporting and enriched chromogenic agar testing with a sensitivity of % and > hour reporting (am j clin pathol, ); both reduced sensitivity and prolonged reporting time negatively impacting the success of mrsa timely isolation. we have shown that capturing % of mrsa isolation days in a modest mrsa prevalence setting ( infections/ , patient days) with a high sensitivity test having a > hour result reporting time did not reduce hospital-wide mrsa disease (ann int med : , ) . others have demonstrated that surveillance in an icu with similar mrsa prevalence, again with a high sensitivity test having day result reporting, did not reduce icu disease until preemptive isolation was initiated (crit care : r , ) . finally, we demonstrated that universal admission surveillance and decolonisation capturing % of possible mrsa isolation days had a dramatic impact by reducing % of all in-hospital infections from mrsa. future research in this area should focus on better defining those patients that benefit from mrsa screening and the role of decolonisation in these programs. clostridium difficile infection (cdi) is a toxin-mediated intestinal disease and extraintestinal manifestations are exceptional. clinical outcomes can range from asymptomatic colonisation to mild diarrhoea and more severe disease characterised by inflammatory lesions and pseudomembranes in the colon, toxic megacolon or bowel perforation, sepsis, shock, and death. the main clinical symptoms, secretory diarrhoea and inflammation of colonic mucosa, can be in great part explained by the actions of two large protein toxins, toxin a (tcda) and toxin b (tcdb). both toxins are cytotoxic, destroy the intestinal epithelium and decrease colonic barrier function by disruption of the actin cytoskeleton and tight junctions resulting in a decreased transepithelial resistance allowing fluid accumulation. in addition, c. difficile toxins also cause release of various inflammatory mediators which affect enteric nerves, sensory neurons and promote inflammatory cells, adding to the fluid secretion, inflammation and transmigration of neutrophils. some experimental evidence points also to possible extraintestinal action of c. difficile toxin b. in zebrafish embryos tcdb caused damage and edema in cardiac tissue and in hamsters the same toxin caused lung damage. only recently efficient systems have been developed to genetically manipulate c. difficile. comparison of knock-out mutants producing only one of both toxins have shown that tcdb-positive-only mutants retain the ability to kill hamsters, whereas tcda-positive-only mutants were not virulent for hamsters. these results are in concordance with epidemiological findings that naturally occurring a-b+ strains still cause the entire spectrum of cdi, but are not in concordance with effects observed after intragastric challenge of hamsters with purified toxins tcda and tcdb. the role of the third toxin produced by c. difficile, binary toxin cdt in the development of human disease is not well understood. cdt was shown to have enterotoxic effect in rabbit ileal loop assay, but natural strains producing cdt but neither tcda nor tcdb colonised animals but were not lethal in hamsters. comparative genomic analysis will most likely reveal additional factors involved in pathogenesis and in increased virulence (including cell surface layer proteins, sporulation characteristics and antibiotic resistance). additionally, the role of the host immune response in cdi has just started to be better understood. since , there has been an escalation in rates of clostridium difficile infection (cdi) with epidemic c. difficile (pcr ribotype /north american pulsed-field type [nap ]) responsible for outbreaks of severe infection in north america and europe. while fluoroqinolone resistance and over-use are thought to be driving the epidemic, the ageing population and improved case ascertainment are contributing to the dramatic increase in cases. other factors may also be important, such as the increase in prescription of proton pump inhibitors. in the netherlands, since , there has been an increase in prevalence of human cdi with ribotype strains usually found in animals. these infections were in a younger population and more frequently community acquired. there was alarm when it was reported that % of retail beef samples in canada contained c. difficile. the figure is higher in the usa where more than % of packaged meats (beef, pork and turkey) from arizona stores contained c. difficile. most animal isolates of c. difficile produce binary toxin, and both pigs and cattle harbour pcr ribotype a strain that, like ribotype , also produces more toxins a and b, and binary toxin. in the eastern part of the netherlands where > % of pig farms are located, > % of human isolates are now ribotype , and human and pig strains of c. difficile are highly genetically related. it has been suggested that the overlap between the location of pig farms in the netherlands and the occurrence of human ribotype infections involves a common source. that source is likely to be the environment. the upsurge in cdi has prompted diagnostic companies to try to either improve current tests or develop new ones. laboratory diagnostic methods can be divided into groups; traditional faecal cytotoxin detection (with or without culture), enzyme immunoassays (eias) and molecular methods. faecal cytotoxin detection is specific but lacks sensitivity, culture is sensitive but lacks specificity. new eias should find a niche in medium sized laboratories. current in-house pcr methods have the potential for great sensitivity and specificity but have been available only in larger laboratories. new commerciallyavailable platforms will make this methodology more accessible to smaller laboratories. whatever method is chosen, it is necessary for the laboratory to have as fast a turn-around-time as possible, particularly in an outbreak situation. d. lévy-bruhl°(saint-maurice, fr) in , the advisory board on immunisation (abi) has been asked to make recommendations to the ministry of health regarding the inclusion or not in the french immunisation schedule of the soon to be licensed first hpv vaccine. the main elements considered in the establishment of the benefit-risk balance of routine hpv vaccination were: on the benefit side: -the very significant potentially preventable burden of diseases; -the very high efficacy of the vaccine against persistent hpv / infections in naive subjects; -the expected additional impact on other hpv / related lesions and cancers; -the fact that vaccination, by preventing the pre-cancerous lesions, has the advantage over screening to reduce the cost and anxiety related to their detection and management; -the available data in favour of a satisfactory safety profile; -the benefit of vaccination for the women not covered by the opportunistic screening program. on the "risk" side: -the high cost of vaccination; -the unknown duration of protection; -the need for continuation of screening, even for vaccinated women; -the fact that the majority of residual cervical cancers could be prevented by the organisation of the screening program; -the risk of a decrease in compliance to screening for vaccinated women; -the low benefit if vaccinated and screened women were the same. a cost effectiveness analysis, carried out on a multi cohort markov model, showed that, over a years period, the impact of vaccinating % of years old girls or of organising the screening were comparable (reduction of cancer deaths close to %). however, the cost-effectiveness ratio of the vaccination was higher than that of the screening organisation, resp. , and , € per life year saved (at a % discount rate). on the basis of the economical analysis, the screening organisation was therefore the first priority. however, if both interventions were implemented, the overall reduction in cervical cancer deaths was estimated at %. the cost-effectiveness of the addition of vaccination on the top of the organisation of the screening appeared acceptable ( , € per life year saved). based on those results, the abi issued in march a recommendation to include the hpv vaccination in the immunisation schedule for years old girls, together with a catch up for to years old women not having started their sexual life more than one year ago. the vaccine cost has been reimbursed since july . clinical microbiology − is outsourcing the way to go? s the (r)evolution of clinical microbiology in europe − is it good or bad? laboratory medicine in general and clinical microbiology in particular is presently subject to rapid (r)evolution. are we aware? are we in command? do we know where we are going? should we oppose or cooperate? do we have a choice? do we recognise a driving force other than money? is it good, bad or just plain necessary? and are we gaining or losing? it is not one evolutionary process -it is several parallel processes with varying emphasis in different areas. there are at least four distinctive major trends over the last years; the gradual formation of bigger and bigger units (concentration), the amalgamation of many different laboratory services into one (laboratory medicine), accreditation and an explosion of professional proficiencies and backgrounds of staff in microbiological laboratories. personally i have withstood the first two, with pleasure succumbed to the latter. a recent th trend, outsourcing microbiology services to large private consortiums, is splitting clinical microbiology into a purely analytical high-throughput money-saving activity, often leaving the consultative, clinical part of microbiology and health care infection control adrift. what is driving the evolution? not only cost-saving but also our inability to recruit medically trained microbiologists, the need to broaden the knowledge base of microbiology laboratories, automation, the development of new techniques and apparatus common to many laboratory disciplines, computerised medicine, political trendiness, power struggles, and much more. there is much to be gained by both concentration and amalgamation but much to be lost as well and many consider the heart and soul of clinical microbiology at risk. over a period of years, rational high-throughput production has won over consultation and personalised microbiology. that may be fine for the production of negative hiv-antibody/antigen analysis as for the screening of blood-donors but certainly not for the bacteriological cultures taken in conjunction with a hip replacement. or when it comes to understand and advise on the intricacies of antimicrobial resistance development. in other cases "outsourcing" and/or "amalgamation" mean that blood cultures are sent to x-town, cmv-antibodies to y-town and everything else to z-ville. when that happens clinical microbiology is lost. there are several instances where concentration, amalgamation and/or outsourcing of clinical microbiological services, alone or with other services, have meant that the tie between clinical microbiology and infection control has been severed and that many, both small and large hospitals have lost the personalised service so necessary to control outbreaks of multi-resistant bacteria and other health care related infections. a good service requires a strong knowledgeable and enthusiastic champion. a service which encompasses too many branches of laboratory medicine cannot be expected to champion each and every one with equal strength and fervour. and when outsourced to "big companies", there is no "clinical", only "microbiology". in "medical microbiology" broke out from "laboratory medicine" in uems. we are now striving towards a strong "medical microbiology" service in europe. it will have many facets, much strength, some weakness, great opportunities, but many threats. escmid certainly intends to help shape microbiology in europe. the optimal organisation of microbiology laboratories in european metropolis is an evolutionary task, driven by the evolution in laboratory tasks, laboratory technologies, communication technologies, regulations and financial issues. in the past five-ten years, medical and societal query for a more rapid and refined detection and identification of pathogens and antimicrobial resistance determinants coincided with the expansion of internet-based and remote tools for communication, an unprecedented revolution in laboratory technologies and new financial constraints. the concentration of laboratory workforces into one unique laboratory is one way to address these apparently contradictory issues. the tertiary medical school hospital system in marseille, a -million metropolitan area in france, comprises four hospitals for a total of , beds. the system had once four microbiology laboratories which have been progressively embedded into a unique, , acts per year, laboratory which deals with bacteriology, virology and environmental microbiology and hygiene. the medical staff comprises of , the ingenior staff of , technical staff of and support staff of persons for a total of persons. this organisation allowed reducing labour time for routine microbiology, to develop prospective and sophisticated time-consuming diagnostic methods and to develop advanced diagnostic methods such as molecular methods (real-time pcr-based tests, sequencing, and mass spectrometry identification) and new generation serology. new, sophisticated technologies such as automated serology and mass spectrometry were corner-stones on which to base the constant diminution of routine labour time and the development of time-consuming tasks such as fastidious organisms' isolation. these evolutions paralleled the exponential increase in the ratio of ingeniors in the laboratory. this paradigm allowed for the constitution of large collections of biological specimens for retrospective analyses, the specialisation of every medical senior in one particular field of internationally recognized expertise and the increase in knowledge output in terms of peer-reviewed papers, patents and grants. implantation of point-of-care in the emergency department, in permanent internetbased connection with the central laboratory, was the last, but not least, evolution of this system. when tuberculosis epidemiology is seen in a global perspective, and the millennium development goals are considered, it is clear that two regions of the world, africa and europe, are severely behind in the control of the disease. in africa, especially sub-saharan africa, the tb problem is closely related to the endemic hiv/aids situation. in europe, especially the eastern part and in parts of the former soviet union, the main obstacle to an effective tb control is related to drug resistant forms of m. tuberculosis. the prevalence of the most severe forms of resistance, mdr-and xdr-tb, is so high that it makes control efforts both extremely complicated and very expensive. unfortunately, increasing levels of drug resistant tb are today also seen in many african countries, and hiv infection is spreading in eastern europe. during the last ten-year period new tools, based on molecular fingerprinting of m. tuberculosis strains, have been increasingly adapted to study tb transmission. with such molecular methods to characterise clinical isolates of m. tuberculosis it is now possible to study the spread of individual strains of the bacteria in detail. the laboratory tools used, rflp, miru/vntr, spoligotyping and others, will be presented and their use exemplified. how molecular epidemiology contributed to the detection and characterisation of a major outbreak of drug resistant tb in the stockholm area will be discussed. molecular characterisation of clinical isolates from different parts of the world has led to an increased recognition of the differences between different families of m. tuberculosis strains. to further describe and understand the role of these differences in the clinical field as well as for tb epidemiology is an ongoing and interesting field of research. an increased understanding of how tb is transmitted will hopefully help in the efforts to control this global health threat both on the local level and in a global perspective. living in the era of increasing tuberculosis drug resistance, the importance of making an early and accurate diagnosis with drug sensitivities has never been greater. the epidemiology of tuberculosis defines the extent of latent disease and the proportion which becomes active. accurate diagnosis is vital if patients are to be treated in a timely manner and to reduce the amount of time infectious individuals go untreated in the community disseminating disease. in many areas of the world, dots programmes are at the forefront of tuberculosis control. however, as a diagnostic this currently relies on sputum smear microscopy which is known to miss % of cases of tuberculosis and provides no data on drug sensitivity. the second major issue around tb is the lack of worldwide diagnostic facilities. there is a need for a simple, low cost, easily implemented diagnostic test. this talk will briefly consider the issues around the diagnosis of latent and active disease which are quite distinct. the focus will be on the diagnosis of active infection. in particular, the use of mods (microscopic observation drug-susceptibility) assay in diagnosis of tuberculosis will be discussed. the potential for using this in resource poor countries will be reviewed as well as the way sophisticated technology maybe harnessed to improve reporting and allow translation to all parts of the world. the important issue of how to distinguish patients with latent and active disease will also be considered. key issues and principles in diagnosis both now and in the future will be reviewed. in terms of treatment, there are main issues. the first is that even short-course therapy is prolonged being a minimum of months leading to issue of compliance. this may result in drug resistance. the massive rise of multi-drug resistant tuberculosis to approximately , cases world-wide with around countries reporting extensively drug-resistant disease means that the need for new approaches to therapy are urgent. the second part of this talk will review different approaches to using current anti-mycobacterial drugs, the emergence of a small number of new drugs such as the diarylquinolones and entirely novel approaches to control and treat tuberculosis. there has been great success and also many threats in the field of infectious diseases during the previous year. the antimicrobial resistance, especially increasing carbapenem resistance among aerobic gram-negative rods and xdr mycobacterial tuberculosis strains are already big threats in some countries and they will probably spread to many other areas all over the world in the future and we will need new drugs for these indications but unfortunately very few new promising drugs seem to be in the pipeline at the moment for these purposes. the virulent clostridium difficile strain spreads rapidly to many new countries and e.g. in finland it killed many times more people compared with mrsa and esbl strains in . however, it is possible to stop its spreading but it needs new thinking in antibiotic use policy and infection control policy in hospitals. clostridium difficile infection has a high relapse rate after metronidazole or vancomycin therapy, but an experimental "stool exchange treatment" is a promising therapy although controlled studies are needed to prove this assumption. an interesting research area during the last years has been the role of infections in the etiopathogenesis of chronic diseases like cancer, atherosclerosis, cardiovascular diseases and many autoimmune diseases. we can fight against many cancers like liver cancer and cervix cancer with virus vaccines and gastric cancer with antimicrobial drugs. also the high incidence of malignant tumours seems to decrease during haart treatment in hiv patients. the role of infections in the etiopathogenesis of cardiovascular diseases and atherosclerosis is complex. it is obvious that infections play a role in the etiopathogenesis of atherosclerosis, stroke and myocardial infarction but the undirected routine antimicrobial treatment is not recommended for these patients but there seems to be subgroups in patients with various cardiovascular diseases which may benefit from antimicrobial treatment. recent studies seem to suggest that there are hla types which protect or make people susceptible for coronary heart disease. the hla type hla-b* seems to be a risk factor for coronary heart disease but it is also a risk factor for chronic chlamydia pneumoniae infection. the feared pandemia due to h n influenza a did not appear during the recent year and the world is now much more prepared to meet the next pandemia which, however, hopefully does not come during the next year. Ø. samuelsen°, c. giske, u. naseer, s. tofteland, d.h. skutlaberg, a. onken, r. hjetland, a. sundsfjord (tromsø, no; stockholm, se; kristiansand, bergen, oslo, førde, no) objectives: the worldwide dissemination of kpc-producing multidrugresistant enterobacteriaceae is worrisome. the first kpc-producing klebsiella pneumoniae in norway was isolated late from a patient after hospitalisation in greece. throughout the following year seven additional kpc-producing k. pneumoniae isolates have been detected in clinical samples from six new patients. the aim of this study was to perform molecular characterisation of the strains and examine their epidemiological relatedness. materials and methods: antimicrobial susceptibility was examined by etest. molecular characterisation was performed by mlst, pfge and sequencing of the blakpc genetic structure. plasmid analysis was carried out by pfge of s nuclease-digested total dna and southern blot hybridisation using a blakpc probe. relevant epidemiological data were collected retrospectively. results: eight kpc-producing clinical isolates of k. pneumoniae have been identified from seven patients in two different regions of norway from the following specimens: blood culture (n = ), urine (n = ), expectorate (n = ), perineal swab (n = ) and wound secretion (n = ). two blood culture isolates with clonally related but different pfgeprofiles were observed in one patient. the detection of kpc-producing k. pneumoniae isolates in norwegian patients was associated with import in four cases after hospitalisation in greece. two patients had been hospitalised at the same hospital in greece. isolation of a kpc-producing isolate in a fifth patient was epidemiologically linked to one of these imported cases and was a case of nosocomial transmission in norway. for the latter two cases no risk factors were identified with respect to recent hospitalisation or travel abroad. molecular analysis of six isolates has shown genetically related pfge-patterns and a common sequence type (st ). st has been associated with dissemination of ctx-m- in hungary. the blakpc gene was localised in tn on a~ kb plasmid. the two most recent isolates are currently undergoing similar analysis. conclusion: the first seven cases of kpc-producing k. pneumoniae in norway are associated with hospitalisation abroad, nosocomial transmission in norway, or urinary tract infections in outpatients without obvious risk factors. the clonal relationship between isolates underlines the existence a biological fit genetic lineage of kpcproducing k. pneumoniae with an epidemic potential. objectives: two recent publications have reported the isolation of kpc producing k. pneumoniae from infections in two patients, one in france and one in sweden, who originally had been hospitalised in greece. since this resistant mechanism had not been identified before in this country, the purpose of this report was to confirm the presence of blakpc producing k. pneumoniae in greece, to assess the extent of its spread and to study the genetic relatedness of the respective bacterial strains and the transferability of the blakpc harbouring plasmids. methods: for a three month period (february to april ) hospitals participating in the greek system for surveillance of antibiotic resistance (www.mednet.gr/whonet) were asked to seek for possible kpc producers among k pneumoniae isolates displaying reduced susceptibility to imipenem (equal or higher than mg/l), a positive hodge test for the presence of carbapenemase and a negative edta synergy test for the presence of metalloenzymes. the presence of blakpc gene in these strains was confirmed by pcr and sequencing. mics to carbapenems were determined by etest. conjugation experiments were carried out both in broth and on agar. the possible absence of ompk porin was detected by pcr. molecular typing was performed by pulse-field gel electrophoresis of xbai-restricted genomic dna. results: ninety two k. pneumoniae clinical isolates (one per patient) from hospitals all over greece were found to harbour blakpc- gene. although colonies present in the inhibition zone made the exact determination of imipenem mic difficult, the absence of ompk porin was always associated with mic of imipenem higher than mg/l. all isolates exhibited resistance to all other drug classes except colistin, tetracycline and tigecycline. pfge analysis revealed that isolates from hospitals displayed more than % similarity and were classified into one pulsotype, whereas the remaining seven isolates belonged into four different pulsotypes. blakpc- gene could not be transferred by conjugation from strains belonging to the main pulsotype. however, it was transferred from strains belonging to three out of the four remaining pulsotypes. conclusion: production of kpc- betalactamase seems to be a new emerging resistance mechanism in klebsiella pneumoniae in greece. blakpc- gene's possible clonal spread imposes the urgent need of implication of infection control practices in the affected hospitals. i. galani, m. souli, e. papadomichelakis, f. panayea, n. mitchell, a. antoniadou, g. poulakou, f. kontopidou, h. giamarellou°(athens, gr) background: until now, carbapenem resistance among klebsiella pneumoniae (kp) clinical isolates in greek hospitals has been attributed to the dissemination of vim- metallo-beta-lactamase. we describe the first outbreak of kpc-producing kp in greece; the first to occur outside the usa or israel. setting: -bed icu of attikon university hospital, athens. methods: kp isolates with an imipenem mic > mg/l and a negative edta-imipenem disk synergy test were submitted to boronic acid disk test, to pcr for a kpc gene with specific primers and sequencing. records from patients colonised or infected with a kpc-producing kp were retrospectively reviewed for clinical and epidemiological data. environmental cultures for kpc-producers were performed. clinical isolates were submitted to molecular typing using pfge. results: from february to november , kp were isolated from patients, ( . %) of which were boronic acid positive and produced kpc- . most of them ( / , . %) were isolated since august. a total of patients were identified as colonised or infected by a kpc producer which in of them belonged to the same genetic clone. the source was faeces ( ), bronchial secretions ( ), blood ( ), cvc tip ( ), urine ( ), pus ( ) and throat ( ). among patients whose medical records were available, median age was , apache ii score; , length of preceding hospital stay; days, total length of stay; days, immunosuppresion was identified in one and crude mortality was %. the kpc-producing kp was more frequently icu acquired whereas in a minority of patients it was already present on icu admission. seventy percent of the patients had previously received a carbapenem for a median of days. environmental colonisation was not identified. ten ( . %) of the kpcproducers from ( . %) patients were identified as the cause of an infection: bacteraemia ( ), ventilator-associated pneumonia ( ) and surgical site infection ( ) and exhibited mic (mg/l) for imipenem, > ; meropenem, > ; gentamicin, ; ciprofloxacin, > ; fosfomycin, > ; colistin, . and tigecycline, . most patients were successfully treated with a colistin-containing combination mostly with a beta-lactam. there was no attributed mortality. isolates from the same bacterial species were typed by pfge or automated ribotyping. kpc-encoding gene was fully sequenced. plasmid preparations and i-ceu digestion of total dna were resolved in agarose gels, blotted and hybridised with a blakpc probe. the blakpc-carrying element (tn ) was amplified with various primer pairs, digested with eag i and sequenced. results: strains each carried kpc- and kpc- . one e. cloacae carried kpc- . k. oxytoca were kpc- -producers and s. marcescens harboured blakpc- , all from usa. great genetic diversity was observed among the isolates ( different types). one clone of e. cloacae was detected in new york state ( ) ( ) . small clusters of and strains were detected among e. coli, e. cloacae, k. oxytoca. plasmids were present in all but isolates. persistence of clones throughout the years was not observed. in isolates the kpc-encoding gene was located in high molecular weight plasmids (> kb). blakpc was located in the chromosome of strains (e. cloacae, e. coli and k. oxytoca) and the location of this gene could not be determined in strains. small plasmids were present in several strains, but did not harbour blakpc. tn carried blakpc in isolates, and the transposon element was conserved. this structure was not detected in strains. conclusions: kpc-encoding genes were most often located in tn among several enterobacteriaceae species collected in usa and israel. this blakpc-carrying element was located in plasmids and on the chromosome. this study highlights the importance of tn in the dissemination of blakpc genes in several genetically diverse bacterial species. blakpc was not associated with tn in only of strains. these strains are under further investigation. objective: to evaluate the carbapenem resistance mechanism in a raoultella planticola bacteraemia isolate recovered from a patient hospitalised in ohio, usa. methods: species identification was performed by vitek and confirmed by s rrna sequencing. susceptibility testing used clsi broth microdilution method. blakpc was amplified and sequenced. the blakpc genetic element (tn ) was amplified and sequenced. plasmid extractions and conjugation experiments were carried out and the isolate was screened for esbl-encoding genes, qnr and qepa. a year old female patient was admitted to a hospital located in akron with a diagnosis of cap in may/ . sputum, paracentesis and blood cultures were negative. urine culture grew e. coli and patient received courses of moxifloxacin, ceftriaxone, azithromycin and meropenem. the patient was discharged and returned after three weeks with respiratory problems. tracheal aspirate grew a multidrug resistant a. baumannii and the blood culture grew the enteric-like gramnegative bacillus. the isolate was identified as r. planticola by the vitek , which was confirmed by s sequencing. r. planticola strain demonstrated resistance against most b-lactams, including carbapenems. screening for kpc-encoding genes was positive and this strain carried blakpc- . fluoroquinolone and aminoglycoside mic values were elevated. kpc- -encoding gene was located in tn , but conjugation experiments failed. esbl and qnr/qepa genes were not detected. conclusions: kpc serine-carbapenemases have been detected in several gram-negative species commonly isolated from clinical specimens. kpc genes are embedded in transposon-like structure usually harboured in conjugative plasmids carrying multiple antimicrobial resistance mechanisms. this is the first report of kpc-producing r. planticola that is an environmental organism related to klebsiella spp. the similarity between these organisms could facilitate the transfer of genetic material. kpc-producing isolates appear to be prevalent among different enterobacteriaceae species in usa hospitals and was detected in an isolates of apparent environmental origin. objectives: it is long known that not all individuals with a specific disease present with the same clinical manifestations, nor do they have identical prognoses or responses to treatments. it has become clear that variations in the human genome are likely to have an impact on these aspects. tank-binding kinase (tbk ) is a central molecule in the induction of a.o. the type i interferon response to pathogens. our goals for this study were ) to investigate the frequency of single nucleotide polymorphisms (snps) in the promoter and coding region of tbk in a dutch caucasian population and ) to search for potential associations between these snps and bloodstream infections. methods: whole blood samples or samples of positive blood cultures were collected and after genomic dna was isolated, pcr and sequencing were performed for snp identification. functional studies included promoter activity measurements using a luciferase assay as well as electrophoretic mobility shift assays (emsa) to study binding of the transcription factor usf to the wt and mutant promoter. snp incidences were studied in a case control study. results: in samples from dutch caucasian healthy volunteers, snps were found with allele frequencies higher than % whereas other known snps had frequencies lower than % in our cohort. two snps (rs and rs ) located in the promoter region were studied in a larger cohort of anonymised patients from the maastricht university medical center with either gram-positive or gram-negative blood cultures. we found that the prevalence of rs was significantly increased in patients with positive blood cultures in comparison with those with negative blood cultures or healthy volunteers. further investigation of this snp showed that it is located just outside a usf -binding site. measuring the promoter activity using luciferase assays, the mutant promoter exhibited a decreased activity of < %. this observation was confirmed by emsa which showed that recombinant usf protein had a reduced binding affinity to the mutant promoter. conclusions: snp rs in the promoter region of tbk has a significant association with gram-positive infections. our results demonstrate that this is likely due to a decreased expression of tbk due to reduced binding of the transcription factor usf to the mutant promoter. our results support recent findings that tbk plays also an important role in the host response to gram-positive infections. objective: lymphocyte apoptosis has been recognized as an important factor contributing to both the onset of sepsis post infection and to the progression into septic shock. animal data suggest that prevention of lymphocyte apoptosis by caspase inhibition stabilises the immune system, improves bacterial clearance and decreases mortality in experimental sepsis. the present study evaluated the potential of vx- , a novel broad caspase inhibitor, as a therapy for sepsis. methods and results: initial characterisation of vx- in a number of enzymatic and cellular assays clearly demonstrated that the compound is a broad caspase inhibitor with potent anti-apoptotic activity in vitro. in vivo, vx- was tested in a murine model of endotoxic shock and a clinically relevant model of peritonitis. in the endotoxic shock model, male cd- mice (n = per group) were administered lps ( mg/kg iv) and survival was monitored for h. vx- administered by repeat iv bolus ( , , and h post-lps) significantly improved survival in a dose-dependent fashion (p < . ). in the rat peritonitis model, adult male sprague-dawley rats (n = per group) underwent caecal ligation and puncture (clp) and survival was monitored over d. continuous administration of vx- by mini-osmotic pump ( . mg/kg/h) immediately following surgery significantly improved survival (p < . ) from % in the control group to % in the compound-treated group. mode of action studies in the rat clp model confirmed that vx- reduced thymic atrophy and lymphocyte apoptosis (p < . ), supporting the anti-apoptotic activity of the compound in vivo. in addition, vx- reduced plasma endotoxin levels (p < . ), strongly suggesting an improved clearance of bacteria from the bloodstream. most importantly, we demonstrated that vx- fully retained its efficacy when dosed hours after insult (p < . ) by improving survival to % versus % in control animals, further highlighting the potential of anti-apoptotic therapy in sepsis. overall these data demonstrate that vx- inhibits lymphocyte apoptosis, improves the clearance of bacterial endotoxin and improves survival in experimental sepsis. importantly vx- improves survival in the clp model when dosed post insult, and therefore represents significant progress in the development of therapeutically viable broad caspase inhibitors for the treatment of this disease. v. vankerckhoven°, s. van voorden, n. hens, h. goossens, g. molenberghs, e. wiertz (wilrijk, be; leiden, nl; hasselt, be) objectives: toll-like receptors function as key regulators of both innate and adaptive immunity. lactobacilli modulate the immune system in different ways. the aim of this study was to examine toll-like receptor (tlr , tlr / and tlr ) signalling induced by clinical and probiotic lactobacillus strains. methods: a total of lactobacillus strains ( l. paracasei and l. rhamnosus) of different origin ( probiotic, faecal, and clinical) were tested for tlr , tlr in combination with tlr , and tlr . tlr signalling was measured as relative il- promotor activation in transfected human embryonic kidney (hek) cells. il- concentrations were measured using an enzyme-linked immunosorbent assay. heat-killed listeria monocytogenes (hklm) was used as positive control in all assays, whereas pam , pam , and lps were used as positive controls for, respectively, tlr , tlr / , and tlr . all assays were performed at least in duplicate. linear mixed model analyses and stepwise model selection were used to identify the statistically significant effects. random effects were used to account for heterogeneity across and homogeneity within isolates. p < . was considered statistically significant. results: hek-tlr and hek-tlr / , but not hek-tlr , cells released il- upon stimulation with uv-inactivated lactobacilli, which was enhanced by co-transfection with cd- . interestingly, the production of il- was shown to be variable for the different lactobacillus isolates. although similar results were seen for all isolates for tlr and tlr / , il- production was significantly higher for tlr ( . log pg/ml) compared to tlr / ( . log pg/ml) (p < . ). no significant differences in il- production were seen between clinical and probiotic isolates. however, l. rhamnosus isolates induced a significantly higher il- production compared to l. paracasei isolates in both cell lines, . and . log pg/ml, respectively (p = . ). intra-isolate correlation was found significant (p < . ). conclusions: our study shows that lactobacilli activate both tlr and tlr in combination with tlr . our results also indicate that heterodimerisation of tlr with tlr does not lead to an improved recognition of lactobacilli. furthermore, taking intra-isolate correlation into consideration proved to be important. finally, our results suggest that differences in immunomodulation by lactobacilli may be related to differential signalling through tlrs, including tlr and tlr / . m.c. gagliardi, v. sargentini, r. teloni, m.e. remoli, g. federico, m. videtta, g. de libero, e. coccia, r. nisini°(rome, it; basel, ch) objective: to gain insights into the mechanisms used by mycobacterium tuberculosis and bacillus calmette guérin to cause human monocytes differentiation into cd negative dendritic cells (my-modc), unable to present lipid antigens to specific t cells. methods: human monocytes infected or not with mycobacteria were induced to differentiate into dc with gm-csf and il- in the presence or absence of p or erk specific inhibitors. kinases activation was detected by western blot using antibodies specific for phosphorylated and non phosphorylated isoforms. differentiation of monocytes into dc and the cd a, cd b and cd c expression was evaluated by flow cytometry and by real time pcr at different time points from infection. functional expression of cd molecules was assessed by recognition of lipid antigens by cd restricted t cell clones. results: we show that mycobacteria trigger phosphorylation of erk and p mitogen-activated protein kinase in human monocytes as well as of activating transcription factor (atf)- . mycobacteria-infected monocytes treated with a specific p inhibitor, but not with a specific erk inhibitor become insensitive to mycobacterial subversion and differentiate into cd positive my-modc, which are fully capable of presenting lipid antigens. data indicate that phosphorylation of p is directly involved in cd inhibition. conclusions: we propose p signaling as a pathway exploited by mycobacteria to affect cd expression, thus representing a novel target of possible pharmacological intervention in the treatment of mycobacterial infections. s. ebert°, s. ribes, r. nau, u. michel (gottingen, de) objective: activin a (act a) is a multifunctional cytokine with roles in the immune system and the inflammatory response. act a levels are elevated in the cerebrospinal fluid of patients with meningitis. microglial cells, the major constituents of innate immunity within the brain, express toll-like receptors (tlrs) recognising exogenous and endogenous ligands. upon stimulation with tlr agonists, primary mouse microglial cells become activated and release nitric oxide (no), cytokines, and also act a, suggesting that they are a source of elevated conclusions: pre-treatment with act a enhances no release from microglial cells activated by agonists of the principal tlrs involved in the recognition of bacteria. these findings provide further evidence for a role of act a in the innate immune response and suggest that act a acts as an pro-inflammatory modulator during infection and inflammatory processes in the cns. insertion sequences (is) are genetic tools that can mediate expression of previously silent genes or be responsible for the overexpression of certain genes (in each case by providing promoter sequences). in addition to be involved in gene transcription levels, is elements also play a very important role for gene acquisition/mobilisation. an is is usually made of of two inverted-repeat sequences (irs) bracketing a gene encoding the transposase which activity enables this entity to replicate and target another sequence. the is-related mechanisms at the origin of antibiotic resistance gene acquisition are diverse, including composite-transposition, rolling-circle transposition, one-ended transposition. is elements may be also involved in gene acquisition by mediating co-integration processes, or recombination events as hypothetized for is in relation with blashv extendedspectrum b-lactamase (esbl) genes originating from the chromosome of klebsiella pneumoniae. the blactx-m esbl genes known to be extremely widespread worldwide are encoded on plasmids, and have been found in association with isecp (acting by one-ended transposition) or iscr (acting by rolling-circle transposition). in that case, iss have played a role in the mobilisation from the chromosome of kluyvera spp. being the blactx-m progenitors and then in their expression. also, genes encoding acquired ampc b-lactamases, being of the blaacc, bladha, and blacmy-types, are mostly found in association with iscr or isecp . sometimes antibiotic resistance genes are mobilised by composite transposons which are made of two copies of a given is bracketing the mobilised fragment. in acinetobacter baumannii, the worldwide disseminated blaoxa- carbapenemase gene is part of a composite transposon structure made of two copies of isaba , forming transposon tn which had mobilised a chromosomal fragment from acinetobacter radioresistens that actually corresponds to the progenitor of blaoxa- . another possibility can be the forming of composite transposon structure bracketed by two different is (sharing similar irs) as observed with the blaper- esbl gene in pseudomonas aeruginosa. this diversity of iss elements at the origin of mobilisation/acquisition of antibiotic resistance genes is therefore responsible for the very efficient dissemination of many of them. s resistance islands − their role in the accumulation and spread of antimicrobial resistance genes historically, multi-antibiotic resistance in many bacterial species was largely attributed to the acquisition of resistance (r)-plasmids encoding one or more resistance determinants. however, over the last decade the r-plasmid paradigm has begun to be challenged. 'resistance islands' comprising large, chromosomally-integrated spans of alien dna harbouring multiple antibiotic resistance genes have been identified in the major hospital pathogens methicillin-resistant staphylococcus aureus (mrsa) and multi-resistant acinetobacter baumannii, and the foodand water-borne diarrhoeal pathogens shigella, salmonella and vibrio cholerae. in addition, comparative genomics analysis of the archetypal haemophilus influenzae conjugative resistance element that had spread worldwide revealed that it belonged to a large syntenic family of integrative islands, members of which could be found in at least other b-and g-proteobacteria. with the exception of the a. baumannii island, these elements can be described as classic self-excising, -circularising and -integrative elements. all three functions are mediated by short island-flanking direct repeats and cognate integrase proteins encoded by the islands. in fournier et al. described an kb a. baumannii island (abar ) which harboured resistance genes packaged within a highly mosaic, integron-rich element that had almost certainly evolved via recombination, transposition and integron-mediated cassette capture from an 'empty' ancestral prototype. abar probably represents a new class of resistance island as it exhibits several features reminiscent of complex nested transposons, suggesting a distinct functional natute. however, despite the widespread distribution of resistance and genomic islands only a minority are known to code for part or all of the conjugative machinery necessary for their dissemination; others have been mobilised by helper plasmids or bacteriophages. regardless, data on the mechanisms of mobilisation of the vast majority of similar nonresistance islands remain sparse. importantly, resistance islands may not consists merely of packages of resistance genes. on the contrary, these diverse and frequently hybrid entities could potentially confer upon their hosts other advantageous traits relating to host-pathogen interaction, virulence, survival in the environment and/or transmissibility, truly justifying the label 'selfish islands' and further explaining their evolutionary success. due to the availability of new techniques, genome sequencing of bacteria has become fast and inexpensive. furthermore, recent methods using paired-end reads located several kb apart in the genome eases the assembling process, even though no reference sequence is available. in a reasonably close future, it should be possible to obtain the fully assembled sequence of a bacterial isolate overnight. the new sequencing techniques generate enormous amounts of genomic data and, thereby, a need for new tools. these should able to quickly analyze genomes and point to zones of interest, prompting further analysis on a reduced number of regions or genes, such as genomic islands. pathogenicity islands, a subset of genomic islands, carry genes such as toxins or resistance genes and have the particularity to be mobile, i.e. they may transfer to other species or strains. thereby, they confer their new hosts a more resistant or infectious phenotype, making this phenomenon particularly important to study. nucleotide composition of genomes is fairly homogeneous inside bacterial genomes. in general, horizontally transferred regions can be spotted due to their particular nucleotide content, because they tend to retain the composition of their original host and don't share the one of their new hosts. to do an analogy with languages, genomes speak dialects, and as one would easily spot a paragraph in finnish in an english text while not knowing finnish, one can spot genomic and pathogenicity islands transfers in a given genome. several techniques relying on various compositional aspects and on different algorithmic methods have been recently developed to detect pathogenicity islands in bacterial genomes. even very simple measures of the genome composition, such as the variation in t vs. a bias (ta skew) can lead to the identification of all known prophages in streptococcus pyogenes. it can even trigger the discovery of a putative ancient genomic island carrying a large number of genes related to pathogenicity in all strains of that species. in conclusion, with the rise of fast and inexpensive genome sequencing, new quick and simple methods are being developed. they take the advantage of the homogeneous nucleotide composition of bacterial genomes to uncover mobile genetic elements carrying genes involved in pathogenicity. in the past years, significant progress has been achieved in the management of chronic hepatitis b with the successive development of six potent antiviral medications (lamivudine, adefovir dipivoxil, pegylated interferon alpha, entecavir, telbivudine and tenofovir). however, the clinical results of antiviral therapy have been limited by the emergence of antiviral drug resistance especially with the first generation of nucleoside analogs (lamivudine, adefovir and telbivudine). furthermore, the unique mechanism of viral genome replication and persistence within infected cells is responsible for viral persistence even after prolonged therapy with the newer antivirals (entecavir and tenofovir). this is the major reason why life-long treatment is envisaged in the majority of patients, which may expose them to long-term risk of developing resistance. the use of in vitro phenotypic assays has been crucial for the characterisation of newly identified resistant mutants and determine their cross-resistance profile. results allowed to understand the different mechanism of viral resistance to lamivudine and adefovir, the mechanism of primary failure to adefovir therapy, the unique mechanism of entecavir resistance, and to characterise the emergence of multi-drug-resistant strains in patients receiving sequential antiviral therapy. the crossresistance profile for the main resistant mutants was determined which allowed to provide recommendation to clinicians for treatment adaptation based on molecular virology data. the understanding of the development of hbv drug resistance has allowed to significantly improve the management of antiviral resistance and to design better treatment strategies to prevent resistance. the current standard of care relies on treatment initiation with antivirals combining a strong antiviral potency and a high barrier to resistance. a precise virologic monitoring is required to measure antiviral efficacy, and to diagnose partial response or viral breathrough at an early stage. this allows to adapt antiviral treatment preferrably using an add-on strategy with a drug having a complementary cross-resistance profile. this strategy has been shown to be efficient in controling viral replication and preventing liver disease progression in the majority of patients. treatment of chronic hepatitis b virus (hbv) infection is aimed at suppressing viral replication to the lowest possible level. in many prospective clinical trials it has been shown that a sustained hbv dna response was correlated with serologic, histologic, or biochemical responses. despite the recent progress in hepatitis b antiviral treatment, it is shown that antiviral drug resistance is inevitable against many of the nucleoside analogs. the emergence of antiviral-resistant strains of hbv leads to viral and subsequently biochemical breakthrough and may lead to disease progression and increased death. most of the data on the clinical impact of antiviral resistant hbv came from the data derived from studies of lamivudine therapy. there is limited data on other hbv antiviral drugs like adefovir. it is shown in several studies that treatment of hbeag-negative chronic hepatitis b with lamivudine effectively suppresses hbv replication and results in biochemical remission and histologic improvement in more than two thirds of patients. however, relapse has occurred in the majority of hbeag-negative patients after the cessation of therapy. there are several studies to support the occurrence of severe hepatic flares, and liver failure after the emergence of lamivudine resistance. several studies, where liver biopsies were taken, demonstrated that histological improvement was reduced in those patients experiencing lamivudine resistance. the clinical outcome for patients with antiviral resistance is related to their age, the severity of the underlying liver disease and the severity of the hepatic flares. on the other hand in a different study it was found that long-term lamuvidine treatment was associated with a reduced chance of developing cirrhosis and hcc in patients without advanced disease but, although resistant mutants reduced the benefits from lamivudine therapy, the outcome of these patients was still better than untreated patients. results of several clinical trials have shown that the addition or substitution of newer antiviral agents can restore suppression of viral replication, normalisation of liver function and reverse histological progression in patients with antiviral resistance. consequently, well-tolerated, potent therapies that offer a strong genetic barrier against the development of resistance are desirable, since antiviral resistance and poor adherence are key risk factors for treatment failure and subsequent reversal of clinical improvement. resistance of enteric fever-causing and non-typhoid salmonella serovars to agents traditionally used to treat these infections in the past shows extensive geographical variation. decreased susceptibility to ciprofloxacin is rapidly increasing all over the world with target alteration and increased efflux being the most important mechanisms behind. infections with such strains often result in extended hospitalisation or even in therapeutic failures. furthermore, it is likely that moderately increased mic values facilitate the development of strains with higher level of resistance, i.e. a pattern described at various locations. screening methods based on quinolone sensitivity testing may fail to indentify decreased fluoroquinolone susceptibility both in typhoid, as well as in non-typhoid salmonella. plasmid mediated quinolone resistance genes are detected increasingly all around the world although neither the frequency nor the variety of genes identified has approached that seen in some other members of enterobacteriaceae. treatment with gatifloxacin or azithromycin are alternative options for invasive and systemic infections caused by strains with decreased susceptibility to ciprofloxacin. at some parts of the world resistance to extended spectrum cephalosporins reached such incidence that may have therapeutic implications particularly when initial, empiric treatment of invasive infections is concerned. resistance is due to plasmid coded ampc type beta lactamases (particularly to cmy- ), and most often to esbls of which usually some of ctx-m types are the frequently encountered ones. carbapenem resistance is still rare, albeit does occur, among salmonella isolates. the recent description of a non-typhoid salmonella strain with the blaimp- gene co-located on a class- integron with several other resistance determinants on a conjugative plasmid is of particular concern. campylobacters exhibit natural resistance to a variety of antimicrobials. the drugs of choice used to be fluoroqunolones or macrolides. however, the current incidence of ciprofloxacin resistance made the former drugs already obsolete or seriously limited their use at several parts of the world. with the exception of few locations the incidence of macrolide resistance is still relatively low and is seen more frequently in c. coli than in c. jejuni. however, strains exhibiting resistance against both groups of drugs have been emerging, particularly in south-east asia. neisseria meningitidis, the meningococcus, is a major cause of meningitis and septicaemia worldwide while neisseria gonorrhoeae, the gonococcus, is responsible for one of the most widespread sexually trasmitted disease. the behaviour of these two species towards antibiotics is very different: resistance in n. gonorrhoeae is now widespread occuring as both chromosomally and plasmid mediated to a variety of drugs, whereas, besides resistance to sulphonamides, n. meningitidis remains largely susceptible to antibiotics used both for therapy and prophylaxis. however, as in the gonococcus, the resistance to antibiotics of n. meningitidis is also evolving, as documented by the ever higher frequency of strains with intermediate resistance to penicillin in many countries. transformation has apparently provided both species with a mechanism by which they can increase resistance to penicillin by replacing part of their pena gene, which encodes pbp , with part of the pena gene of related species that fortuitously produces forms of pbp less susceptible to the antibiotic. n. meningitidis is still at this step, whereas n. gonorrhoeae has acquired also mutation in the pona gene that encodes pbp , mutation in porin ib, increased expression of efflux pump and the tem- b-lactamase plasmid. the emergence and the spread of gonococci fully resistant to penicillin since the second half of the s years led to the recommended use of fluoroquinolones as primary therapy. however, this class of antibiotics became rapidly unefficacious, mainly in asia, due to the emergence of mutations in gyra and parc which are able to block the activity of the quinolones on gyrase and topoisomerase iv. since , cdc no longer recommends their use for treatment of gonococcal diseases. fortunately, the occurrence of quinolone resistant meningococci, due to mutations in gyra, is still rare but even if cases are still few they are of great concern for the epidemic potential of this pathogen and the required prophylaxis of contacts. also for the other antibiotic, frequently used to this aim, rifampicin, some meningococci have showed to be resistant, again for the presence of mutations, in this case in the rpob gene coding for the b-subunit of the meningococcal rna polymerase. the molecular epidemiological identification of clonal clusters for both neisseria species with distinct resistance profiles is required to monitor ongoing trends that may pose problems both in therapy and prophylaxis. l. brookes-howell°, c. butler, k. hood, l. cooper, h. goossens (cardiff, uk; antwerp, be) introduction: grace is a european network of excellence established to focus on antibiotic use for community-acquired lower respiratory tract infection (lrti) and antimicrobial resistance across europe. grace- , the second study to begin within grace, is a large qualitative study that explores the attitudes of clinicians and patients to antibiotic use for lrti and antibiotic resistance. aims: this presentation will focus on clinicians' accounts of the factors that contribute to variation in management of lrti and patient views on when antibiotics are necessary. methods: semi-structured interviews with clinicians and patients were conducted in primary care networks in nine european countries. interviews were audio-recorded, transcribed and, where necessary, translated into english for analysis. themes were identified, organised and compared using a framework approach. results: analysis of clinician interviews shows that, beside clinical findings, factors which influence the management decision for patients can be divided into two main areas. firstly, within each european network there is a group of country specific factors imposed by the system in which consultations take place. these factors include: near patient test usage, self-medication, patients' finances and lack of consistent, local prescribing guidelines. secondly, there is a group of factors, similar across all networks, that relate to personal characteristics of certain groups of clinicians. these include clinicians' professional ethos, self-belief in decision making and attitude towards the doctorpatient relationship. analysis of patient interviews shows that beliefs about antibiotic use tend to draw on clinical factors, namely the severity of specific symptoms (fever and/or coughing). many patients also implied a period of waiting or alternative action required before antibiotics are used − to identify whether the immune system would fight the infection or whether nonantibiotic management was effective before turning to antibiotics. discussion and conclusion: with a greater understanding of the factors that contribute to the decision to prescribe, we discuss ideas to enhance appropriate prescribing. this analysis highlights the need for interventions to be sensitive to factors relating to the systems in which different european networks operate, to target the individual characteristics of specific groups of clinicians and to build on the clinical beliefs already held by patients. o pre-treatment with low-dose endotoxin prolongs survival from experimental lethal endotoxic shock k. kopanakis, i. tzepi, e.j. giamarellos-bourboulis°, a. macheras (athens, gr) objective: clinical trials of immunointervention with anti-endotoxin antibodies in patients with severe sepsis have failed to disclose survival benefit. these failures led us to the assumption that the opposite approach with a low endotoxin stimulus may result to low level immunoaralysis and subsequent survival benefit. this approach was tested in an experimental setting. methods: a total of male c b mice were studied divided into two groups: group a stimulated with the ip injection of sodium saline followed after one day by the ip injection of mg/kg of lipopolysaccharide (lps) of escherichia coli o :h ; and group b stimulated with the ip injection of mg/kg of lps of the same isolate followed after one day by the ip injection of mg/kg lps. lps was diluted in sodium saline and the volume of each injection was . ml. survival was recorded at six hour time intervals. results: survival of group b was considerable prolonged compared with group a (log-rank: . , p: . ) as shown in figure . thirteen mice of group a died ( . %) compared with seven mice of group b ( . %, p: . between group). conclusions: administration of low doses of lps prolongs survival after lethal endotoxic shock. this approach opens a promising novel pathway for immunointervention in sepsis. fragilis isolates with an mxf mic of mg/ml (n = ), mg/ml (n = ) and mg/ml (n = ), which were virulent in the mgp model, were used to determine the efficacy of mxf. for the mgp model, pouches were created by injecting ml of air and . ml of . % croton oil in olive oil under the skin of the back. on day , the air was withdrawn and replaced by ml soft agar. on day , a bacterial suspension was injected into the pouch. infected mice (n = mice/group) were treated with mxf mg/kg iv, b.i.d. for days. this dose simulates the auc of the human mg once-daily mxf iv dosage. efficacy was assessed by the reduction in colony forming units (cfus) in pouch exudates hours post-infection compared with the untreated infection control. results: in the mgp model, mxf, mg/kg b.i.d., displayed good efficacy in term of cfu reduction against all used strains in this study. there were no non-responders in terms of cfu reductions. conclusion: the loss of atle had no impact on the mics of cloxacillin and vancomycin. conversely, the mutant atle(−) strain was less susceptible to bactericidal activity of both antibiotics, supporting the implication of atle in the tolerance of s. epidermidis to cell wall active antibiotics. the loss of atle did not alter the virulence of s. epidermidis in the mouse peritonitis model, whereas it decreased virulence in previously published experiments using an intravenous catheter infection model. therefore, the mouse peritonitis model was suited to compare antibiotics efficacy against atle(+) and atle(−) strains. our results showed that the loss of atle did not alter significantly the activity of cloxacillin and vancomycin in the mouse peritonitis model. this study shows that the loss of atle results in decreased susceptibility to bactericidal activity of cell wall active antibiotics, with no apparent impact on the activity of these antibiotics in the mouse peritonitis model. in infant rat pneumococcal meningitis, ceftriaxone plus daptomycin versus ceftriaxone attenuates brain damage and hearing loss while ceftriaxone plus rifampicin versus ceftriaxone does not d. grandgirard, m. burri, k. oberson, a. bühlmann, f. simon, s.l. leib°(berne, ch) objectives: lytic antibiotics for therapy of bacterial meningitis (bm) increase the release of pro-inflammatory bacterial compounds which, in turns, induce inflammation. exacerbation of the inflammatory response in cerebrospinal fluid (csf) contributes to the development of neurological sequelae in survivors of bm. daptomycin, a nonlytic antibiotic acting on gram-positive bacteria has been shown to decrease inflammation and brain injury vs. ceftriaxone in experimental pneumococcal meningitis. with a view on the clinical application for empiric therapy of paediatric bacterial meningitis we investigated, whether therapies combining daptomycin or rifampicin with ceftriaxone are beneficial when compared to ceftriaxone monotherapy in infant rat pneumococcal meningitis. methods: eleven day old wistar rats were infected by intracisternal injection of s. pneumoniae and animals were treated with daptomycin ( mg/kg, s.c., daily) plus ceftriaxone ( mg/kg, s.c., bid), rifampicin ( mg/kg, i.p., bid) plus ceftriaxone or ceftriaxone alone. csf was sampled at h and h after the initiation of therapy and assessed for concentrations of chemo-and cytokines (mcp- , mip- a, il- b, il- , il- ; il- and tnf-a). a subset of animals was sacrificed h post infection (h pi) and brain damage quantified by histomorphometry. the remaining animals were treated for d and were tested for hearing loss, by assessing the auditory brainstem response (abr) at weeks after infection. results: compared to ceftriaxone alone, daptomycin plus ceftriaxone significantly (p < . ) lowered csf concentrations of mcp- , mip- alpha and il- at h and mip- a and il- b at h after initiation of therapy, led to significantly (p < . ) less apoptosis assessed at h pi, and significantly (p < . ) improved hearing capacity. while rifampicin plus ceftriaxone also led to lower csf inflammation (p < . for il- at h), apoptosis and hearing loss were not significantly different from the ceftriaxone group. conclusion: compared to ceftriaxone monotherapy, daptomycin plus ceftriaxone lowers the level of pro-inflammatory mediators in the csf and reduces hippocampal apoptosis and hearing loss in infant rat pneumococcal meningitis. d. croisier-bertin°, l. piroth, p.e. charles, d. biek, y. ge, p. chavanet (dijon, fr; alameda, us) objectives: ceftaroline (cpt) is a novel, parenteral, broad-spectrum cephalosporin exhibiting bactericidal activity against gram-positive organisms, including methicillin-resistant s. aureus (mrsa) and multidrug-resistant s. pneumoniae, as well as common gram-negative pathogens. the efficacy of simulated human dosing with cpt or ceftriaxone (cro) was evaluated in a rabbit model of penicillin-resistant pneumococcal pneumonia. methods: s. pneumoniae strains were used to induce pneumonia in rabbits: pssp, pisp, and prsp. mics (mg/l) were . / . , / . , and / . for cro and cpt, respectively. the animals were randomised to no treatment (controls), intravenous (iv) cpt human equivalent (he) dosage ( mg/ h), iv cro he dosage ( g/ h), or intramuscular (im) cpt ( or mg/kg) for prsp-infected rabbits. serum levels were measured by microbiological assay and pk data were obtained. evaluation of efficacy was based on bacterial counts in lungs and spleen (per gram tissue). results: − animals/group were tested. for iv cpt/iv cro, mean auc − was / mg.h/l, cmax was / mg/l and cmin was . / mg/l, respectively. bacterial counts in target tissues are listed in the iv cpt and iv cro were highly efficacious against pssp and pisp. iv and im cpt were superior to iv cro against prsp with a quasi sterilisation of lungs and spleen. combined results from the iv and im studies indicated that %t > mic for cpt of % and % were associated with % and % bacterial count reductions, respectively. in this rabbit model of penicillin-resistant pneumococcal pneumonia, cpt administered iv (with he dosing) or by im administration was more effective against prsp than iv cro. r. endermann°, d. hoepker, k. merfort, m. glenschek-sieberth (wuppertal, de) objective: moxifloxacin (mxf) is approved in the usa and other countries for the treatment of complicated intra-abdominal infections (ciais). we compared the efficacy of mxf with piperacillin/tazobactam (pip/taz), a commonly used treatment for ciais, in three different models: ( . c. clp model: survival over days was significantly higher in the mxf group than in the pip/taz group (p < . ). conclusions: using humanised dosages, mxf had greater antimicrobial activity and provided higher survival rates that pip/taz in three different models for ciai. m. nairz, i. theurl, a. schroll, m. theurl, s. mair, t. sonnweber, g. fritsche, r. bellmann-weiler, g. weiss°(innsbruck, at) mutations in hfe predispose to hereditary haemochromatosis type i, a frequent genetic disorder characterised by progressive parenchymal iron deposition and eventual organ failure. since hfe mutations are associated with reduced iron levels within mononuclear phagocytes, we hypothesized that hfe deficiency may be beneficial in infections with intramacrophage pathogens. using hfe+/+, hfe+/− and hfe−/− mice in a model of typhoid fever, we found that animals lacking one or both hfe alleles are protected from systemic infection with salmonella typhimurium, displaying prolonged survival and improved bacterial control. this increased resistance can be referred to an enhanced production of the siderophore-binding peptide lipocalin and the reduced availability of iron for salmonella engulfed by hfe deficient macrophages. this effect is mediated via stimulation of lipocalin -dependent iron export from infected cells since hfe−/− macrophages concurrently knocked out for lipocalin are unable to efficiently control the infection or to withhold iron from intracellular salmonella. correspondingly, infection of hfe+/+ and hfe−/− mice with siderophore deficient salmonella abolishes the protection conferred by the hfe defect. thus, by inducing the formation of the iron-capturing peptide lipocalin , the hfe mutation harbours a genetically determined immunological advantage towards infections with intracellular pathogens such as salmonella. i. koutelidakis, a. kotsaki, p.d. carrer, k. louis, a. savva, e.j. giamarellos-bourboulis°(thessaloniki, athens, gr) objective: the majority of clinical trials of immunointervention in severe sepsis have failed to disclose survival benefit. a likely explanation may be administration of therapy when immunoparalysis of the septic host supervenes. in an attempt to reverse immunoparalysis, injection of mononuclear cells was attempted in experimental sepsis by multidrugresistant pseudomonas aeruginosa (mdrpa). methods: peripheral blood mononuclear cells (pbmcs) diluted in rpmi were isolated from five healthy human volunteers after gradient centrifugation over ficoll. × /kg of one mdrpa live or heat-killed isolate from one patient with severe sepsis was injected intraperitoneally for bacterial challenge. a total of male c b mice were studied divided into four groups: group a (n = ) pre-treated with rpmi and challenged after one hour with live isolate; group b (n = ) pretreated with × pbmcs/kg and challenged after one hour with live isolate; group c (n = ) pre-treated with rpmi and challenged after one hour with heat-killed isolate; group d (n = ) pre-treated with × pbmcs/kg and challenged after one hour with heat-killed isolate. survival was recorded for mice of groups a and b and for all mice of groups c and d. six mice of groups a and b were sacrificed six hours after challenge. blood was collected from the lower vena cava and tnfalpha and il- were estimated in serum by an enzyme immunoassay. bacterial growth of liver and lung at the same time was assessed. results: median survival of group a was hours and of group b hours (log-rank: . , p: . ). nineteen animals of group a died ( %) compared with eight animals of group b ( %, p: . ). four animals of group c died ( %) compared with nil animals of group d ( %, log-rank: . , p: . ). median serum tnf-a of groups a and b at sacrifice was and pg/ml respectively (p: . ). respective values for il- were and pg/ml (pns); for liver bacterial cells . and . log cfu/g (pns); and for lung bacterial cells . and . log cfu/g (pns). conclusions: allogeneic transplantation with pbmcs prolonged survival in experimental sepsis by mdrpa. its mechanism of action was related with a) blockade of cell wall structures as shown by survival experiments with heat killed isolate; and b) reversal of immunoparalysis as evidenced by increase of serum tnf-a. this approach creates a promising novel perspective for immunointervention in sepsis. a. marangoni°, c. nanni, m. donati, r. aldini, d. di pierro, s. trespidi, s. accardo, s. fanti, r. cevenini (bologna, it) objectives: chlamydia trachomatis is one of the world's major causes of sexually transmitted diseases of the cervix and urethra and it is a major agent of pelvic inflammatory disease. genital tract infection of female mice with chlamydia muridarum closely mimics acute genital tract infection in women. aim of this study was to assess the predictivity of ga-chloride small animal positron emission tomography ( o inadequate statistical power of published comparative cohort studies on ventilator-associated pneumonia to detect mortality differences between the compared groups m. falagas°, v. kouranos, a. michalopoulos, s. rodopoulou, a. athanasoulia, d. karageorgopoulos (athens, gr) objective: comparative cohort studies are often conducted to identify novel therapeutic strategies or prognostic factors for ventilator-associated pneumonia (vap). we aimed to evaluate the statistical power of such studies to provide statistically and clinically significant conclusions. methods: we searched in pubmed and scopus for comparative cohort studies evaluating the mortality of patients with vap. we calculated for each of the included studies the statistical power to detect the observed difference in mortality between the compared groups (observed power), as well as expected, clinically relevant, effect sizes (expected power). we identified ( prospective) comparative cohort studies on vap as eligible for inclusion in this analysis. the median observed power of these studies was . % [interquartile range (iqr), . − . %]. the median expected power was . % (iqr, . − . %) for a risk ratio for mortality of . between the compared groups; . % (iqr, . − . %) for a risk ratio of . ; and . % (iqr, . − . %) for a reduction in mortality from % to %. all expected power measures were significantly lower than the observed power. the statistical power of most cohort studies to detect the observed difference in mortality between compared groups of patients with vap is low. the power is even lower when expected, clinically relevant, differences in mortality are considered. for a wiser utilisation of resources allocated to research, we favour the conduction of cohort studies with larger sample size so that potential differences between the compared groups are more likely to be shown. objective: to clarify issues regarding the frequency, prevention, outcome, and treatment of patients with ventilator-associated tracheobronchitis (vat), which is a lower respiratory tract infection involving the tracheobronchial tree, while sparing the lung parenchyma. methods: we performed a systematic review and meta-analysis of relevant available data, gathered though searches of pubmed, scopus, and reference lists, without time restrictions. a conservative random effects model was used to calculate pooled odds ratios (or) and % confidence intervals (ci). results: out of the initially retrieved articles, papers were included. frequency of vat was . %. selective digestive decontamination was proved an effective preventive strategy against vat. presence, as opposed to the absence, of vat was not associated with higher mortality (or: . , % ci . − . ). administration of systemic antimicrobials (with or without inhaled ones), as opposed to placebo or no treatment, in patients with vat was not associated with lower mortality (or: . , % ci . − . ). most of the studies providing relevant data noted that administration of antimicrobial agents, as opposed to placebo or no treatment, in patients with vat was associated with more ventilator-free days and lower frequency of subsequent pneumonia, but without shorter length of intensive care unit stay or shorter duration of mechanical ventilation. conclusions: approximately one tenth of mechanically ventilated patients suffer from vat; an infection potentially prevented by the implementation of selective digestive decontamination. antimicrobial treatment of patients with vat may protect against the development of subsequent ventilator-associated pneumonia. degranulation. subsequently, allergen specific ige to chlorhexidine was demonstrated and skin prick/intradermal testing was positive to chlorhexidine, confirming the diagnosis of chlorhexidine-precipitated anaphylaxis in each patient. a detailed review of the case-notes revealed that each patient had manifest evidence of minor cutaneous reactions to pre-operative chlorhexidine use that had not been ascribed to chlorhexidine at the time. discussion: fda issued a public health notice [ ] following st description of anaphylaxis to chlorhexidine coated central venous catheter. a recent case cluster has also been reported from another cardiac centre in the uk [ -cases over a -month period]. references to be presented. it is interesting that these reports of chlorhexidine anaphylaxis have all occurred in patients undergoing cardiac surgery. these patients receive multiple exposures to chlorhexidine during their pre-operative investigations and preparation. this has increased recently as a result of the drive to reduce the incidence of hospital-acquired infections. we wish to postulate that these patients have been sensitised by repeated topical exposure to chlorhexidine and have exhibited anaphylaxis when this allergen was presented to the patient in the form of the chlorhexidine coated central venous catheter. type i strains of helicobacter pylori possess the cag pathogenicity island to deliver virulence factors. cag is a specialised type iv secretion machinery that is activated during infection and comprises genes originated from a distant event of horizontal transfer. after translocation the effector protein caga is phosphorylated on tyrosine residues restricted to a previously identified repeated sequence called d . this sequence is located in the c-terminal half of the protein and contains the five amino acid motif epiya, which is amplified by duplications in a large fraction of clinical isolates. tyrosine-phosphorylation of caga is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. in addition, we observed that two members of the src kinases family, c-src and lyn, account for most of the caga-specific kinase activity in ags cell lysates. translocated caga interacts with the zo- and jam host-cell proteins causing disruption of the apical junctional complex. transfection of the caga gene into polarised epithelial cells induces disruption of cell-to-cell contacts and altered morphology. strikingly caga-expressing cells become migratory and invasive penetrating into collagen gel. the study of different portions of the molecule revealed the presence of two distinct functional domains and both are necessary to induce abnormal cell differentiation through interactions with host cell morphogens. cell polarity and invasion have been suggested to contribute to both early and late stages of cancer formation. these results suggest a mechanism by which caga may acts at the early stage of tumorigenic progression causing loss of cell polarity, increased cell motility and invasiveness of epithelial cells. after a period of years of silence, a disease with an unpronouncable name, "chikungunya" (chik), has recently become a medical reality and reached the public throughout the world. conclusion: low mhla-dr expression after septic shock independently predicts ni. this promising biomarker may be of major interest in identifying patients at increased ni risk who could benefit from targeted and tailored therapy aimed at restoring immune functions. pneumonia, the leading infectious cause of death in the us, kills more people annually than aids, tuberculosis, meningitis and endocarditis combined. from a wide range of observational studies of communityacquired pneumonia (cap), only half of the cases had an aetiologic agent identified. streptococcus pneumoniae was consistently the predominant bacterial aetiology. this lecture will primarily focus on the innate immune response to pneumococcal pneumonia. toll-like receptors (tlrs) are key molecules that recognize pathogen associated molecular patterns (pamps) and induce an inflammatory response. pneumolysin, an intracellular toxin found in all s. pneumoniae clinical isolates, is an important virulence factor of the pneumococcus that is recognized by tlr . although tlr is considered the most important receptor for gram-positive bacteria, tlr does not play a decisive role in host defence against s. pneumoniae pneumonia; likely, pneumolysin-induced tlr signalling can compensate for tlr deficiency during respiratory tract infection with s. pneumoniae. besides tlr and tlr , tlr contributes to an effective host defence against s. pneumoniae in the airways. the importance of tlr signaling for host defence against pneumococcal pneumonia is illustrated by the fact that mice lacking the common tlr adaptor protein myd are highly susceptible to this infection. activation of tlrs results in the production of proinflammatory cytokines. there is ample evidence that underlines the importance of tumour necrosis factor (tnf) and interleukin (il)- in host defence in bacterial pneumonia: in a murine s. pneumoniae pneumonia model, treatment with a neutralising anti-tnf mab strongly impaired antibacterial defence. in addition, il- a receptor type i deficient mice infected with s. pneumoniae displayed an increased bacterial outgrowth. of considerable interest, treating il- receptor deficient mice with a neutralising anti-tnf antibody made them extremely susceptible to pneumococcal pneumonia. infection of the lower airways by s. pneumoniae is associated with complex interaction between the pathogen (e.g. cell wall components, pneumolysin) and the host (e.g. tlrs, cytokines). these interactions play a crucial role in the outcome of this clinically important infection. severe bacterial pneumonia remains uncommon unless specific conditions exist that tip the balance between the host and pathogen in favour of the microorganism. such conditions include: persons at the extremes of age; exposure to especially virulent organisms; patients with concomitant illness impairing pulmonary clearance mechanisms; and immunocompromised hosts. pathogens overcome an array of innate and acquired host defences to successfully invade the host. the known virulence traits of three common respiratory pathogens (streptococcus pneumoniae, staphylococcus aureus, and pseudomonas aeruginosa) will be briefly reviewed. the capsular polysaccharide of pneumococci is the major anti-phagocytic virulence trait but many other factors contribute to disease pathogenesis including the critically important exotoxin known as pneumolysin, bacteriocins, adherence factors, choline binding proteins, lipoteichoic acid, iron, manganese and magnesium transporters, pili, competence and biofilm capacity, and virulence genes that promote invasion and impair clearance once the organism has entered the blood stream. s. aureus is notorious for the numerous a/b type toxins, cytotoxins, and superantigens it generates during the course of invasion. staphylococci deploy a complex series of quorum sensing signals that coordinate adhesin and invasion genes within biofilms or between planktonic organisms and likely contribute to the success of this pathogen. p. aeruginosa produces an array of extracellular exotoxins and cytotoxins delivered by type iii secretion systems. these include elastase, phospholipases c, a series of apoptotic and anti-phagocytic exotoxins, along with an alginate capsule and an unusual and variable lps structure that participate in microbial invasion. the pathogen expresses at least three interacting, quorum sensing systems to coordinate virulence and biofilm formation. a detailed understanding of these virulence factors is now providing therapeutic options to control these respiratory pathogens. surface expressed and extracellular toxins of pneumococci have been selected as new vaccine targets. inhibitory peptides and small molecule inhibitors of quorum sensing and biofilm formation are under investigation for staphylococcal and p. aeruginosa infections. these innovative and non-antibiotic treatment strategies are gaining greater importance as progressive antibiotic resistance threatens the management of these severe bacterial infections in the future. brucellosis, possibly the commonest zoonotic infection worldwide, has troubled humans since antiquity. recent years have seen the expansion of the animal reservoir of the disease to a wide spectrum of wildlife species, extending to marine mammals, and the recognition of novel brucella species. furthermore, animal and human disease has re-emerged in numerous countries which were brucellosis-free, and currently the most important endemic foci include near east and central asia. complex socioeconomic and political factors may be incriminated for these alterations in endemicity. the complex mechanisms by which brucella evades immune response and survives intracellularly are progressively clarified. novel diagnostic techniques as real time pcr may shed light in the life cycle of brucella inside the human host; preliminary studies have indicated that the pathogen may persist in latent form for years after apparent clinical cure, in asymptomatic individuals. treatment principles have not evolved significantly. the expert guidelines issued recently under the name of "ioannina recommendations" support the need for a six-week combined treatment that includes traditional antibacterials and is modified accordingly in serious complications as spondylitis and central nervous system involvement. the road to the development of a vaccine for humans seems long though. anthrax is ancient diseases and relatively a forgotten disease in western world until when spores were mailed in usa causing five deaths. currently, human anthrax is seen most commonly in agricultural regions of the world where anthrax in animals is prevalent, in which countries of middle east, in africa, central asia and south america. it is also an endemic disease in turkey. human cases may occur in an agricultural or an industrial environment. the infection is an occupational hazard of workers who process hides, hair, bone and bone products, and wool and of veterinarians and agricultural workers who handle infected animals. the main route of transmission is contact with or ingestion of contaminated meal with or inhalation of bacillus anthracis spores. leptospirosis is a very old disease that has been known for more than a hundred years and possibly even longer since the time of hippocrates. it remains a major cause of illness in many tropical and subtropical countries and thus in travellers. it has also been identified as a zoonosis in europe and north america. it is a disease that can surprise us because the clinical presentations are not always typical. in recent years, pulmonary and other atypical presentations have been more widely recognised. there is no effective vaccine but chemoprophylaxis is effective in selected populations. prompt recognition and early institution of appropriate treatment as with most other infectious diseases appear to be critical in ensuring a good outcome for our patients. there are interesting new developments in diagnostics and molecular epidemiology but clearly there are many challenges remaining in this field. objectives: the spread of carbapenemase genes within gram negative bacteria is of great cause for concern. in , the first report of a blaoxa- gene outwith acinetobacter baumannii was reported in acinetobacter genospecies . we had also identified a genospecies isolate encoding a blaoxa- -like gene, and the aim of this study was to examine the genetic environment of the gene to investigate the mobilisation between species. methods: restriction analysis of rrna was used to confirm identity to the species level. susceptibility to imipenem and meropenem was determined through the plate doubling dilution method. screening by pcr for blaoxa- -like, blaoxa- -like, blaoxa- -like and blaoxa- -like genes was carried out. analysis of the genetic environment surrounding the blaoxa- -like gene was conducted by sequencing inverse pcr products and gene-walking fragments. the structure of the surrounding sequence was confirmed using internal primers, which were also used to screen other blaoxa- -like positive isolates in our collection. results: restriction analysis confirmed the isolate belonged to acinetobacter genospecies . the isolate showed reduced susceptibility to imipenem and meropenem with mics of mg/l for both antibiotics. the isolate was negative for a blaoxa- -like, blaoxa- -like or blaoxa- -like gene, but positive for a blaoxa- -like gene. analysis of the genetic environment of the blaoxa- -like gene revealed the gene was within a novel genetic structure. upstream of the blaoxa- -like gene was the left-hand end of an isaba element, interrupted by an isaba element. the elements contained putative promoter sequences. downstream was an arac and a lyse gene, followed by a sequence similar to the re element described previously. following this was a complex region containing the right-hand end of an isaba tnpa gene, interrupted by an incomplete tnpa gene with % similarity to isaba , itself interrupted by an isaba sequence. this region was followed by a second blaoxa- -like gene. all other blaoxa- -like positive isolates in our collection were negative for isaba upstream of blaoxa- . this study is the first to report multiple copies of a blaoxa- -like gene in an acinetobacter genospecies isolate, and has identified a novel structure containing two blaoxa- -like genes and two isaba sequences. the isaba elements may be responsible for the duplication of the blaoxa- -like gene. objective: acinetobacter baumannii is an important nosocomial pathogen with wide intrinsic resistance. however, due to the dissemination of the acquired resistance mechanisms; such as extended-spectrum beta-lactamase (esbl) and metallo betalactamase (mbl) production, multidrug resistant strains have been isolated more often. per- was first detected in turkey and was found to be widespread among acinetobacter spp. and p. aeruginosa. since then, per- has been discovered in other countries, and most recently found in northern italy and in korea. in this study, the presence of per- type esbl was investigated in caftazidime resistant a. baumannii strains isolated from bloodstream infections by pcr and also the clonal relatedness of the isolates were investigated by random amplified polymorphic dna (rapd) and pulsed field gel electrophoresis (pfge) in all per- producing a. baumannii strains. methods: a. baumannii strains isolated from bloodstream infections was included in this study. the isolates were identified as a. baumannii by conventional methods and phoenix bd automated system system (becton dickinson diagnostic systems, sparks). ceftazidime resistance was determined by e-test. per- genes were screened by these clusters encode: (i) resistance genes and transporters plausibly involved in drug efflux ( transporters of the mfs, dmt, abc, rnd, mop and acr families were unique of drug resistant strains and absent in the susceptible sdf strain); (ii) pili and fimbriae systems related to biofilm formation and motility; (iii) haemolysin-and haemagglutininrelated proteins differently distributed among the four genomes, (iv) iron uptake and other metabolic genes. conclusion: genome comparison identified unique features of a. baumannii epidemic clones and provided novel insights into the genetic basis of multidrug resistance and pathogenesis in this species. this study may contribute to understand the concept of infection, invasiveness and colonisation in the emergent pathogen a. baumannii. hard to swallow − emerging and re-emerging issues in food-borne infection (symposium arranged with efwisg) s mrsa in food products: cause for concern or case for complacency? in first, a switch from intravenous-to oral medication ( - ); second, education programs for interns/residents and physicians and the release of a new antimicrobial formulary ( - ); third, a restriction note was printed on all laboratory rapports ( - ) and fourth, active monitoring and giving feedback on prescriptions ( - ). susceptibility patterns for e. coli including ciprofloxacin, cefuroxim, ceftazidim, co-trimoxazole and tobramycin from hospitalised patients were analyzed starting in . statistical analyses were performed using segmented poisson regression models to look at effect of interventions on resistance (both sudden stepwise changes and changes in trends). bayesian model averaging was used to account for model uncertainty. results: before the start of the interventions the resistance rate was increasing by an average of . % per year. the interventions resulted in a significant reduction of quin use from on average prescribed daily doses to pdd per month. in the best fitting poisson model for the resistance data, a significant stepwise decrease was found to be associated with interventions and . however, there was substantial uncertainty in the model choice, and after accounting for this there was no conclusive evidence in support of any particular intervention, although there was evidence that at least one of the interventions was associated with the observed reduction in resistance. there were no stepwise decreases or decreasing trends in resistance rates to other antimicrobials during the study period. conclusion: many mds prescribe antibiotics often and believe their practice may have an effect on antibiotic resistance. results indicate that mds value information, interventions and surveillance in order to support responsible use of antibiotics. there is an ongoing effort in germany to address these findings at the national level e.g. by establishing a surveillance system for antibiotic resistance and antibiotic usage. table) . . ir for pn, er and tt were always higher in children (ch) than in adults (ad). significant differences were found for pn ( ), er ( er ( , er ( , er ( , er ( , , tt ( tt ( , . generally, cp-ir was higher in ad than in ch. ir was lower in the north (n) than in the south (s). significant differences: pn ( pn ( , , er ( er ( , er ( , , tt ( ) . both n and s knew a deceasing ir tendency: pn= n ( . − . ), s ( . − . ); cp= n ( . − . ), s ( . − . ); tt= n ( . − . ), s ( . − . ). er increased in the n ( . − . ). total outpatient antibiotic use (did) decreased from . ( ) to . ( ) and increased to . ( ) . did for pn and fq increased, mls stabilised and tt decreased. conclusions: since - an ir decrease was noted for pn, cp and tt. er-ir increased further over the years. the decrease paralleled the start of public campaigns on antibiotic use. ir rates remain higher in ch than in ad. the n/s difference became less marked. objectives: parachlamydia acanthamoebae is a new recognized member of the order chlamydiales. growing evidences suggest that this bacteria may have a pathogenic role in humans causing respiratory diseases. it has also been recently identified as an agent of bovine abortion and may be a cause of miscarriage in women. in contrast, little is known about the pathogenic role of rhabdochlamydia crassificans, another related chlamydiales. molecular diagnostic tools are useful to detect these obligate intracellular bacteria because of their inability to grow on conventional culture media. the aim of this work was (i) to develop a real-time pcr for the diagnosis of rhabdochlamydia infection and (ii) to study respiratory secretions of newborns for the presence of parachlamydia and rhabdochlamydia dna. methods: a new quantitative real-time taqman pcr (q-pcr) to be used on abi prism was developed. the q-pcr was then blindly applied to consecutive respiratory samples (endotracheal or nasopharyngeal secretions) taken from critically-ill newborns admitted in the neonatology ward of our university hospital. these samples were also tested using a previously developed parachlamydiaspecific pcr. results: most newborns ( / ) were premature (median gestational age: . weeks; range: . − . ). initial respiratory distress syndrome was present in % of them. positive pcr results were obtained in / ( %) patients ( parachlamydia, rhabdochlamydia, both species) at a median of . days (range: - ) after birth. when compared to the control group ( patients with negative pcr), these newborns had a significantly worse primary adaptation and a higher incidence of resuscitation maneuvers at birth (table) . duration of noninvasive mechanical ventilation and stay in neonatology ward were also significantly longer. a fatal issue was observed in infected cases, as compared to no death in controls (p = . ). gestational age at birth as well as the incidence of pulmonary or systemic infections did not differ between cases and controls. conclusion: a high prevalence of parachlamydia and rhabdochlamydia dna was observed in respiratory secretions of premature critically-ill newborns. the presence of dna of these microorganisms was associated with a worse primary adaptation, a more severe respiratory distress syndrome and a trend towards a higher mortality. their pathogenic role should be further investigated. the genus kingella consists of species, k. kingae, k. oralis and k. denitrificans. all are gram negative, sometimes difficult to stain, rod shaped bacteria that are normal respiratory and genitourinary flora. they are slow-growing and fastidious. although improved recovery was shown when using fan or peds-f blood culture bottles, the majority of these infections remain undetected, especially in pre-treated patients. we report the use of real time polymerase chain reaction (rt-pcr) assays for detection of k. kingae and s. aureus in paediatric osteoarticular infections. methods: synovial fluid samples from patients, month and years of age, were collected over months ( / to / ). the samples were from knees, hips, ankles, elbows, shoulders, wrists and femur abscesses. after automated dna/rna extraction, specimens were subjected to hour pathogen-specific rt-pcr. samples were inoculated onto sheep blood and chocolate agar as well as a peds-f bottle. final species identification and antimicrobial susceptibilities were determined by phoenix (tm). results: patients ( specimens) had positive culture and/or rt-pcr, resulting in an overall positivity rate of %. s. aureus was the predominant pathogen accounting for specimens of patients ( mrsa, mssa) and. % of positive specimens ( patients) were due to k. kingae (n = ). among children − years (n = ), k. kingae was the predominant pathogen accounting for positive patients ( %), followed by mssa in patients ( %). the positivity rate for this age group was %. only children > years ( and years) were positive for k. kingae. mrsa was the predominant pathogen in − year olds, and mssa was evenly distributed among children − years old. culture detected only of specimens positive for k. kingae and of s. aureus. other pathogens were detected by culture only. the use of these molecular assays enhances detection of organisms, especially for k. kingae ( % vs. % for culture). additionally, faster identification (tat hrs) allows for rapid targeted therapy. this improvement in tat could lead to shorter hospital stays in about % of cases. results: genotyping revealed a high degree of diversity, indicative of a panmictic bacterial population. further, there was no association between genotype and colonisation frequency, or year of isolation. pcr screening for virulence genes revealed an incidence of % for uspa , % for hag, % for uspa and % for uspa h. no significant difference was observed in the prevalence of virulence-associated genes between isolates originating from children who were colonised only once or children colonised on all occasions (p = ). pcr-rflp analysis of uspa , hag and uspa showed many gene variants, with no association between pcr-rflp patterns and colonisation frequency, or year of isolation. conclusion: even in relatively localised geographical settings, the genotypic diversity of m. catarrhalis isolates colonising children is large, with no yearly pattern of genotype predominance. children serially colonised with m. catarrhalis isolates appear to clear a particular genotype only to become subsequently colonised with a different genotype. the incidence of virulence genes in this relatively localised study group is remarkably similar to that reported in global m. catarrhalis isolates, possibly indicating that similar selection pressure exists for m. catarrhalis at both the local and global level. virulence gene variation appears to be high, even in this relatively restricted geographical group. these results could have consequences for vaccines designed against virulence genes. a. naessens°, i. foulon, a. casteels, w. foulon (brussels, be) objectives: to evaluate the epidemiology of cytomegalovirus in pregnancy and to evaluate the risk for delivering a child with congenital cmv (ccmv). methods: between - , unselected mother-infant pairs were included. in the mother a serological screening was performed consisting in the detection of cmv igg and igm antibodies at the first prenatal visit and at birth. in the neonate cmv urine culture was performed to diagnose congenital infection. when a pregnant woman was found to have a second trimester spontaneous abortion or a death in utero, an investigation for possible congenital cmv infection was carried out. results: serological screening at the first prenatal visit showed no immunity in women, evidence of past infection (igg positive igm negative) in women ( . %) and in women ( . %) both igg and igm antibodies were detected. after investigation of stored and follow up samples from these patients, could be classified as having a primary cmv infection during pregnancy, patients had previous immunity before the current pregnancy and from patients the type of the maternal cmv infection could not be determined. follow-up serology of the women without immunity revealed a seroconversion in of them ( . %). a total of ( . %) congenital infections (ccmv) were diagnosed. the incidence of the ccmv among the different groups of women are summarised in the table. conclusion: ccmv infection occurs in . % of our population of pregnant women. ccmv was considered to be due to a primary maternal cmv infection in % of the infants; % due to a recurrent maternal cmv infection and in % the type of maternal infection could not be determined. the risk for a seronegative pregnant woman of acquiring cmv during pregnancy is . %. the transmission risk after a maternal primary infection is %. women with prior immunity have a very low risk ( . %) for ccmv, this risk increases to % when igm are find in women with know prior immunity. the risk for women with undetermined infectious status in early pregnancy to give birth to a congenitally infected neonate is . %. this report provides the first data on rotavirus epidemiology and disease burden in norway. further studies are needed to assess the economic impact of rotavirus disease and the cost-effectiveness of vaccination to inform decisions on introduction of rotavirus vaccines into the national program of childhood immunisation. pseudomonas aeruginosa may colonise the lungs of cystic fibrosis patients over years but may also cause acute infections in mechanically ventilated patients and immuno-compromised hosts within a matter of days. despite aggressive antibiotic treatments the organism is rarely eradicated. instead p. aeruginosa adapts to its host environment by developing resistance mechanisms and changing its lifestyle and virulence properties. focusing on mechanically ventilated patients, we will detail the dynamics of resistance emergence and persistence of p. aeruginosa lung populations during antibiotic therapy. we further discuss how p. aeruginosa populations evolve naturally in the absence of any antimicrobial treatment within the lungs of intubated patients by changing their virulence properties. the relevance of these findings both with respect to concepts of social evolution and the development of novel anti-infective strategies will be highlighted. the genome of p. aeruginosa encodes many potential efflux systems. however, only a few of them appear to play a significant role in antibiotic resistance. in this respect, the mex (for multiple efflux) systems are of particular interest because of their ability to extrude a wide range of antimicrobials. these polyspecific machineries result from the assembly of (i) a drug/proton antiporter, (ii) a periplasmic adaptor protein, and (iii) an outer membrane gated channel. it is now well established that the constitutive expression of the tripartite pump mexab-oprm provides p. aeruginosa with a relatively high intrinsic resistance to quinolones, blactams (except imipenem), tetracyclines, macrolides, chloramphenicol, trimethoprim, and novobiocin. this protective mechanism is potentiated by the poor permeability of the outer membrane and activity of another pump, mexxy/oprm, whose expression is induced by substrates targeting the ribosome (e.g., tetracyclines, macrolides, aminoglycosides). accumulating reports indicate that multidrug resistant mutants upregulating one or both of these systems are quite common in the clinical setting. such mutants, which are readily selected by sub-optimal treatments with fluoroquinolones, b-lactams or aminoglycosides, tend to accumulate various resistance mechanisms without loosing the wildtype pathogenicity of p. aeruginosa. whether the low resistance levels (mic x -to -fold) conferred by efflux may promote second-step mutants with altered drug targets (gyra, gyrb, parc) or derepressed ampc b-lactamase has not been confirmed in vitro. in the specific context of cystic fibrosis (cf), a recent study from our laboratory showed that the mexxy/oprm pump can be responsible for much higher resistance levels to aminoglycosides ( -to -fold). this increased efficacy of the system partially results from adaptive mutations in the mexy gene. in contrast, subpopulations deficient in mexab-oprm tend to emerge during long-term colonisation of cf airways. while easily selected in vitro on selective media, mutants overexpressing other mex systems (mexcd-oprj, mexef-oprn, mexghi-opmd, mexjk/oprm, mexvw/oprm) have been rarely described in cf and non-cf patients. some data support the notion that up-regulation of mexcd-oprj or mexef-oprn might be detrimental to the virulence of p. aeruginosa. in conclusion, therapeutic strategies based on efflux inhibitors should target the mexab-oprm and the mexxy/oprm systems in priority. european aspects of malaria s rapid diagnostic tests for malaria: twenty years to convince . . . prompt diagnosis and treatment of malaria are critical factors in reducing morbidity and mortality. microscopy has long been the gold standard for malaria diagnosis, but the newer rapid diagnostic tests (rdts) now offer considerable advantages, especially so in endemic countries. after close to twenty years of development and operational research, the diagnostic performance of rdts is now established in all settings. meta-analyses have clearly demonstrated equivalence of rdts over expert microscopy to detect parasites, and clear superiority over routine microscopy. actually, one of the major reasons that have delayed successful implementation of rdt in endemic areas was the use of poor quality microscopy that has impeded reliable measurement of sensitivity and specificity and undermined confidence of health workers in rdts. other factors were poor product performance, inadequate methods to determine the quality of products and a lack of emphasis and capacity to deal with these issues. for the potential of rdts to be realised, it is crucial that high-quality products that perform reliably and accurately under field conditions are made available and that quality insurance is performed on all steps of the procedure. in achieving this goal, the shift from symptom-based diagnosis to parasite-based management of malaria can bring significant improvement for the management of fever in endemic areas. for travelers returning in temperate climates with fever, rdts have also the potential to improve diagnostic procedures, especially so in hospitals where reliable microscopy is not available out of hours. in patients with no danger sign or significant thrombopenia, a negative rdt is sufficient to exclude malaria and allows waiting − hours for performing or reading the microscopy slide. rdts should be repeated every − hours for three consecutive days if fever persists and in the absence of alternative diagnosis. rdts represent a revolution in the fight against malaria and will tremendously help to manage appropriately patients with fever, especially so when malaria is declining and hence other causes of fever increasing. the ambitious deployment that is foreseen in the coming years in africa through large grants from the global fund should contribute to achieving the millennium goals. fever is the key symptom of malaria among returning travellers ( %). headache, chills, myalgia, sweating and lack of a focus are frequently recorded, but non-specific. nausea and vomiting are often seen in children. the differential diagnosis of other infections, mainly of viral origin, is further difficult because (dry) cough and (mild) diarrhoea are often present. laboratory findings (thrombocytopenia, low or normal leucocyte count) can be helpful in the assessment of mild to moderate malaria. clinical signs and symptoms, e.g. fever, may be mitigated in semiimmune patients (visiting friends and relatives, foreign visitors) seen in non-endemic countries who represent the majority of cases diagnosed in industrialised countries. caution is warranted in assessing such patients as many of them may no longer be exposed to malaria in their countries of origin, thus no longer partially protected and also at risk of suffering from severe complications. up to % of all imported malaria cases may be severe, presenting with jaundice, impaired consciousness to coma, acute renal failure, and, in the course of events, acute respiratory failure. delay in diagnosis and start of treatment is partly responsible for fatality rates of % and more in some countries. if you don't look for them, you won't find them: anaerobes revisited s anaerobic microbiota of the mouth − friend or foe? anaerobes form a major part of the commensal microbiota in the digestive tract where they constitute an integral component of the function on mucosal surfaces. in the mouth, teeth create a unique, non-shedding environment for bacteria to attach and to form biofilms. there is an age-related succession order of species in bacterial colonisation of the mouth, and once established, individual anaerobic species tend to remain as members of the oral microbiota. the agerelated pattern of the colonisation of anaerobic bacteria is partly connected with the development (or loss) of the dentition. interactions between different bacteria residing in the same microenvironment influence the composition of the microbiota − or the development of pathologic conditions. although commensal bacteria are regarded beneficial to the host, some anaerobic members of the oral microbiota contain characteristics potentially detrimental for the health status of an individual. molecular means of characterisation have resulted in increased knowledge about the "normal" microbiota of the mouth and in detection of new species and genera as well as phylotypes, which can be associated with infectious situations in the mouth. oral infections are multifactorial and polymicrobial in nature, and their aetiologic organisms originate mainly from the oral resident microbiota. the involvement of anaerobes is most obvious in infections of root canals, periodontal tissues, and tissues surrounding erupting wisdom teeth where typical anaerobic findings are gram-negative rods. in addition, gram-positive anaerobic cocci and non-spore-forming gram-positive anaerobic rods are common in odontogenic infections. on some occasions, anaerobes of localised dentoalveolar infections can spread to adjacent tissues and even to the bloodstream, resulting in severe complications in extraoral sites. interestingly, a relatively limited number of anaerobic species are involved in clinically severe infections, however, microbial findings seem to vary depending on geography. concomitant with the increase in the number of immunosuppressed patients, the number of opportunistic infections caused by commensal anaerobes may increase. identification to the species level will help to establish associations between individual anaerobic species and specific disease states. studies on the bacteriology of diabetic foot infections (dfi) have yielded varied and often contradictory results. the role of anaerobes is particularly unclear, often because the type and severity of the infection is poorly defined, recent antibiotic therapy is unknown, and specimen collection and culture techniques are inadequate. when optimal collection, transport, and culture techniques are used, multiple organisms including aerobes and anaerobes are usually recovered from severe dfi. interactions within these polymicrobial soups lead to production of virulence factors, such as haemolysins, proteases, collagenases, and short chain fatty acids, which promote inflammation, impede healing and contribute to the chronicity of the infection. to better define the bacteriology of diabetic foot infections, we analyzed our data from a large prospective u.s. multicentre trial of patients with moderate to severe infection that required initial parenteral antibiotic therapy and used optimal post-debridement sample collection, transport and culture procedures. of the culture-positive specimens (of total), only . % were pure cultures while . % yielded or more organisms. a total of anaerobes (range − , average . , per specimen) were recovered from % of patients, with gram-positive cocci (gpc) accounting for . % of all anaerobic strains. s is culture still the gold standard, really? tremendous technological advances are made in culture-independent methods of detection and identification of human bacterial pathogens, such as pcr or hybridisation of their genomic dna. yet, time honoured pastorian bacterial culture in liquid and solid nutritive media still remains the gold standard for the laboratory diagnosis of a majority of bacterial infections. this unusual robustness of a th century technology stems from its unmatched operational characteristics: . broad range of detected agents, depending on adequate combination of media/incubation conditions; . unlimited source of clonal population for individual isolate, allowing versatile characterisation of antibiotic susceptibility and/or pathogenic factor production and/or epidemiological subtyping; . possibility of storage/bio-banking of cells for complementary clinical testing, research and diseases surveillance collections; . proof of pathogenic role of agent at the time of viable cell isolation from the site of infection, in contrast to false-positive results with molecular tests (tissue translocation or persistence of bacterial dna, soluble antigen,. . . ). major drawbacks of bacteriological culture include long turn-around time, cost and labour/skill intensity. these are partly alleviated by new technologies, including automated processing, physical/chemical growth detection and rapid molecular fingerprinting (maldi-tof, raman spectrometry, s rdna snp detection). it is likely that the next decade will see a complete redefinition of the place of direct detection methods and culture-based confirmation methods in clinical bacteriology, enabling a rejuvenation rather than elimination of culture as a daily diagnostic tool. the advent of real-time pcr revealed instrumental to the successful implementation of molecular methods in routine clinical microbiology laboratories. automated nucleic extraction platforms can now be coupled to robotic handling for large-scale detection and quantification purposes, mostly in virology. i will review here the attempts of implementing home-brew and commercial nucleic-acid based detection methods directly from blood samples and highlight hopes and pitfalls. i will then expand on two promising nucleic acid amplification methods: lamp (loop mediated isothermal amplification) and a protein-free method called dnazyme. these isothermal amplification methods share several strengths: robustness across highly diversified physico-chemical conditions, versatility in assay development and minimal requirements (if any) for sample preparation. they will definitely compete against current real-time pcr assays and might become a novel standard, due to lower costs and improved performances. the ribosomal rna (rrna) approach to microbial evolution and ecology has become an integral part of microbiology. rapidly growing databases exist that encompass besides the s rrna sequences of almost all validly described bacteria and archaea also numerous s rrna sequences of so far uncultivated microbes, directly retrieved from the environment by pcr or metagenomics. based on the patchy evolutionary conservation of rrna genes oligonucleotide probes can be designed in a directed way with specificities ranging from species up to large evolutionary entities like phyla or even domains. when such probes are labeled with fluorescent dyes or the enzyme horseradish peroxidase they can be used to identify single microbial cells by fluorescence in situ hybridisation (fish) directly in complex environmental samples. an update on recent applications and methodological improvements will be given which includes the identification of small bacterial cells by catalyzed reporter deposition (card)-fish. with optimised methods and proper controls fish yields exact cell numbers and spatial distributions for defined bacterial populations also in highly complex mixed microbial communities. r. amann & b.m. fuchs ( ) nature reviews microbiology : - . quick and reliable species identification of microorganisms is of great importance in medical microbiology. several bacterial and fungal species can be identified only using laborious and time-consuming methods. furthermore, in many cases misidentification occurs due to e.g. limited biochemical reactivity, different morphotypes or limited information in reference panels. in this talk, matrix-assisted laser desorption/ionisation time-of-flight (maldi-tof) mass spectrometry will be presented as a method for species identification. this technology applies protein pattern matching based on mass spectrometry. during the identification process, a mass pattern is generated for each organism. the subsequent comparison of this pattern with a database comprising reference patterns derived from well-characterised reference strains leads to species identification. as examples, the identification of various nonfermenting bacterial strains isolated from clinical specimens in comparison to partial s rdna sequencing will be shown. moreover, speed, accuracy in comparison to other methods, and inter-and intra-laboratory reproducibility of maldi-tof ms-based species identification will be discussed. o trends in invasive streptococcus pneumoniae serogroup sequence types in belgium t. goegebuer, k. van pelt, j. verhaegen, j. van eldere°(leuven, be) objectives: s. pneumoniae serogroup (sg ) isolates frequently cause invasive pneumococcal disease, particularly in children. from onwards a marked increase in sg isolates was observed; overall prevalence increased from . % ( - ) to . % ( ) ( ) ( ) ( ) . we determined the sequence types (st) in sg isolates in order to better understand trends in sg resistance and spread. methods: as national reference centre, we receive all invasive isolates from more than of laboratories in belgium. randomly chosen sg isolates from all ages from to were analysed via multi-locus sequence typing (mlst) as described by enright & spratt (microbiol. ; : − ) . we also included data on strain characteristics and patient characteristics. results: different sequence types (st) were identified: st (n = ), st (n = ), st (n = ), st (n = ), st (n = ), st (n = ), st (n = ), st (n = ), and st (n = mutations usually increase the mic slightly, but enhance the probability of further mutations. efflux pumps like pmra reduce antibiotic concentrations in the bacterial cell, enabling longer survival. we hypothesised that efflux positive bacteria are more likely to develop resistance than efflux negative bacteria. the following questions were addressed: . do the efflux pump inhibitors reserpine and verapamil reduce the mutation frequency? . do fluoroquinolone-susceptible efflux positive pneumococci exhibit higher parc or gyra qrdr mutation frequencies than efflux negative isolates? . does efflux phenotype impose a fitness cost? methods: matched efflux positive and negative pneumococcal isolates with identical or similar genotype according to multi-locus sequence typing collected by the german community acquired pneumonia network capnetz were analysed (n = ). strains tigr and r were included as efflux negative controls. ciprofloxacin (cip) mics and efflux phenotype were measured by agar dilution method, for efflux detection reserpine ( mg/l) was added and a fourfold decrease in mic was considered as efflux positive. mutation frequencies were determined by plating bacterial suspensions onto agar with and without cip. after incubation colonies were counted and the ratio of cfu/ml yielded the mutation frequency. equally, the mutation frequency was determined adding different concentrations of verapamil ( , , , , mg/l) or reserpine ( . , . , , , mg/l). biological fitness was calculated as the maximum slope of growth curves recorded in a microtitre plate reader. results: ) even at low concentrations, reserpine clearly reduced the mutation frequency of efflux positive and, to a lesser extent, efflux negative pneumococci when exposed to cip (figure ); verapamil exhibited this effect merely at high concentrations. ) efflux positive isolates produced more frequently mutants ( / ) than efflux negative isolates ( / ) (p = . , fisher's exact test). ) efflux phenotype had no measurable impact on the biological fitness. conclusion: a positive efflux phenotype increases the qrdr mutation frequencies in the presence of fluoroquinolones and this effect can be inhibited by very low concentrations of reserpine. as a matter of concern, efflux is not associated with decreased biological fitness. background: use of fluoroquinolone (fq) has been associated with increasing fq resistance in s. pneumoniae. because respiratory fqs (levofloxacin (levo) and moxifloxacin (moxi)) are first line therapy for serious respiratory infections, increasing fq resistance (fqr) in sp is a concern. levo targets parc, and moxi targets gyra, which may permit differentiation of degree of selective pressure. we examined fq use, and changes in the prevalence of fqr and qrdr mutations in canadian isolates of sp. methods: cbsn is a canadian collaborative network of microbiology laboratories that has performed surveillance for antibiotic resistance in sp since . antimicrobial resistance is performed in a central lab to clsi standards. we sequenced qrdr regions of all fqr isolates and a stratified sample of fq susceptible isolates. population fq use was obtained from ims canada. results: from to , fq use increased from to rx/ pop/yr; levo use from to rx/ pop/yr, and moxi use from to rx/ pop/yr. isolates were available for testing. levo r rates increased from in to . % in then remained stable until ( . % in ). moxi r rates increased to . % in , then stabilised ( . % in ). the prevalence of parc only mutations has not increased significantly in the last decade (see table) . the prevalence of isolates with both parc and gyra mutations increased until , but has decreased in . the first gyra only mutant was detected in ; the prevalence of gyra only mutants since then has increased, but remains very low ( / , . % in ) . conclusion: despite increasing use of respiratory fqs, fqr in pneumococci is very low and not increasing in canada. the prevalence of isolates with parc mutations is decreasing. isolates with mutations in gyra alone remain extremely rare, suggesting that moxi exerts minimal selective pressure for resistance. in streptococci, two well characterised macrolide resistance have been described: target modification and active drug efflux. target site modification is mediated by the erm genes -erm(b), erm(a), erm(c)which confers the mlsb phenotype. target modification by mutations in s rrna as well as mutation in l and l ribosomal proteins have also been reported. expression of mef(a) genes activate an efflux mechanism responsible for m-type resistance we characterised a clinical isolate of s. agalactiae mb gbs exhibiting the mlsb phenotype and tetracycline resistance. in this study, we determined the resistance genes, their association, and their localisation and mobility by conjugation. methods: the macrolide and tetracycline resistance genes were confirmed by pcr. the association between macrolide and tetracycline genes was investigated by long-pcr and sequencing. conjugation experiments were performed by filter matings. the genetic localisation of resistance genes was determined by endonuclease i ceui -followed by pfge and southern blot. the hybridisation study was performed using three specific probes for the s and s rrna genes, for erm(b) and tet(o) genes. results: s. agalactiae mb gbs carried erm(b) and tet(o) genes on the same amplicon of kb in size. the nucleotide sequence analysis of the entire product was identical to the peoc of kb from pediococcus acidilactici that contains four orfs, of which orf and orf encode a putative resolvase and topoisomerase type i, respectively. the endonuclease i ceui method, that easily distinguishes between plasmid and chromosomal localisations as i-ceui only cuts chromosomal dna, revealed the localisation of resistance genes on the plasmid. all attempts to transfer erm(b)-tet(o) structure by conjugation from s. agalactiae mb gbs to og ss e. faecalis as recipient failed. conclusion: our results show the first case of the association between erm(b) and tet(o) genes on the unique mosaic structure in s. agalactiae, probably on the plasmid, as demonstrated by the i ceui-assay. further studies are on going to characterise the entire genetic element carrying resistance genes. o improving influenza pre-analytic collection systems: alternative collection systems to inactivate, preserve, or extract influenza for rapid testing s. castriciano°, k. luinstra, m. ackerman, a. petrich, m. smieja (brescia, it; hamilton, ca) objectives: in this study, alternative influenza sample collection systems were evaluated for potential use in a pandemic situation. the objectives were to develop: ) a non-temperature dependent swab collection and transport system, that inactivates influenza virus infectivity but preserves cell morphology and nucleic acid (na) for the detection of suspected influenza infections and/or ) a system compatible with direct na testing without the need for purification prior to detection by a rapid real-time rt-pcr. methods: flocked nasopharyngeal swabs (nps) collected in utm (u) were compared to nps collected in a cymol (c), m-swab (m) or dry (d) flocked swab collection system (copan, italia). cymol is an alcoholbased medium that preserves cells for dfa testing. the m-swab contains ul of medium and ul of glass beads, and requires no na purification step. shell vial culture was used to assess influenza virus inactivation after minutes exposure to the collection media. a mockinfected influenza a virus sample was absorbed to duplicate swabs then placed into the collection systems. the infected collection media were held at rt for minutes and then inoculated in duplicate into shell vial culture and stained after hours. influenza a stability and na recovery after mock infection of each collection system was assessed after , , and days (d) at ºc, − ºc, room temperature (rt) and ºc. aliquots of infected collection media were extracted by easymag and ul of purified na tested by a quantitative influenza a rt-pcr on the roche lightcycler. m-swab collected samples were also tested directly or after boiling, without na purification. results: shell vial culture found that influenza a virus was inactivated after minutes exposure to the c medium but not when exposed to the u and m media. influenza a was detected by dfa from the u and c cell smears. quantitation of influenza a rna was constant after , and d in u, c, m and d collection systems at − , ºc and rt. the quantity of rna recovered declined significantly after d at ºc in all collection systems. m with boiling yielded data comparable to the easymag extraction. the copan cymol medium inactivates influenza infectivity, preserves cells and stabilises rna up to days at − , ºc and rt. cymol medium is a potential alternative for safe sample collection during a pandemic influenza situation. the m-swab presents a rapid testing alternative. luminex respiratory viral panel in respiratory specimens from children r. selvarangan°, s. selvaraju, d. baker, k. estes, l. hays, d. abel, s. hiraki (kansas city, us) objective: luminex respiratory viral panel (rvp) is a multiplex pcr capable of detecting and differentiating twelve different respiratory viruses and their subtypes; influenza a (flu a) (subtypes h and h ), influenza b (flu b), respiratory syncytial virus (rsv) (subtypes a and b), adenovirus (adv), parainfluenza (piv ), parainfluenza (piv ), parainfluenza (piv ), human metapneumovirus (hmpv) and rhinovirus (rhv). the aim of this study was to evaluate the analytical performance characteristics of rvp assay and to evaluate its ability to detect respiratory viruses from nasopharyngeal aspirates obtained from children. method: analytical sensitivity, specificity, accuracy and precision of the luminex rvp assay were determined using control viral stocks and respiratory specimens previously tested by rmix shell vial culture. result: luminex rvp assay reliably detected atcc viral stocks of flu a, flu b, rsvb, rhv and piv in the range of e- to e- tcid /ml. no cross reactivity was noted with cmv, hsv, hhv , ebv, vzv, piv , cornoavirus e and oc . among respiratory specimens previously characterised by culture specimens were accurately detected with overall accuracy of %. the median coefficient of variation in mean fluorescent index values of signals from replicate analyses of influenza a, b and rsv was % ( % to %). the clinical specimens tested by rvp assay included culture positive and culture negative specimens. respiratory viruses isolated from the culture positive specimens include the following; adv, flu a, flu b, rsv, piv , piv , piv , hmpv and rhv. rvp assay detected all of the respiratory viruses except one each of rsv, piv and piv virus with overall sensitivity ranging from % to % for the different respiratory viral groups. among the culture negative specimens respiratory viruses were detected by rvp of which were subsequently confirmed by repeat analyses. conclusion: luminex rvp assay is a highly sensitive and specific test useful in the detection of commonly encountered respiratory viruses in respiratory specimens. the addition of rvp assay to the viral testing algorithm of respiratory infections in children provides rapid results, improves diagnostic yield and may result in decreased antibiotic usage, reduced diagnostic testing and reduced hospital stay. m. savvala, i. daniil, i. berberidou, a. koutsibiri, a. stambolidi, m. papachristodoulou, n. spanakis, d. petropoulou°, a. tsakris (athens, gr) objective: in developed countries, viruses, particularly noroviruses, are recognized as the leading cause of acute gastroenteritis. we determined the aetiology, prevalence and seasonal distribution of viral gastrointestinal infections in hospitalised patients with acute diarrhoea. methods: during one-year period (november -november ), a total of faecal specimens were collected from children, premature neonates and adults who were hospitalised with symptoms and signs of acute gastroenteritis. stool samples were tested for the presence of rotavirus, adenovirus, astrovirus and norovirus. rotavirus, adenovirus and astrovirus antigen detection was performed by chromatographic immunoassays (rotavirus and adenovirus, vikia ® -biomerieux, france; h&r astrovirus-vegal farmaceutica, spain). noroviruses were detected by an enzyme immunoassay (ridascreen ® rbiopharm, germany) and confirmed by reverse transcription-pcr. data were analyzed for seasonality of infection and possible transmission mode. the overall incidence of viral identification in acute diarrhoeal stool was % ( of patients). fifty one viral antigens were detected one patient with positive antigen detection is suffering from a disease of unclear aetiology. so, an association of replication of cihhv- with the disease might be considered. in contrast, the other patient did not show any symptoms at the time of antigen detection. this patient shows a special mode of acquisition of cihhv- (by bmt) possibly resulting in differences in the immunological priming and response. in addition, in the latter patient cihhv- is restricted to blood cells. two other patients did not show antigen expression. so, it is unclear how the transcription and translation of viral genes is influenced? furthermore, is there a pathophysiological impact of viral replication in individuals with cihhv- ? objectives: several case studies have reported on meningo-encephalitis caused by a primary epstein-barr virus (ebv) infection. we aimed to investigate the viral loads, and the inflammatory characteristics of this thus far poorly defined disease entity. we evaluated all cases from - , in which an ebv polymerase chain reaction test (pcr) was requested on a cerebro spinal fluid (csf) sample. primary infection was defined as a clinical presentation with sore throat/pharyngitis/malaise in combination with lymphocytosis, and detectable heterophile antibodies or positive ebv igm antibodies. patients with proven neuroborreliosis served as control group. leukocyte response and ebv viral loads in csf, and serum were compared between primary ebv and neuroborreliosis cases. results: we identified six cases with a primary ebv infection (median age: , male: ) with neurological symptoms ranging from meningeal signs to encephalitis. these were compared to patients with neuroborreliosis (median age: , male: ). in four out of six patients with a primary ebv infection with neurological symptoms ebv dna was detected in csf and in serum, whereas all neuroborreliosis cases were ebv pcr negative in both compartments. viral loads were lower in csf as compared to serum. in blood, leukocytes, lymphocyte, and monocyte counts were significantly increased as compared to the neuroborreliosis cases (see table ). specific for vp and vp genes, using pools of g and p type specific primers. all strains (niv/brv/ , niv/brv/ , and niv/brv/ ) were not typeable for the vp and vp genes. after purification by "qiaquick gel extraction kit" (qiagen, germany), the vp , vp , vp , and nsp first amplicons of the borv-a strains were subjected to sequence analysis with automated sequencer abi xl dna analyzer (applied biosystems, usa). phylogenetic analysis was performed using mega version . . objectives: dengue is a flavivirus and is among the most widely-spread viral diseases. our previous report demonstrates existence of live dengue virus in blood and urine even in the convalescent postfebrile period. in some cases, excretion in the patient's urine can be detected as late as days after the onset of illness. this goes along with the model of west nile virus, another type of flavivirus, which can be excreted in the urine for months after acute infection in both animal studies and human case report. here we report a pilot study to address a magnitude of such findings. methods: between april and october , paediatric and adult febrile patients suspected of dengue infection were enrolled. diagnosis of dengue was based on standard specific serology on paired sera. patients with negative serology served as controls. blood and urine specimens were collected at several time points. whole blood was separated into plasma and peripheral blood mononuclear cells (pbmc). these have been aliquoted and used for earlier studies and some stored in freezers. available plasma, pbmc, and urine were processed and inoculated into aedes aegypti. surviving mosquitoes at days after inoculation were employed for viral detection by dengue-specific rt-pcr. indirect fluorescence antibody (ifa) staining of mosquito heads was performed on all positive rt-pcr specimens, except for the one from pbmc (awaiting ifa result). results: and cases of primary and secondary infections, respectively, and negative controls were included. these translated into and early and late dengue specimens, and and early and late negative-control counterparts, respectively. dengue virus were isolated in some blood and urine specimens as late as days after the onset of illness. no virus was isolated from control specimens. all but positive rt-pcr specimens also demonstrated positive ifa. out of negatives were from early-phase specimens. conclusion: our study demonstrates prolonged survival of dengue virus after clinical recovery. this finding has pathologic and epidemiologic significance, adding a potential role of urine in the transmission of the disease. spread of the virus to humans might occur through infectious urine with help from arthropod vectors. this research could provide new insights into our understanding of the pathogenesis of denv infection. isolation of dengue virus from blood and urine specimens during early (days − after onset of illness) and late (days − ) phases of infection (specimens with dengue isolated/total specimens for mosquito inoculation) early phase late phase plasma / ( %) / ( %) pbmc not performed / ( %) urine / ( %) / ( %) all specimens / ( %) / ( %) dna copies) (< - ) † ; n = <dl all <dl ebv pcr in serum (dna copies) (< - ) † ; n = <dl all <dl data presented as median (interquartile range). *p < . ; † p < . ; comparison by mann-whitney u-test. csf: cerebrospinal fluid. ebv: epstein-barr virus y. achermann°, c. spormann, c. kolling, c. remschmidt, j. wüst, b. simmen, m. vogt (baar, zurich, ch) objectives: elbow arthroplasty is increasingly used for treatment of rheumatic or posttraumatic arthritis. data on epidemiology, characteristics and treatment outcome of elbow prosthetic joint infection (pji) is limited. furthermore, no validated therapeutic algorithm exist as for hip or knee pji. we analyzed all pji, which occurred in a cohort of implanted elbow prostheses during a -year-period. methods: between / and / , all cases with implanted elbow prosthesis at our institution were retrospectively included. elbow pji was defined as visible purulence, acute inflammation on histopathology, sinus tract or microbial growth in periprosthetic tissue. patients were regularly follow-up at outpatient orthopedic visits and were in addition recently contacted by phone. a kaplan-meier survival analysis was performed. results: during the study period, elbow prostheses were implanted. overall, of cases ( %) developed elbow pji (median age y, range − y, % males). among them, ( %) had a rheumatic disorder and ( %) a posttraumatic arthritis. infections were early ( months), were delayed ( − months) and were late ( months). more infections were acquired intraoperatively (n = , %) than haematogenously (n = , %). the following pathogens were cultured: staphylococcus aureus (n = ), coagulase-negative staphylococci (n = ), streptococcus agalactiae (n = ), corynebacterium sp. (n = ), enterococcus sp. (n = ), enterobacter cloacae (n = ), mixed infections (n = ) and culture-negative (n = ). treatment approaches included débridement with implant retention (n = ), one-stage exchange (n = ), two-stage exchange (n = ), resection arthroplasty ( ) and antibiotics only (n = ). at follow-up, ( %) patients were free of infection (median follow-up time . y, range . − . y) and ( %) had a relapse (median time to infection . y, range . − . y). one patient died due to infectious endocarditis with secondary haematogenous elbow pji. the relapse-free survival ( % confidence interval) was % ( %- %) after year, % ( %- %) after years, % ( %- %) after years and % ( %- %) after years. the infection incidence after elbow arthroplasty was higher ( %) than reported after hip (< %) or knee arthroplasty (< %). most infections ( %) manifested early ( months). underlying rheumatic disorder were common in elbow pji ( %). the relapse-free survival after elbow pji was %, %, % and % after , , and years. e.a.n. oostdijk°, a.m.g.a. de smet, h.e.m. blok, e.s. thieme groen, j.a.j.w. kluytmans, m.j.m. bonten (utrecht, breda, nl) introduction: selective digestive tract decontamination (sdd) and selective oropharyngeal decontamination (sod) aim to eradicate gram-negative bacteria (gnb) from the intestinal en respiratory tract in intensive-care-unit (icu) patients. we aimed to quantify effects of sdd and sod on bacterial ecology in icus that participated in a clustered group-randomised cross over study in the netherlands, in which sdd, sod or standard care (sc) was used during consecutive periods of months (nejm ; : ) . the order of periods was randomised per icu. methods: point prevalence surveys of rectal and respiratory samples were performed once monthly in all patients in icu (receiving or not receiving sod/sdd). selective media were used to detect gnb and mic testing was done for ceftazidime (cft), ciprofloxacin (cip) and tobramycin (tob). effects of sdd on rectal carriage were determined by comparing data from sdd months in each icu to data from all months preceding and following (in which sod or sc was given). combined effects of sdd/sod on respiratory tract carriage were determined by comparing data from months sdd/sod (in any order) to data from the sc months before and afterwards. results: rectal and respiratory tract samples were analysed. during sdd average proportions of patients colonised with gnb resistant to either cft, tob, cip were %, % and % and these proportions were %, % and % during the post-intervention period (p < . in each case). this effect is most explicit for cft with resistance levels increasing from . % in the last month of sdd to . % in the st month after sdd (p < . ). during sdd and sod resistance levels in respiratory tract samples were < % for all three antibiotics, but prevalence increased gradually (for cft and tob resistance; p < . for trend). after discontinuation of sdd and sod average proportions increased to levels > % for all three antibiotics (p < . in each case). cft resistant isolates in rectal samples mainly included enterobacteriaceae not being escherichia coli and klebsiella spp, whereas tob and cip resistant isolates mainly included e. coli. conclusion: sod and sdd have marked effects on the bacterial ecology in an icu with a rapid and persistent increase in resistance after intervention. antibiotic resistance remains a major concern associated with these infection control measures. o throwing caution to the winds? three cases of anaphylaxis to chlorhexidine coated central venous catheters from a regional cardiac centre in northwestern england this blaoxa- -producing clone showed resistance to several b-lactams (including imipemem), susceptibility to ceftazidime, netilmicin and minocycline, and variable susceptibility to meropenem, cefepime, and aztreonam. mics for colistin and tigecycline ranged from > mg/l and from . − mg/l, respectively. all oxa- -producing isolates presented the isaba downstream of the blaoxa- gene. hybridisation assays revealed a plasmidic location for the blaoxa- gene with ca kb. plasmid sequencing showed an isaba -like truncated at the end upstream of the blaoxa- gene, a fact that may explain the observed negative carbapenemase-production bioassay. conclusion: blaoxa- -carrying a. baumannii is, apparently, more ancient than initially imagined. although undetected from onwards, the fact that it possessed a non-expressible gene, due to alterations in the promoter region, suggests that this information might have been incorporated from a still unidentified source. twenty-seven ( %) were male. isolates were recovered from respiratory secretions ( isolates, . %), blood ( , . %), urine ( , . %), catheter ( , . %) and other secretions ( , . %). only ( . %) of patients received appropriate antimicrobial therapy either with polymyxin b ( . %), ampicillin-sulbactam ( . %) or tigecycline ( . %). overall -day mortality of patients with crab was %. mortality rates were . per -patient/day. these rates were significantly higher among patients who have not received appropriate therapy ( . per -patient/day) compared with those who have received it ( . per -patient/day; p = . ; figure ). in the cox regression model only receiving appropriate treatment (hazard ratio [hr] . ; % confidence interval [ci] . − . ); p = . ) was independently associated with -mortality. positive blood culture for crab remained in the final model (hr . ; % ci . − . ; p = . ). all isolates submitted to pcr were positive for blaoxa- . all these isolates were susceptible to polymyxin b and tigecycline. conclusion: high -day mortality occurred in this icu outbreak. many patients did not receive appropriate therapy, which significantly increased mortality. other clinical risk factors for mortality in this outbreak are currently under investigation. acinetobacter baumannii in norwegian strain collections reveal major discrepancies to phenotypic identification and the presence of carbapenemase-producing clonal lineages baumannii isolates the per- gene was identified in ( %). the similarity of the bands were calculated according to "dice smilarity coefficients" and all per- positive isolates were found as clonally related. conclusion: in our study the prevalence of per- was lower than the previous studies. but the presence of high ceftazidime resistance rates among these isolates may indicate the presence of other beta-lactamases. dna analysis by pfge and rapd revealed an outbreak caused by a unique clone. detection of clonal related isolates among different services may be because of the treatment of these patients at the same services before and this may explain the spread of per- positive strains.o resistance genomic islands related to abar are common in acinetobacter baumannii strains belonging to european clone i l. krizova°, m. maixnerova, l. dijkshoorn, a. nemec (prague, cz; leiden, nl) objective: acinetobacter baumannii strains belonging to european (eu) clone i are commonly resistant to multiple antimicrobial agents. a number of resistance genes were recently detected on an -kb genomic resistance island (abar ) inserted in the atpase gene of eu clone i strain aye. the aim of this study was to assess the presence of abar related structures in epidemiologically unrelated strains of eu clone i. methods: the study set included multi-drug resistant (mdr) strains of eu clone i collected in european countries in - and genotypically unique, fully susceptible strains. using pcr, all strains were investigated for the presence of the atpase gene and for nine genes found to be associated with abar . furthermore, the strains were tested for the disruption of the atpase gene using pcr primers directed against the and ends of this gene. strains with the disrupted gene were investigated for the presence and structure of the atpase gene-abar connecting regions using pcr mapping and rflp. pcr primers were derived from the known sequence of strain aye. results: all strains were positive for the atpase gene. the susceptible strains had an intact atpase gene whereas all mdr strains failed to produce the expected amplicon in the atpase disruption test. all eu clone i strains yielded positive results for the atpase gene-abar connecting regions, the structure of which corresponded to those of aye. these findings suggest the presence of atpase integrated elements in clone i strains, the integration of which had invariably taken place at the same locus site. none of the abar -associated resistance genes were found in any of the susceptible strains. in contrast, the mdr strains harboured the following abar -associated genes (% positive strains): aacc ( ), aada ( ), aadb ( ), apha ( ) stra ( ), mera ( ), teta ( ), cat ( ), the gene encoding heavy metal detoxification protein ( ). individual mdr strains carried from one to nine abar -associated genes in different combinations. there was a good correlation between the content of resistance genes and resistance phenotypes. conclusion: genetic structures related to abar are common in strains belonging to eu clone i. the heterogeneity of resistance patterns in this clone is likely to result from the variations in the content of abar related structures. supported by grant / / of the grant agency of the czech republic. objectives: to study the differences in mutation frequency and evaluate the possible correlations between drug resistance development and mutation rate in acinetobacter baumannii (ab). the mutation frequency (mf) of rifampicin (rif) resistance was used as a surrogate measure of differences in mutation rate and for detection of the presence of mutator phenotype. -and -fold higher when larvae were infected with atcc and sdf, respectively. thus, the sdf genome was used as reference genome to identify functions acquired by pathogenic strains with a possible role in antibiotic resistance and pathogenicity. sixty-two clusters, corresponding to almost cdss, were identified in the acicu and aye genomes (and partially in atcc ) that were absent in sdf. this study found that targeted interventions that reduce the use of quin were associated with a decrease of the quin resistance rate in e. coli. e. velasco°, w. espelage, i. noll, a. barger, t. eckmanns (berlin, de) objectives: growing populations of older and immunocompromised patients, changes in epidemiology and unchecked use of antibiotics can led to a rise in consumption as well as resistance to certain treatments. medical doctors (mds) often have an important role alongside contributing factors. we conducted a national survey of mds in germany on their behaviours and expectations for intervention. we aimed to assess md behaviours with and influences on antibiotic prescribing and the potential for related interventions that address antibiotic resistance.methods: a representative sample comprised , mds with differing practice specialties, from both stationary and ambulatory settings (respectively: % and % internists, % and % general practitioners, % and % surgery, % and % ear/nose/throat, % and % paediatrics, % and % urology, % and % gynaecology, % and % dermatology, % and < % other) in federal states. we developed study questions to capture baseline information on mds and their practice with antibiotics. questions also focused on selected influences that may affect behaviour in practice. other questions solicited opinions about interventions that may improve practice. mailed questionnaires were distributed to participants via state medical associations. results: among survey respondents (n = , ; response rate = %), % reported that they prescribe antibiotics daily, and % indicated they do so at least weekly. of all surveyed mds, % reported that they think their own prescribing practice has an influence on antibiotic resistance in their region. of all mds, % found it "important" to continually improve use of antibiotics through industry independent experts providing consultation, audits and feedback. of all mds, % found it "important" to have provision of regional coverage of antibiotic resistance with appropriate feedback for practicing mds, and % found it "important" to have provision of antibiotic regulations of prescriptions with appropriate feedback for practicing mds. (results in table .) -a not all results shown and remaining percentages are as follows: a closed three category scale was used for options "yes", "no", "do not know". a closed four category scale was used with options "very important", "important", "less important" and "not important". a closed five category scale was used for options "daily", "weekly", "monthly", "seldom" and "never". a closed five category scale was used for options "strongly agree", "agree", "neutral", "disagree" and "strongly disagree". objectives: to investigate the mlsb and tetracycline resistance and the emm gene distribution among the invasive streptococcus pyogenes (gas) strains. methods: between january and december , a total of strains responsible for invasive infections for adult patients were sent to the french national reference center for streptococci to be studied. antibiotic susceptibility testing was done by disk diffusion method according to the ca-sfm guidelines. mics were determined by e-test method. streptococcal emm sequence was done according to the cdc protocol. detection of macrolide and tetracycline resistance genes: erm(b), erm(tr), mef(a), tet(m), tet(o), tet(k), and tet(l) was performed by pcr. results: among the streptococcus pyogenes invasive strains; more than ten different emm-types were identified. the most frequent emm sequence types were emm , emm and emm . a total of strains ( %) were resistant to erythromycin. erythromycin resistance prevalence had decreased during the three years period ( . %- , . %- , . %- ) . had an mlsb constitutive ( strains) or inducible ( strains) phenotype due to erm(b) or erm(tr) resistance gene. with the m phenotype and mef(a) gene were susceptible to clindamycin. among the ( . %) tetracycline resistant isolates tet(m), tet(o) and tet(l) genes were detected in , and strains, respectively. tetracycline resistance prevalence had also decreased during the three years period ( . %- , %- , . %- ) . conclusion: most of the invasive french gas isolates remained erythromycin and tetracycline susceptiple during three years. nontheless, the resistance rates have had the tendency to decrease slightly. taking into account the resistance trends helps to guide the therapy for penicillin-allergic patients. objectives: during a survey on antimicrobial susceptibility in betahaemolytic group c and g streptococci (gcgs) from portugal, a macrolide resistance rate higher than previously reported in other european countries was found ( %) among s. dysgalactiae subsp. equisimilis isolates. to gain further insights into the resistance mechanisms involved and the clonal structure of the resistant population, we undertook the phenotypic and molecular characterisation of macrolide resistant s. dysgalactiae subsp. equisimilis isolates and compared it with the susceptible population. methods: antimicrobial susceptibility testing and macrolide resistance phenotype were determined by disk diffusion. all the macrolideresistant isolates were further characterised by mic testing and genotype determination by pcr. a combination of emm typing and pulsed-field gel electrophoresis (pfge) was used to type the population and the simpson's index of diversity (sid) with % confidence intervals was calculated as previously described.results: a total of isolates were resistant to erythromycin (mic range, to > ug/ml). the vast majority of isolates presented a mlsb phenotype (n = ) and carried the erm(a) gene (n = ), while the mefencoded m-phenotype was expressed by only isolates. among resistant isolates, distinct emm types were found distributed by pfge clusters that overlapped with the main clusters detected in the susceptible population. the emm types stg , stg , stg and stg accounted for approximately two thirds of the resistant isolates. pfge did not always separate neither macrolide-resistant from susceptible isolates nor erm(b) and mef(a) from the prevailing erm(a) isolates. the sids of emm and pfge calculated for resistant isolates were not statistical different from the overall population. the two most prominent mls resistant lineages were one with stg /erm(a) isolates (n = ) and stg /mef(a) (n = ), and another including stg /erm(a) (n = ).conclusion: although most of the resistant isolates presented a mlsb phenotype and carried an erm(a) gene, molecular typing revealed extensive diversity in both emm types and pfge clones. macrolide resistance had a polyclonal origin, with resistance emerging among most susceptible clones. monitoring of macrolide resistance patterns in s. dysgalactiae subsp. equisimilis is essential as this pathogen is increasingly recognised as an important human pathogen. a.s. simões°, r. sá-leão, s. nunes, n. frazão, a. tavares, h. de lencastre (oeiras, pt) while performing pneumococcal nasopharyngeal colonisation surveillance studies among children attending day care centres (dcc) in portugal, we observed that the rate of strains with penicillin mic ug/ml more than tripled from . % in to % in (p = . ). the aim of this study was to characterise the isolates recovered in which had a mic to penicillin ug/ml. methods: pneumococci were isolated and identified on the basis of selective growth on gentamycin blood agar plates, optochin susceptibility, colony morphology, and alfa-haemolysis. susceptibility to antimicrobials agents was performed according to the clsi recommendations and definitions. strains were serotyped by the quellung reaction and/or multiplex pcr using specific primers for each serotype. pulsed-field gel electrophoresis (pfge), after restriction of the total dna with smai, was performed to compare genetic backgrounds. results: sixteen of the isolates belonged to serotype , three were serotype a and one was of serotype a. strains of serotype were also resistant to sulfamethoxazole-trimethoprim and belonged to a single pfge cluster identified as clone spain v st . the penicillin resistant serotype strains were isolated in two dcc, from nine children vaccinated with the -valent pneumococcal conjugate vaccine (pcv ), four non-vaccinated children and three children with unknown vaccination status. five of these carriers had received antibiotics recently. in these two dcc the overall proportion of children vaccinated with pcv was %; % of the children had received antibiotics within the previous month and % had received three or more courses of antibiotics in the last six months. since the introduction of the pcv in portugal, in june , the proportion of penicillin resistant pneumococci recovered from colonisation has been stable (c.a. %). the sudden increase in the levels of penicillin resistance observed in the surveillance study was found to be largely due to the dissemination of clone spain v st serotype variant in two dcc with high consumption of antibiotics. the observations suggest a combination of high antibiotic selective pressure and transmission rates resulting in an outbreak-like situation with a penicillin resistant vaccine type clone being disseminated among children in day care despite use of pcv . background: beside target mutation, active efflux is another common resistance mechanism to fluoroquinolones (fq) in s. pneumoniae. two main efflux systems have been described so far, namely pmra (member of the mfs superfamily) and the two abc transporters pata/patb. we have studied the inducibility of pmra, pata and patb genes expression when bacteria are exposed to subinhibitory concentrations of fq. we used a wild-type sensitive strain (atcc ), two clinical strains resistant to fq (sp and sp ), and two efflux mutants (sp and sp ; selected in vitro after exposure to ciprofloxacin [jac , : - ] ). mic were determined according to clsi. induction was obtained by growing bacteria in todd-hewitt broth added by half the mic of each fq (cip, nor, lvx, mxf, gmf) for h at ºc in % co atmosphere. expression levels of pmra, pata and patb genes were determined by real-time pcr. reversibility of induction was tested by re-cultivating bacteria for h in drug-free medium. results: antimicrobial susceptibilities for cip and mxf and gene expression at basal level and after exposure to these fq are shown in the in women with single infection, the most common hpv types were hpv- and hpv- , followed by hpv- , hpv- , hpv- and hpv- , whereas in women with multiple infections hpv- was the most commonly detected type, followed by hpv- , hpv- , hpv- and hpv- . a different distribution of hpv types and a higher rate of multiple infections were observed in young vs. older women, suggesting the existence of a natural selection of hpvs which preserve a better fitness. high-risk hpvs were detected in all high-grade cervical intraepithelial lesions, with hpv- , hpv- , hpv- , and hpv- as the most frequent types. however, hr-hpv types were detected also in a high rate of women with a negative pap test as well as in women with a negative cervical biopsy, suggesting the need to improve the accuracy of available cervical cancer screening tests. the results of this study, which provide information on the epidemiology of hpv infection and type distribution in women from south italy, should be taken into consideration in the implementation of local vaccination programs. objectives: chromosomal integration of the hhv- genome (cihhv- ) into the human genome occurs in − % of healthy individuals and leads to persistently high levels of hhv- pcr copy numbers in blood and tissue. consequently, this may be interpreted as persistent active hhv- infection. although hhv- mrna has been detected in a few individuals with cihhv- , there is no evidence of replication of viral particles up to now. viral cultures have shown negative results. so, cihhv- is thought not to be linked to any disease. methods: we performed hhv- antigen detection in pbmcs of individuals with fish proven cihhv- by means of antibodies directed against hhv- variant a and b (indirect immunoperoxidase staining). results: in unrelated female adolescents (both with cihhv- variant a) we detected hhv- antigen. one patient is suffering from recurrent parotitis since years and from hypoimmunoglobulinaemia. the other patient ( a) was treated with allogeneic bone marrow transplantation (bmt) for acute myeloid leukaemia (aml) and acquired cihhv- from the healthy donor. so, cihhv- is only found in blood cells. in the latter patient only symptoms attributable to the post bmt course have been observed (prolonged mixed haematological chimerism, protracted mucositis, transient hypertension and transient neuropathy). at the time of antigen detection years after bmt the patient was clinically well. in individuals (a girl after fatal myocarditis and her healthy father − both with variant b) no hhv- antigen has been detected. discussion: up to now cihhv- is considered not to cause any disease. for the first time we show the expression of hhv- antigen, which indicates the replication of viral particles. this might have a pathophysiological impact. sixty-seven % of cases with ebv meningo-encephalitis have detectable viral dna amounts in csf and serum, whereas neuroborreliosis patients do not. cases with primary ebv meningoencephalitis have increased systemic leukocytosis, with higher lymphocyte, and monocyte levels compared to neuroborreliosis patients.o incidence of post-herpetic neuralgia in treated and untreated patients with herpes zoster followed for year in an italian prospective cohort: preliminary results g. parruti°, f. sozio, c. rebuzzi, m. tontodonati, e. polilli, a. agostinone, a. manna, f. di masi, a. consorte, g. congedo, l. cosentino, d. d'antonio, l. pippa, l. manzoli, c. granchelli (pescara, chieti, it) objectives: a large prospective cohort of patients with herpes zoster (hz) was enrolled between may and june in pescara, italy, with a planned -year follow-up after clinically and/or molecularly assessed diagnosis. aim of the study was to evaluate predictors of prolonged acute course and/or incidence of post-herpetic nevralgia (phn). methods: data from all enrolled patients were collected by a network of general practitioners. suspected cases and patients with intense acute pain were referred to our institution for immediate evaluation. clinical and demographic information was mandatory at baseline, as photographs of enrolled patients. for uncertain cases, varicella-zoster virus (vzv) antibodies and vzv dna pcr on plasma and/or vesicular eluates (whenever available) were performed. follow-up data were collected at outpatient control visits or by phone calls at , , and months after onset of hz. phn was diagnosed when pain persisted or relapsed at least one month after complete clearing of dermatomeric lesions. adverse events other than pain were classified according to who grading scale and reported if . all statistical calculations were performed by stata . software package. results: patients were enrolled, ( . %) females, with a mean age of . years, -year follow-up data being now available for . hz was localised at thorax in . % and head in . %; pain in the acute phase was reported as intense or very intense by ( . %) patients; ( . %) patients were referred for molecular diagnosis as clinically uncertain, ( . %) being confirmed as vzv-related cases. forty eight ( . %) patients were not prescribed any antiviral drug at diagnosis by referring physicians, in spite of extensive support in the study plan. during follow-up, ( . %) patients reported any type of adverse event (at a mean of . ± . days), including ( . %) patients reporting phn. phn was significantly more frequent in untreated vs treated patients ( . % vs . %, p = . ), as were total adverse events ( . % vs . %, p = . ). untreated patients did not significantly differ from those treated by age ( . % vs . %, p = . ) and sex (females vs males . % vs . %, p = . ), whereas they complained for more intense pain ( . % vs . %, p = . ) at presentation. conclusion: our study confirms the importance of early diagnosis and prompt antiviral treatment at the onset of hz in order to minimise the risk of phn. methods: faecal specimens (n = ) from apparently healthy and diarrheic calves (aged < year) were collected per-rectally and investigated for detection of group a rotavirus by antigen capture elisa (generic assay, germany). elisa positive specimens (n = ) were investigated further for molecular characterisation. genotyping of borv-a strains was carried out on dsrna extracted from % pbs faecal suspensions by a nested and/or heminested rt-pcr key: cord- -ilsenpus authors: mihalov-kovács, eszter; martella, vito; lanave, gianvito; bodnar, livia; fehér, enikő; marton, szilvia; kemenesi, gábor; jakab, ferenc; bányai, krisztián title: genome analysis of canine astroviruses reveals genetic heterogeneity and suggests possible inter-species transmission date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: ilsenpus canine astrovirus rna was detected in the stools of / ( . %) samples, using either a broadly reactive consensus rt-pcr for astroviruses or random rt-pcr coupled with massive deep sequencing. the complete or nearly complete genome sequence of five canine astroviruses was reconstructed that allowed mapping the genome organization and to investigate the genetic diversity of these viruses. the genome was about . kb in length and contained three open reading frames (orfs) flanked by a ′ utr, and a ′ utr plus a poly-a tail. orf a and orf b overlapped by nucleotides while the orf overlapped by nucleotides with the ′ end of orf b. upon genome comparison, four strains (hun/ / , hun/ / , hun/ / , and hun/ / ) were more related genetically to each other and to uk canine astroviruses ( – % nt identity), whilst strain hun/ / was more divergent ( – % nt identity). in the orf b and orf , strains hun/ / , hun/ / , and hun/ / were related genetically to other canine astroviruses identified formerly in europe and china, whereas strain hun/ / was related genetically to a divergent canine astrovirus strain, ita/ /zoid. for one canine astrovirus, hun/ / , only a . kb portion of the genome, at the ′ end, could be determined. interestingly, this strain possessed unique genetic signatures (including a longer orf b/orf overlap and a longer ′utr) and it was divergent in both orf b and orf from all other canine astroviruses, with the highest nucleotide sequence identity ( % and %, respectively) to a mink astrovirus, thus suggesting a possible event of interspecies transmission. the genetic heterogeneity of canine astroviruses may pose a challenge for the diagnostics and for future prophylaxis strategies. astroviruses (astv), family astroviridae, are non-enveloped viruses with a diameter of - nm and with a typical star-like shape. the genome is a single strand of positive sense, rna of . - . kb in size, containing three overlapping orfs (orf a, orf b and orf ) with a poly(a) tail (mendez and arias, ) . orf a encodes a serine c type of viral protease. orf b is separated from orf a by a heptameric frame shift signal (aaaaaac) and encodes the viral rna polymerase. orf encodes an -kda polypeptide, which functions as the capsid precursor. astv infection is associated with gastroenteritis in many animal species and humans, and they are also associated with extra-intestinal diseases, such as nephritis in chickens, hepatitis in ducks and shaking syndrome in minks (imada et al., ; fu et al., ; blomström et al., ) . the evolution of astvs is driven by mechanisms of genetic drift, recombination and, possibly, inter-species transmission (finkbeiner et al., ; de benedictis et al., ; martella et al., ) . so far, astvs are classified into two distinct genera, mamastrovirus (mastv) and avastrovirus (avastv) with mastv (mammalian) species and avastv (avian) species listed officially by the international committee on taxonomy of viruses (ictv) (bosch et al., ) . however, taking advantage of broad-range pcr primers for astvs and metagenomic protocols, several novel astvs have been identified in a number of mammalian and avian species (chu et al., ; finkbeiner et al., ; bosch et al., ; de benedictis et al., ) . using the classification criteria adopted http://dx.doi.org/ . /j.virusres. . . - /© elsevier b.v. all rights reserved. in the th ictv report that is based on genetic analysis of the full-length orf , an additional mastv and avastv candidate species have been defined recently (schultz-cherry, ) . astv-like particles have been detected only occasionally in dogs by em, either alone or in co-infection with other enteric viruses (williams, ; marshall et al., ; vieler and herbst, ; toffan et al., ). more recently, astvs have been identified in dogs with enteric signs and characterized molecularly, suggesting that the detected viruses may represent a distinct astv species (toffan et al., ) . also, a canine astv, strain ita/ /bari, was successfully adapted to replicate in vitro on mdck cells, and astv-specific antibodies were detected in convalescent canine sera (martella et al., a (martella et al., ,b, . the prevalence of astv infection seems higher in dogs with enteric disease than in asymptomatic animals (martella et al., a,b; zhu et al., ; caddy and goodfellow, ; takano et al., ) . also, monitoring of natural infection by astv in dogs has revealed that the acute phase of gastroenteritis overlaps with peaks of viral shedding (martella et al., ) . at present, limited and partial information on canine astv genomes is available in genbank. this limited amount of information seems to suggest that canine astvs are genetically heterogeneous (martella et al., a,b; caddy and goodfellow, ) , thus posing a challenge for the diagnostic and for the understanding of the genetic and biological properties of these viruses in dogs. in this study, the complete or nearly complete genome sequence of five canine astvs and the partial genome sequence of one canine astv were determined and analysed, providing information on the genome organization and genetic diversity of these viruses. during samples were collected from diarrheic and nondiarrheic dogs from a hungarian shelter. there was no age restriction. a total of samples obtained from animals were tested for astv by using a pan-astrovirus specific primer set (chu et al., ) as described elsewhere (mihalov-kovács et al., ) and (from dogs) were randomly selected for viral metagenomics. fecal samples were diluted : in pbs (phosphate buffered saline) and homogenized. the homogenates were centrifuged at g for min and the supernatant was collected for extraction of viral rna (zymo directzol viral rna extraction kit, zymo research). templates for deep sequencing were prepared as described previously (mihalov-kovács et al., ) . in brief, viral rna samples were denatured at • c for min in the presence of m random hexamer tailed by a common pcr primer sequence (djikeng et al., ) . reverse transcription was performed with u amv reverse transcriptase (promega, madison, wi, usa), m dntp mixture, and × amv rt buffer at • c for min following a min incubation at room temperature. then, l cdna was added to l pcr mixture to obtain a final volume of l and a concentration of m for the pcr primer, m for dntp mixture, . mm for mgcl , × taq dna polymerase buffer, and . u for taq dna polymerase (thermo scientific, vilnius, lithuania). the reaction conditions consisted of an initial denaturation step at • c for min, followed by cycles of amplification ( • c for s, • c for s, • c for min) and terminated at • c for min. we subjected . g of random pcr product to enzymatic fragmentation and adaptor ligation (nebnext fast dna fragmentation & library prep set for ion torrent kit, new england biolabs, ipswich, ma, usa). the barcoded adaptors were retrieved from the ion xpress barcode adapters (life technologies, carlsbad, ca, usa). the resulting cdna libraries were measured on an qubit . device using the qubit dsdna br assay kit (invitrogen, eugene, or, usa). the emulsion pcr that produced clonally amplified libraries was carried out according to the manufacturer's protocol using the ion pgm template kit on an onetouch v instrument. enrichment of the templated beads (on an ion one touch es machine) and further steps of presequencing setup were performed according to the page of -bp protocol of the manufacturer. the sequencing protocol recommended for ion pgm sequencing kit on an chip was strictly followed. to determine the actual end genomic sequence of two astv strains, a race protocol was used, following the manufacturer's instructions (invitrogen ltd). the amplicons were visualized on . % agarose gel, and cleaned up with qiagen qiaquick gel extraction kit, according to the manufacturer's instructions. the amplicons were cloned using topo xl pcr cloning (invitrogen ltd) and the clones were subjected to sequencing in both directions using big dye v . chemistry on a xl instrument from applied biosystems (foster, ca). for sequencing accuracy, a minimum of three independent clones for each fragment type were selected for sequencing in both directions using the universal m f/r primers. the first strand iii kit (invitrogen ltd) was used to generate cdna from the poly-a tailed rna target, as described previously (martella et al., a,b) . forward primers were designed about kb upstream of the terminus of the genome of the various astv strains and used with a reverse poly-t oligonucleotide with a anchored tail. the race products were sequenced on ion torrent pgm. sequence data generated by the ion torrent pgm were trimmed and analysed by the clc bio software (www.clcbio.com). the same software package was utilized to map sequence reads to reference eucaryotic viral sequences retrieved from genbank. sequence editing, annotation and analysis were carried out using geneious software v . . (biomatters ltd, new zealand). open reading frames (orf) were predicted with the orf finder. multiple alignments were prepared and manually adjusted, whereas phylogenetic analysis was performed by using the mega software (tamura et al., ) with a neighbor-joining method, jukes cantor genetic distance model and bootstrapping over replicates. mean amino acid genetic distances were computed using the p-distance method of mega . the protein sequence analysis and classification were prepared with the aid of embl-ebi website (http://www.ebi.ac.uk/interpro/). transmembrane domains were determined using phobius web server (käll et al., ) . nuclear localization signals (nls) were predicted by http://www.moseslab. csb.utoronto.ca/nlstradamus/. astv genomic sequences were deposited in the gen-bank with the following accession numbers: hun during , stool specimens were screened for astvs using broad-range astv-specific primer set. of these, seven tested positive; out of diarrheic animals ( %) and out of non-diarrheic animals ( %). the difference between groups was not statistically significant (chi-square value, . ; p = . ). a subset (n = ) of samples was selected from animals for viral metagenomics to detect virus diversity in canine fecal specimens. among these samples specimens contained eukaryotic origin viral sequences (fig. ) . by viral metagenomics (table ) , astv rna was initially identified in out of fecal specimens ( . %). however, the proportion of astv specific reads ranged from out of ( < . %) to out of ( . %) eukaryotic viral sequence reads, respectively, and five samples that had very low astv sequence read numbers (range, - ) were not considered for further processing. other enteric viruses detected in the stools included parvovirus (in samples), coronavirus (in samples), rotavirus (in samples), picornavirus (in samples), gyrovirus (in sample), adenovirus (in sample) and calicivirus (in sample). overall, by either the pan-astrovirus rt-pcr or by the viral metagenomic screening, we identified astv rna in / ( . %) samples. interestingly, some samples negative in rt-pcr generated astv-specific reads by deep sequencing, whilst, in turn, some samples containing astv-specific reads tested negative by the panastrovirus rt-pcr, indicating a weak correlation between the two methodologies (table ) . for further analysis the complete, coding complete and partial genome sequence was determined for four, one and one astv strains, respectively. in general, the length of the coding sequences ranged between and nucleotide (nt), with a gc content of . - . %. the total genome length, considering the and utrs, ranged between and nt. the utr was sequenced completely for strains hun/ / , hun/ / , hun/ / and hun/ / , and ranged between and nt in length. the utr was sequenced completely for all the six strains and ranged between and nt in length. the genome of strain hun/ / was nt long, excluding the polya-tail. the utr and utr were, respectively, nt and nt long. the genome of strain hun/ / was nt long and it was organized similarly to strain hun/ / , even if the utr was nt longer. the genome of strain hun/ / was nt long and it differed for the longer utr region ( nt) from the other canine astvs. despite several attempts, for strain hun/ / , it was not possible to generate the actual utr sequence. the genome of this strain was organized in an identical manner to the canine astv strain gbr/ /gillingham. the genome length of the astv strain hun/ / was nt long, excluding the polya-tail. the utr was nt in length, with an additional in-frame atg codon located nt upstream (bases - ) of the atg codon of orf . the utr was nt long. only the partial genome ( nt in length) of strain hun/ / was determined. the virus possessed an early stop codon in the orf , resulting in a longer ( nt) utr ( table ) . the coding sequences showed the typical organization of astv genome. there were three overlapping orfs, coding the non-structural and structural proteins. the predicted genome organization of the viruses is shown in fig. and summarized in table . the complete orf a was nt long for all canine astvs, except strain hun/ / , and codes for a aa long polyprotein. for strain hun/ / , an in-frame start codon is located nt upstream of the orf start codon (the length of the orf is / depending on the start). a ribosomal frameshift signal, the heptameric (slippery) aaaaaac sequence, was recognized at nt - ( / in hun/ / depending on the start used), with the orf b initiating after the slippery sequence. the predicted kda nsp a protein, encoded by orf a, contained conserved domains typical for the astv serine protease between aa and aa. five transmembrane domains were identified on the predicted orf a protein at residues aa - , aa - , aa - , aa - and aa - . a possible coiled coil structure was also found at residues aa - and aa - (fig. ) . upon sequence comparison, strains hun/ / , hun/ / , hun/ / and hun/ / displayed - % nt identity to each other and - % nt to the canine strains uk/ /lincoln and uk/ /gillingham, whilst strain hun/ / displayed only % nt identity to the other canine astvs. identity to non-canine astvs was markedly lower ( - % nt) (table ) . the complete orf b was nt long in all canine astvs, except the partially sequenced strain, hun/ / , where a nt long portion was determined. the orf b begins with the last nucleotide of the slippery heptamer and with a − frameshift with respect to orf a. also, the downstream stem loop structure was highly conserved among canine astvs (caddy and goodfellow, for the orf a and orf , the orf length is calculated considering the presence of an additional start codon. this value is also calculated for the orf b/orf overlap. a partial sequence. b an additonal atg codon is present nt upstream (bases - ) of the orf a initiation codon. c based on the additional orf a start codon, the orf a-predicted polyprotein could be aa longer; -not available. - % nt ( - % aa) to the other canine astvs. strain hun/ / displayed the highest identity to a mink astv ( % nt and % aa), whilst identity to other canine astvs was lower ( - % nt and - % aa) ( table ) . the length of orf is variable among the canine astv strains. in all but one strains analysed in this study, the orf b and orf overlapped by nt. in strain hun/ / the overlapping was nt in length. in addition, there was a second in-frame atg start codon, located nt upstream of the start codon homologous to other mamastrovirus genomes. only in strain hun/ / , this additional in-frame start codon was located nt upstream of the first start codon. during the rna replication, orf is expressed from a sgrna (walter and mitchell, ; mendez et al., ) . the sgrna promoter sequence has a highly conserved nucleotide sequence motif and is present upstream of the orf start codon (mendez et al., ) . the highly conserved nucleotide stretch upstream of orf , atatggaggggaggaccaaaaaattgcgatggc, believed to be part of a promoter region for synthesis of sgrna, was completely conserved in the sequence of canine astv strains except for the sequence of strain hun/ / , that was ctttggaggggaggaccaaagcagcgctatggc. the orf was / nt long for strain hun/ / and hun/ / , / nt long for strain hun/ / , / nt long for strain hun/ / , / nt for strain hun/ / and / nt for hun/ / ( table ). the terminal nt stretch of orf and the adjacent utr (s m) is highly conserved among most astvs and similarities have been observed in the sequence and folding of the utr of viruses from other viral families, suggesting that it is relevant for astv genome replication (monceyron et al., ) . the conserved s m sequence ccgcggccacgccgagtaggaacgagggtacag overlaps the aa c terminus of the capsid (srghae), that is highly conserved in several mammalian astvs, including the canine astvs (fig. ) . however, in strain hun/ / the highly conserved s m stretch was found downstream of the orf termination codon, in the utr. as a consequence, the aa c terminus of strain hun/ / was tlstkh. upon sequence comparison, the hungarian canine astvs were found to differ markedly. strains hun/ / and hun/ / were highly related to each other ( % nt and % aa identity) and to chinese castvs ( - % nt and - % aa identity) as well as to the uk strain gbr/ /lincoln ( - % nt and - % aa). strain hun/ / displayed the highest identity ( - % nt and - % aa) to three italian strains (ita/ - , ita/ - , ita/ and to the uk strain gbr/ /gillingham. strain hun/ / displayed the highest identity ( % nt and % aa) to the italian strain ita/ /zoid. strain hun/ / displayed the highest identity ( % nt and % aa) to the italian strain ita/ /zoid, while sequence identity to other canine astvs ranged from to % at the nt level and - % at the aa level. interestingly, strain hun/ / displayed the highest identity ( % nt and % aa) to a mink astv (table ) . phylogenetic trees were constructed using the nucleotide alignments of orf a, orf b and orf (fig. ) . the orf a and orf alignments were based on the full-length orf sequences and also included a selection of sequences of mammalian astv strains. the orf b alignment included nt at the end ( aa at the c terminus of the protein). in all phylogenetic trees, the majority of canine astvs formed a distinct monophyletic group. in the orf a-based tree (fig. a) , only five canine astv strains generated in this study and two astv strains from uk could be included, as information on the orf a is not available for other canine astvs. although monophyletic, the canine astvs segregated in two distinct branches, with strain hun/ / in the basal position. when enlarging the number of analysed sequences, in the orf b (fig. b) , this pattern of segregation was confirmed. four strains (hun/ / , hun/ / , hun/ / , and hun/ were grouped with the majority of canine astvs identified in european and extra-european countries, whilst strain hun/ / clustered with the highly virulent strain ita/ /zoid. interestingly, strain hun/ / was distantly related to all other canine astvs and it was grouped with astvs identified in minks and in californian sea lions. the unique evolutionary relationship of this canine strain was confirmed in the orf (capsid)-based tree (fig. c) . this gene is considered as the fundamental target for classification and characterization of astvs. when observing in detail the large monophyletic group of canine astvs which classified into species (schultz-cherry, ), it was possible to identify clearly sub-clustering patterns, with at least sub-clusters. all chi-nese strains, the uk strain gbr/ /lincoln and the hungarian strains hun/ / and hun/ / formed a unique group, with more than % nt identity in the full-length orf . a second group included three italian strains (ita/ - , ita/ - , and ita/ , the uk strain gbr/ /gillingham and strain hun/ / , with more than % nt identity to each other. a third group encompassed the virulent strain ita/ /zoid and strain hun/ / ( % nt identity to each other). other canine strains (gbr/ /braintree, gbr/ /huntingdon, hun/ were intermingled between these three main sequence groups and, as supported by sequence comparison (table ) , they clearly appeared as distinct genetic sub-lineages. astvs have been reported repeatedly in dogs across the world, although the association with the disease, prevalence rates and information on the genetic diversity are still largely unknown. in this study, we could neither confirm nor refute an association between astv infection and diarrhea in dogs. furthermore, we observed discrepancies between results obtained by the panastrovirus pcr assay and those derived from viral metagenomics. however, none of these methods were developed for routine diagnostic procedures and we do not have information about the actual sensitivity of these assays. at present it seems more appropriate to consider these methods, originally developed to detect viral diversity, as independent approaches that help understand the viral community and diversity in clinical/pathological specimens. thus, our prevalence data for astv must be interpreted with caution, even if the detection rates of astvs fell in the ranges reported in the literature. for example, canine astv rna has been detected in - % of dogs with enteritis and - % of aysmptomatic dogs in studies in italy ( (takano et al., ) , france (grellet et al., ) and uk (caddy and goodfellow, ) . even if those studies differed in the number, provenience and age of the animals enrolled in the investigations, in most studies the detection rates were found to differ significantly between symptomatic and asymptomatic dogs, suggesting a possible role of astvs as enteric pathogens of dogs. also, the onset and evolution of the clinical signs have been found to correlate with the patterns of virus shedding and with seroconversion in two dogs naturally infected by canine astvs (martella et al., ) . yet, experimental studies will be needed to demonstrate firmly the association of canine astvs with gastroenteritis. sequencing of the orf a, orf b and orf regions revealed significant sequence variation in the castvs detected in this study. five strains were grouped with other canine astvs identified worldwide, but further separated into multiple capsid (orf ) sub-clusters. by parallelism with what is observed in human astvs (mastv species ), that are classified antigenically in serotypes, it is possible to speculate that the genetic heterogeneity observed in canine astvs might account for a significant antigenic diversity (caddy and goodfellow, ) . weak antigenic cross-reactivity has been observed in immune electron microscopy between strain ita/ /bari and ita/ /zoid virus particles and the sera of convalescent dogs (martella et al., ) . however, isolation in vitro of these viruses is fastidious and only one strain, ita/ /bari, has been adapted to grow in vitro thus far (martella et al., a,b) , thus hampering a precise evaluation of the antigenic relationships among different canine astvs. interestingly, one hungarian strain, hun/ / , resembled a highly virulent canine astv, ita/ /zoid, associated with enteric disease in an adult and young dog in italy (martella et al., ) . the question whether some canine astv strains may differ in their biological properties in terms of fitness or virulence remains open. even more interestingly, one of the canine astvs identified in hungary, hun/ / , was found to be genetically distantly related from all other canine astvs identified worldwide. although we were successful to determine only the partial ( . kb) 'end of the genome, the virus displayed unique genetic signatures, such as a longer orf b/orf overlap ( nt), and a longer utr. also, this strain markedly varied from all the other canine astvs in the highly conserved promoter region for synthesis of subgenomic rna, located upstream of the orf (walter and mitchell, ; mendez et al., ) . conversely, all other canine astvs analysed in this study displayed a conserved genomic architecture, which was shared with all currently canine astvs for whom the complete or partial (orf b and orf ) genome sequence is available (martella et al., a (martella et al., ,b, takano et al., ) . in addition, upon sequence comparison and phylogenetic analysis, strain hun/ / differed markedly from other canine astvs concerning the partial orf b and the full-length orf (table ) . according to the th ictv report (http://talk.ictvonline.org/files/ictv official taxonomy updates since the th report/m/vertebrate-official/ ), for mastvs the mean aa genetic distances (p-dist) range from . to . %, and from . to . %, respectively, between and within groups (species). using our set of orf sequences, the p-dist of strain hun/ / was calculated to range between . and . . accordingly, this strain appears distantly related from any other known mammalian astv and may represent a novel astv species. it remains to be established whether strain hun/ / is actually a canine virus, or it is the result of inter-species transmission from an unidentified animal source. although astvs are regarded as viruses with a robust host-species restriction, several pieces of evidences in mammals (including humans) indicate that the host species barrier may be permeable in some occasions, and that heterologous viruses may stably adapt to the new host (finkbeiner et al., (finkbeiner et al., , nagai et al., ) . moreover, occasional transmission of astvs is thought to have occurred between avian and mammalian species in both directions (sun et al., ; pankovics et al., ) . in summary, data about canine astvs is currently scarce. nonetheless, the genetic heterogeneity among circulating astv strains is evident representing some challenge for laboratory diagnosis. this diversity may also be challenging for future prophylaxis strategies, complicating the design of future vaccines, as it is currently unknown whether antigenic cross-reaction exists among and cross-protection is elicited by different canine astv strains. further efforts are needed to better understand the biology of canine astvs and large-scale structured surveillance programs should be initiated to elucidate the relative veterinary importance of various canine astv types. the authors declare no conflicts of interest. the study was supported by the momentum program awarded by the hungarian academy of sciences. s.m. was a recipient of the jános bolyai fellowship (awarded by the hungarian academy of sciences). f.j. was supported by tÁmop ( . . .a/ - - - - ) and Únkp- - -iii, new excellence program of the ministry of human capacities. g.k. received funding from Únkp- - -iii, new excellence program of the ministry of human capacities. the funders had no role in study design, data collection, analysis and interpretation, or the decision to submit the work for publication. detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics nineteen new 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with and without diarrhoea astroviruses in dogs detection and characterization of canine astroviruses enteric disease in dogs naturally infected by a novel canine astrovirus analysis of the orf of human astroviruses reveals lineage diversification, recombination and rearrangement and provides the basis for a novel sub-classification system astroviruses characterization of human astrovirus cell entry enteric viral infections of sheltered dogs in hungary candidate new rotavirus species in sheltered dogs molecular characterisation of the '-end of the astrovirus genome full genome analysis of bovine astrovirus from fecal samples of cattle in japan: identification of possible interspecies transmission of bovine astrovirus detection of a mammalian-like astrovirus in bird, european roller (coracias garrulus) astrovirus research. essential ideas, everyday impacts, future directions detection and characterization of avastrovirus associated with diarrhea isolated from minks in china detection of canine astrovirus in dogs with diarrhea in japan mega : molecular evolutionary genetics analysis version . genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea electron microscopic demonstration of viruses in feces of dogs with diarrhea astrovirus infection in children astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups isolation and characterization of canine astrovirus in china key: cord- - qenzjiu authors: shorter, john r.; maurizio, paul l.; bell, timothy a.; shaw, ginger d.; miller, darla r.; gooch, terry j.; spence, jason s.; mcmillan, leonard; valdar, william; pardo-manuel de villena, fernando title: a diallel of the mouse collaborative cross founders reveals strong strain-specific maternal effects on litter size date: - - journal: g (bethesda) doi: . /g . . sha: doc_id: cord_uid: qenzjiu reproductive success in the eight founder strains of the collaborative cross (cc) was measured using a diallel-mating scheme. over a -month period we generated , litters, and provided , weaned pups for use in different published experiments. we identified factors that affect the average litter size in a cross by estimating the overall contribution of parent-of-origin, heterosis, inbred, and epistatic effects using a bayesian zero-truncated overdispersed poisson mixed model. the phenotypic variance of litter size has a substantial contribution ( %) from unexplained and environmental sources, but no detectable effect of seasonality. most of the explained variance was due to additive effects ( . %) and parental sex (maternal vs. paternal strain; . %), with epistasis accounting for . %. within the parental effects, the effect of the dam’s strain explained more than the sire’s strain ( . % vs. . %), and the dam’s strain effects account for . % of total variation explained. dams from strains c bl/ j and nod/shiltj increased the expected litter size by a mean of . and . pups, whereas dams from strains wsb/eij, pwk/phj, and cast/eij reduced expected litter size by a mean of . , . , and . pups. finally, there was no strong evidence for strain-specific effects on sex ratio distortion. overall, these results demonstrate that strains vary substantially in their reproductive ability depending on their genetic background, and that litter size is largely determined by dam’s strain rather than sire’s strain effects, as expected. this analysis adds to our understanding of factors that influence litter size in mammals, and also helps to explain breeding successes and failures in the extinct lines and surviving cc strains. size in wild populations. a controlled laboratory environment is therefore necessary to measure genetic effects on litter size (jinks and broadhurst ; roberts ; bandy and eisen ; hoornbeek ; peripato et al. ; gutiérrez et al. ; varona and sorensen ) . the collaborative cross (cc) and its eight founder strains are an important resource for studying complex traits, for establishing mouse models for human disease, and for understanding the mouse diversity outbred (do), which originated from the cc (ferris et al. ; chesler ; rogala et al. ; rasmussen et al. ; gralinski et al. gralinski et al. , schoenrock et al. ; maurizio et al. ) . the cc and its founder strains are also useful for studying reproductive ability due to their established record of breeding successes and failures. for example, litter size in the early cc lines shows a steady decline over the first six generations of inbreeding (philip et al. ). in addition, nearly % of all cc lines have become extinct, primarily due to subspecific genomic incompatibilities (shorter et al. ) . although standard reproductive phenotypes have been measured, mostly in male cc founders (odet et al. ) , the genetic and non-genetic control over cc breeding success has never been thoroughly characterized, and is likely to contain vital information that may be used to improve cc breeding in the future. given the growing popularity of the cc as a community resource, we investigated reproductive ability across the eight founders of the cc to better understand the genetic and non-genetic factors that affect their fertility and breeding. using an · diallel design, we measured weaned litter sizes from , litters arising from crosses across four years of breeding. adapting a recently developed statistical model of diallel effects (lenarcic et al. ) , we quantified both the genetic contributions that shape litter size and the contributions of several environmental factors. our results provide a detailed account of breeding patterns across and between the eight founders. this experiment also informs us about genetic combinations that are highly or marginally productive in the cc, helping us to better understand cc fertility problems and line extinction. the mouse inbred strains used in these experiments are the eight founder strains of the collaborative cross (cc) (collaborative cross consortium ) . the founders of the cc include five classical strains, a/j (aj), c bl/ j (b ), s /svimj ( s ), nod/shiltj (nod), nzo/ hlltj (nzo), and three wild-derived strains, cast/eij (cast), pwk/ phj (pwk), and wsb/eij (wsb). mice originated from a colony maintained by gary churchill at the jackson laboratory, and were transferred to the fpmv lab at the university of north carolina (unc) in . the original colony also produced most of the g breeders that populated the inbred funnels at ornl, tau and geniad (srivastava et al. ) . all mice described here were reared by the fpmv lab at unc. mice were bred at the unc hillsborough vivarium from - and bred at the unc genetic medicine building (gmb) vivarium from - . a total of , litters resulting from crosses between , individual dams and , individual sires were born from all eight inbred crosses and of reciprocal f hybrid crosses, excluding hybrids between nzo·cast and nzo·pwk, which are known unproductive crosses (chesler et al. ) . the directions of all crosses are described as female by male (i.e., dam.strain · sire.strain), unless otherwise noted. litter size and sex were determined at weaning by visual inspection. animals were kept on a -hour, -hour light/dark schedule with lights turned on at : am; temperature was maintained at °- °with relative humidity between - %. mice were housed in standard · -cm ventilated polysulfone cages with standard laboratory grade bed-o-cob bedding. water and purina prolab rmh were available ad libitum. mouse chow was supplemented with fenbendazole (feb ) two weeks before and two weeks after transportation to the gmb facility to eliminate possible pinworms. selamectin treatment was dropped onto the coats of mice before transfer to remove mites from the cages. these treated cages were not opened until after their arrival at unc gmb. all animal rearing and breeding was conducted in strict compliance with the guide for the care and use of laboratory animals (institute of laboratory animals resources, national research council , https://www.ncbi.nlm. nih.gov/books/nbk /). the institutional animal care and use committee of the university of north carolina approved all animal use and research described here. testing environmental interactions significant environmental interactions were determined using anova, using jmp software (jmp, version . sas institute inc., cary, nc, - . tested effects included a season effect (average weaned litter size in each month over every year), non-seasonal factors using a yearby-month effect (average weaned litter size for all months across all years), and a litter order effect (average weaned litter size in each subsequent dam litter). we performed the anova on litter size counts from the eight inbred matings only, because of their robust sample sizes throughout the four years of breeding. to estimate the overall contributions of heritable factors affecting litter size in our population, we adapted a previously published linear mixed model, bayesdiallel (lenarcic et al. ) , which performs this estimation for continuous phenotypes, to the setting of a discrete count-based phenotype. to do this we reimplemented the bayesdiallel gibbs sampler as a generalized linear mixed model (glmm) using the r software package mcmcglmm (hadfield ) . let y i be the number of pups born to litter i, where y i is a positive integer (zeros are not observed). for any categorical variable x, let the notation x½i indicate the value of x relevant to i: in particular, let h½i denote litter i's batch h ¼ ; . . . ; ; let r½i denote its parity order r ¼ ; . . . ; ; and let ðj; kÞ½i denote its parentage, defined by maternal strain j and paternal strain k, where ðj; kÞ f ; . . . ; g . the effect of parental strains on y i was modeled using an overdispersed zero-truncated poisson (ztp) regression (data scales of the model in brackets): where ztpoisðl i Þ denotes a poisson distribution with an expected value e½y i ¼ li e l i but is conditional on having observed that y i ¼ (appendix a), g is the link function gðxÞ ¼ logðxÞ, with inverse g ðxÞ ¼ e x , that relates l i to a latent scale ℓ i , and h i is a linear predictor on that latent scale with an error term e i $ nð ; s Þ providing overdispersion. the linear predictor h i is composed of the following: an intercept m; a litter (parity) order effect, modeled as the combination of a fixed effect slope, ra, and a random effect with an independent level for each litter order, i.e., order r $ nðra; t order Þ, where a is a fixed effect and t order is the variance of the random deviations around ra; a batch effect, batch h $ nð ; t batch Þ; and a linear predictor, d t ðj;kÞ b, modeling the effect of the parental strain combination ðj; kÞ. the contribution of parental strains, d t ðj;kÞ b, is equivalent to the 'fullu' (full, unsexed, 'babmvw') model described in (lenarcic et al. ) , namely, where subscripted variables a; m; b; v; w model the effects of specific strains or strain-pairs, b inbred models an overall effect, i fag is an indicator and equal to if a is true and otherwise, s fag is a sign variable equal to if a is true and otherwise. in more detail, the a ("additive") class represents strain-specific dosage effects, with a ; . . . ; a modeled as a j $ n stz ð ; t a Þ, where n stz is a normal distribution subject to the sum-to-zero constraint p j a j ¼ [using the approach of crowley et al. ( ) , appendix a]. the m ("parental sex") class represents parent-of-origin effects, modeled as m j $ n stz ð ; t m Þ, where a positive value of m j implies that strain j increases litter size more when inherited through the maternal line, with the difference between maternal and paternal being m j (since the design matrix entry for m j is coded þ for dam and for sire). the effect of being inbred is composed of an overall (fixed) effect b inbred and strainspecific (random) effects b j $ n stz ð ; t b Þ. epistatic effects are modeled as strain-pair specific deviations, with "symmetric" effects v jk $ n stz ð ; t v Þ representing the overall deviation from the rest of the model induced by the (unordered) strain combination j with k, and "asymmetric" effects w jk $ n stz ð ; t w Þ modeling a further deviation induced by differences in parent-of-origin. to provide directional parent-of-origin effects for maternal and paternal strain we defined, by reparameterization of the additive and parental sex effect, the following two additional types of effect: for example, dam b ¼ a b þ m b is the b -specific dam (maternal strain) effect and sire b ¼ a b m b is the b -specific sire (paternal strain) effect. posterior samples of these quantities were obtained as a post-processing step by reparameterizing posterior samples of a j and m j . obtaining these as a post-processing step, rather than explicit modification of equation , preserves the original bayesdiallel model and therefore has no effect on the effect estimates (or variance projection estimates) for the other diallel categories. (parameter definitions summarized in table s .) the decomposition of diallel effects in equation and its dam vs. sire reformulation have parallels in earlier diallel literature. a reduced version of the decomposition composed of additive (a) and symmetric epistasis (v) estimate, respectively, the generalized combining ability (gca) and specific combining ability (sca) described by sprague and tatum ( ) ; including also the (reciprocal) asymmetric epistasis (w) term recapitulates griffing's method , model (griffing ). the inbred penalty (b inbred , b) is comparable to dominance measures defined in, for example, hayman ( ) . the parental sex effects (m) are akin to the maternal effects of topham ( ) and the "extranuclear" effects in the "bio" model of cockerham and weir ( ) and zhu and weir ( ) , and the bio model's reparameterization to indirectly estimate dam and sire effects has been described in, for example, lynch and walsh ( ) . the estimation of dam and sire effects directly, as explicit model parameters for the diallel, was proposed by robinson ( , ) . the main differences between these earlier analyses and ours are the simultaneous inclusion of all parameter groups, the use of a bayesian random effects framework to allow all these parameters to be fitted, and the extension to modeling a non-gaussian response. priors were chosen to be minimally informative. for fixed effects (m, a, and b inbred ) we used nð ; · Þ. for variances of random effects (s , t a , t m , t b , t v , t w , t order , t batch ) we used an inverse gamma distribution with scale and shape both equal to . . posterior effect estimates are presented as posterior mean (and median), and the % highest posterior density (hpd) interval. in order to stably estimate the contribution of each variance class to the overall phenotype, we used the diallel variance projection [varp; crowley et al. ( ) ]. this is a heritability-like measure that partitions the overall phenotypic variance of an idealized future diallel experiment into additive, parent-of-origin (parental sex), inbred (dominance), epistatic, and other random/fixed effects categories in the diallel. rather than being based on estimated variance parameters (e.g., t a ; t b ; . . .), which are typically ill-informed by the data and thus both uncertain and sensitive to priors, the varp uses the estimated effects themselves, both fixed (e.g., b inbred ) and random (i.e., the best linear unbiased predictors, or blups, such as a ; a ; . . .), since these are well-informed, precise and regularized by shrinkage. the varp calculation involves ratios of sums-of-squares in similar fashion to r but for an idealized diallel that is both complete and balanced. as an r -like measure, the varp is reportable for both fixed and random effects, and includes confidence intervals arising from posterior uncertainty in those effects' values. varps were calculated both from the ztp model described above, and, for comparison, from the standard gaussian-outcome bayesdiallel model (using the bayesdiallel software) (appendix a). the standard bayesdiallel model was applied to our data after litter size was subject to a variance-stabilizing transformation, the square root, this corresponding to the linear mixed model approximation to the ztp, diallel analysis of sex ratio to model diallel effects on sex ratio we recast the bayesdiallel model as a binomial glmm. letting y i be the number of males out of a total of n i pups for litter i, we model where p i is the expected proportion of males predicted for litter i, the link function is gðxÞ ¼ logitðxÞ ¼ logðxÞ=ð xÞ, with inverse link g ðxÞ ¼ logit ðxÞ ¼ expitðxÞ ¼ e x =ð þ e x Þ, and ℓ i , which represents p i on the latent scale, is modeled using the bayesdiallel hierarchy as in equation . additional details about our statistical modeling approaches are provided in appendix a (litter size) and appendix b (sex ratio). file s contains all breeding data used for the analysis in this study. file s contains the scripts and software used for the analysis in this study. litter size is affected by housing facility but not season we bred litters in an · inbred diallel of the cc founder strains, and generated all eight inbreds and of possible reciprocal f hybrids (figure , figure s -s ). the eight inbred strains, displayed across the diagonal, were mated at higher frequencies both for maintenance of inbred strains and propagation of the diallel. for all genetic inbred and hybrid crosses, we recorded the following information: mated pairs, wean dates, litter size at weaning, including total and sex-specific counts (file s ). this diallel cross was originally designed and maintained for the generation of f mice for several experimental projects (koturbash et al. ; aylor et al. ; mathes et al. ; kelada et al. ; didion et al. ; collaborative cross consortium ; calaway et al. ; crowley et al. ; phillippi et al. ; odet et al. ; crowley et al. ; morgan et al. ; percival et al. ; shorter et al. ; oreper et al. ; maurizio et al. ) . as a result, certain reproductive measurements such as time between litters and maximum number of offspring per cross were necessarily biased by experimental breeding requirements. average weaned litter size, however, is a reproductive trait that should be well-estimated independently of these factors. we measured litter size and report the mean number of weaned pups per litter for the viable crosses in the diallel (figure ) . a wide distribution of litter sizes was observed, ranging from an average of . weaned pups for wsb·wsb crosses, to an average of . weaned pups for nod·pwk crosses, with an overall mean of . weaned pups per litter. examining average litter sizes of the inbred strains across all years, we found neglible evidence of consistent seasonal effects (f ; ¼ : p = . ) ( figure s ) but positive evidence of non-seasonal patterns: a modeled 'year.month' covariate significantly affected litter size (f ; ¼ : p , . ; see also figure s ). the non-seasonal effect could be driven by the relocation of mice between vivariums, which occurred in february . the housing facility may have an impact on litter size as well. to test this, we measured average litter size differences between the two housing facilities and found that two founder strains, s and wsb, had significantly larger average litter sizes at the hillsborough facility than at unc gmb with a difference of . to . weaned pups per litter (p , . ) for s , and . to . weaned pups per litter (p = . ) for wsb. the six other founder strains did not significantly differ in their average litter sizes between the two facilities, but tended to have smaller weaned litters at unc gmb (see discussion). for average litter size across the diallel, we tested for an effect of litter number (birth parity number, for a given mating pair) on the number of pups weaned in each litter. previous research suggests that the first litter can be significantly smaller than subsequent litters due to various biological factors (de la fuente and san primitivo ) . this effect was consistent in the diallel: we observed that there was significant reduction in litter size in the first litter (p , . ) compared to the overall linear effect that parity has on reducing litter size ( figure s ). litter size is moderately heritable, and maternal effects account for the majority of explained variation to estimate founder strain effects on litter size, we used a bayesian regression model that decomposes the phenotypic variation in the diallel into genetic and parent-of-origin contributions (lenarcic et al. ). using this model, the percentage of the variance in litter size explained by diallel effects was . %, with additive effects explaining . % (varp[additive]; this gca-like measure being related to narrow sense heritability), parent-of-origin effects (varp[parental.sex]) accounting for . %, the fact of being inbred (varp[inbred.overall]) at . %, and strain-by-strain interactions (varp[epistatic.symmetric] + varp[asymmetric.epistatic]) at . % (figure a) . in more detail, we present estimates for all modeled diallel effects as posterior means and highest posterior density (hpd) intervals ( figure b ). parameters are divided into two groups: general effects and strain pair-specific effects. general effects comprise strain-specific additive effects (additive), strain-specific and overall inbred (inbred), and strain-specific parent-of-origin (parental sex) effects. strain pair-specific (epistatic) effects are the effects that arise specifically in crosses of two heterologous strains, with 'v' referring to symmetric epistatic and 'w' referring to asymmetric epistatic effects (pairwise parent-of-origin effects). under the general effects, we see significant positive additive effects on average litter size from b , nod, and nzo and significant negative additive effects on litter size from cast, pwk, and wsb strain dosages. a similar pattern is seen in the parental sex effects, where b and nod dosages have a significant positive effect on average litter size, whereas cast, pwk, and wsb have significant negative effects. the overall "inbred" effect is negative, indicating that inbred status decreases figure diallel crossing scheme and weaned pup distribution. the number of litters observed per cross is given by the integers, with the largest sample sizes, along the diagonal, corresponding to the production of inbred parental strains. column and row sums are given along the bottom row and rightmost column, respectively. a total of , litters were evaluated for this analysis, resulting in a total of , weaned pups. the shading within each box corresponds to the average number of weaned pups per litter in each cross, with averages ranging from . to . pups per litter. litters for which no pups survived until weaning were not included in our analysis. the symbol "·" is used to indicate incompatible crosses that do not produce any litters. average litter size, regardless of parental strain. each strain also has an individual inbred effect in addition to the overall inbred effect. when the individual inbred effects are taken into account with the overall "inbred" effect, inbred litters are on average slightly smaller than their heterozygous counterparts. for strain pair-specific epistatic effects, there are a few marginally noteworthy effects, with the most prominent being a negative asymmetric epistatic effect for pwk·nod. to more clearly differentiate the contributions of the mother vs. the father strain, we reparameterized the bayesdiallel model to capture effects specific to dam.strain and sire.strain (figure ). b and nod dams increase litter sizes by more than . fold, by an average of . and . pups, respectively, regardless of sire. cast, pwk, and wsb dams tend to decrease average litter size by . , . , and . pups. the sire effect is similar, with nod and nzo sires having larger litters and cast sires producing smaller litters. as expected, we see that the dam.strain has a much larger influence on the variation of litter size compared with the sire.strain ( . % vs. . %). we examined genetic and non-genetic effects on the average sex ratio per litter and found no evidence that sex ratio was skewed ( figure s ; figure s ). the overall mean for sex bias, quantified as number of male pups weaned divided by the total number of weaned pups, was . , and did not significantly differ from our expectation of a : sex ratio (binominal test, two-tailed p = . ). for the eight inbred founders, s is the only strain that departed significantly from expectation, with slight reduction of males . : . (binominal test, two-tailed p = . ). however, correction for multiple testing shows no significant sex ratio bias of any inbred strain. the outbred crosses have substantially fewer litters and offspring than the inbred matings, leading to less balanced sex ratios; however, when multiple testing is accounted for, they also show no significant deviations from expected sex ratios. we have investigated factors influencing litter size in the eight cc founders and their f hybrids using a new extension of the bayesdiallel model. we note that litter size is a component of reproductive performance, but distinct from total strain productivity; a study on total strain productivity would need to take into account litter size, numbers of litters, maximum reproductive age, and pup survival until breeding. our results illustrate how mammalian litter size in a full diallel design is influenced by genotypic and environmental variation. these results present new information on cc founder strains' reproductive performance, show that maternal effects and the environment play a large role in litter size variation, and provide no evidence for seasonality effects on litter size in a controlled animal facility. the results also address some of the factors that contributed to line extinction and breeding problems in generating the cc (chesler et al. ; philip et al. ; collaborative cross consortium ; shorter et al. ) . we estimated that genetic (additive, inbred, and epistatic) effects on average litter size explained . % of variation, suggesting that most of the phenotypic variation arises from unexplained environmental effects. compared with the overall average litter, we observed substantial positive effects of b and nod strains, from both additive genetic and parent-of-origin parameters, and substantial negative effects of pwk and wsb (figure ) . we also observed, as expected, that lower litter size was associated with being inbred (figure a ). these estimates are likely driven by the unique selection history of these inbred lines, and comparisons should be limited to these eight founder strains, and the cc and do populations. during the g and g out-crossing generations of the cc, mean litter size was lower for crosses involving wild-derived strains, cast, pwk and wsb (philip et al. ) . a similar pattern is observed here, but these effects are determined to be specifically through the maternal strain, with cast, pwk and wsb having negative "dam" effects on strain-specific additive, parental sex, and inbred effects, and (right) epistatic effects between each pairwise cross. for each parameter, thin and thick horizontal lines represent % and % highest posterior density (hpd) intervals of effects, respectively, and vertical break and dash give posterior median and mean, respectively. the effects are in relation to an overall mean litter size of . ( % hpd: . - . ). the gray vertical lines indicate zero. effects are shown as the log, or latent, scale effects on the mean litter size attributable to each strain or strainpair and inheritance group, where values are centered at for each random effect class. intervals that exclude zero have non-negligible effects on the mean litter size. labels with "v" or "w" refer to symmetric or asymmetric epistatic effects, respectively. colored bars indicate corresponding variance classes in (a) and (b). litter size (figure ). it is likely that selection pressure in classical lab strains is associated with larger litters compared with the wild-derived strains. additionally, two of the wild-derived strains, cast and pwk, are from a different subspecific origin than the other six cc founders. this likely contributes to decreased productivity through subspecific incompatibilities (shorter et al. ) . we identify and report environmental factors that may influence litter size. the breeding of this diallel was performed across two different vivariums over the course of years, and we see a significant effect from housing facility. the hillsborough facility was associated with larger litters for all strains, especially s and wsb. the two facilities have many different factors that could explain these differences. the hillsborough facility housed multiple species, including dogs and mice, had smaller rooms that held approximately mouse cages, was remotely located in a rural area, had different laboratory personnel, had cage changes once a week, and was supplied with filtered well water. the diallel breeding at the gmb facility took place in a large central room, contained only laboratory mice, is located in a basement of a large seven story research building on campus, has cage changes every other week, and is supplied with filtered city water. it is possible that one or more of these factors, independently or in combination, affected productivity. another finding was that seasonality, which has previously been shown to influence litter size and frequency of litters in mammals (drickamer ) , did not seem to significantly impact litter size in this study. this is likely due to consistent lightdark cycles and temperatures as well as a steady diet. we did observe a significant effect on litter size after the transfer of the mice to the unc gmb facility (february ), which reduced overall litter sizes from march to june . this may have been due to the use of fenbendazole during the time of the transfer. other factors, such as the sex of the laboratory personnel interacting with the animals, are generally known to influence rodent behavior and could contribute to some of the environmentally-induced variation we observed (sorge et al. ) . last, we measured sex ratio across all inbred and outbred crosses. despite some departures from equality at the nominal significance threshold (alpha = . ), no associations with founder strain dosage remain significant after correction for multiple testing. recent work has suggested a potential for bias in sex ratio driven by the male germline in mus musculus (conway et al. ; macholán et al. ; cocquet et al. ; ellis et al. ; cocquet et al. ; turner et al. ; larson et al. ) , particularly in inter-subspecific hybrids that are mismatched for copy number of x-and y-linked genes expressed in postmeiotic spermatids. although there are no previous observations that suggest a bias in sex ratio in the cc or its founders, it remains an important characteristic to measure in a study on reproductive productivity. to estimate heritable effects on liter size and sex ratio we extended the original bayesdiallel model of lenarcic et al. ( ) in two new ways. first, to better understand and distinguish the effects arising from female and male parents, we reparameterized our strain-specific additive and parental-sex effects such that we could provide estimates of maternal strain and paternal strain effects separately. in the original bayesdiallel model, maternal and paternal strain effects are split into "additive" effects, which consolidates the effects they have in common, and "parental sex", which models any remaining deviation between the two. recognizing that additive effects from the sire are essentially wiped out by the additive effects, we instead collapsed the additive and parental-sex effects into "dam.strain" and "sire.strain" effects in a postprocessing step on the posterior output. this allowed us to run the original bayesdiallel model while also viewing our data from the perspective of dam strain and sire strain contributions. second, we reimplemented the original mcmc sampler, designed for modeling a continuous outcome variable, in a general package mcmcglmm (hadfield ) in order to model count and binary responses. litter size, as measured, is most naturally distributed as poisson, with zero-truncation owing to the fact that only successful litters were recorded. sex ratio is most naturally modeled as a binomial, with an underlying (male) proportion between and . although it would be possible to obtain an approximate analysis by transformations to normality using the original bayesdiallel (and we do this for litter size for some otherwise hard-to-obtain quantities), we found such approximations to be inadequate for reliable estimation of higher order effects in the case of litter size and deeply flawed in the case of sex ratio. although there is a computational cost, and added complexity to determining variance contributions, this new implementation achieves several objectives: ) we no longer break the assumptions in the original model regarding normally distributed errors; ) we easily accommodate overdispersion in our data; and ) we can select from a large number of glmms models that more closely resemble the forms of our data observations. in addition, we believe this flexibility will be appealing to many other researchers who would like to model non-gaussian distributed phenotypes using diallel designs, and we have provided the code in an r package litterdiallel (https://doi.org/ . /zenodo. ). overall, these results have implications for other avenues of future research. future multiparental research populations should test for strain incompatibilities, reproductive phenotyping, and other health traits in a full diallel before the recombinant inbreeding begins (odet et al. ) . these future research populations should also use non-related wild-derived individuals from the same subspecific origin in order to increase genetic diversity without introducing hybrid incompatibilities. this work was supported in part by the following grants from the national institutes of health: p gm , p hg / p mh , r hd (fp-mv), t hd (jrs), t ai (plm), r gm (plm, wv), and r gm figure dam.strain and sire.strain variance contributions and estimates of effects on weaned litter size. these effects are a reparameterization of additive and parental.sex effects from the previous analysis. estimates for the maternal ("dam.strain") and paternal ("sire.strain") effects on litter size, as calculated from the additive and parental sex parameters in figure , with hpd intervals defined correspondingly. (wv). the collaborative cross project is also supported by the university cancer research funds granted to lineberger comprehensive cancer center (mcr ccri). we collected data on litter size at weaning for genetic crosses of inbred lines, across four years of breeding. we use zero-truncated poisson (ztp) regression for modeling our data. this type of regression is explicit in its framework accounting for discrete observations, flexible in its ability to use linear mixed models on the latent scale, and allows for parameterization of excess variance observed, in a way that standard poisson regression does not. we account for the zero depletion in our data by using ztp regression instead of standard poisson regression, since we exclude observations of birth cohorts where no pups survived to weaning. in figure , the distribution of the observed data ðlitter sizeÞ is displayed for the wsb·wsb inbred mating, along with simulated data from a zero-truncated poisson distribution based on the data mean. for the ztp, the first two moments (mean and variance), for values y i . , are given by: the density for the ztp, for every count x ; ; :::, is given by: the relationship between the latent, expected, and data scales of the ztp regression model are illustrated by the toy example shown in figure . ztp frequencies were calculated using the r package countreg (zeileis and kleiber ) . diallel effect estimates are obtained using an mcmcglmm (hadfield ) implementation of bayesdiallel (bayesdiallel-glmm, henceforth), with : · iterations, : · iterations of burn-in, and thinning by (saving only every / th iteration), to obtain independent samples with minimal autocorrelation, and plotted as highest posterior densities (hpds) in the results. the effects estimated from the bayesdiallel-glmm model are transformed from the latent scale to the (expected) data scale via the inverse link function, i.e. expða aj Þ, for interpretability of effects on the original data scale. to avoid the problem of interpretability in transforming variance parameter (and variance projection) estimates from the latent to the observed data scales, we instead calculate and report variance projections, as calculated using the gaussian version of bayesdiallel. in order to account for heteroscedasticity (unequal variance) of the model residuals that arises from the approximately ztpoisson nature of the data, we use a variancestabilizing transformation (vst) (yu, ) and run bayesdiallel again (gaussian) to obtain variance projections on the modeled data. the varps that are calculated from these parameter estimates are an approximation of the variance contributions that we would observe in the glmm bayesdiallel model. we model the male pup counts and female pup counts jointly, using the bayesdiallel linear model, formulated for binomial glmm regression. this model directly considers genetic effects on the imbalance in male vs. female pups by parameterizing the number of males and number of total pups (or the total, and the fraction of males in the total). the model is elaborated in the methods section of the main manuscript. the proportion of weaned pups that are male, or equivalently, the proportion of weaned pups that are female, is approximated by a binomial distribution. in our data, the mean and the variance of male ratio are . and . , respectively. to generate the upper and lower % boundaries, as shown in figure s , for the expected phenotype under the null hypothesis of male pup proportion = . , we used the qbinom function in the stats package in r. to generate the upper and lower % boundaries, as shown in figure s , for the expected phenotype under the null hypothesis of male pup proportion = . , we used the qbinom function in the stats package in r. genetic improvement of litter size in pigs genetic analysis of complex traits in the emerging collaborative cross prenatal and postnatal effects in mouse lines selected for body weight and litter size: performance of postnatal dams and growth of progeny on the fitness functions relating parental care to reproductive value mouse phenome database: an integrative database and analysis suite for curated empirical phenotype data from laboratory mice genetic architecture of skewed x inactivation in the laboratory mouse out of the bottleneck: the diversity outcross and collaborative cross mouse populations in behavioral genetics research the collaborative cross at oak ridge national laboratory: developing a powerful resource for systems genetics quadratic analyses of reciprocal crosses a genetic basis for a postmeiotic x vs. y chromosome intragenomic conflict in the mouse the multicopy gene sly represses the sex chromosomes in the male mouse germline after meiosis the genome architecture of the collaborative cross mouse genetic reference population the components of genetic variance in populations of biparental progenies and their use in estimating the average degree of dominance estimation of average 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collaborative cross epigenetic mechanisms of mouse interstrain variability in genotoxicity of the environmental toxicant , -butadiene the composite regulatory basis of the large x-effect in mouse speciation a general bayesian approach to analyzing diallel crosses of inbred strains genetics and analysis of quantitative traits genetic conflict outweighs heterogametic incompatibility in the mouse hybrid zone? architecture of energy balance traits in emerging lines of the collaborative cross bayesian diallel analysis reveals mx -dependent and mx -independent effects on response to influenza a virus in mice genetic architecture of fitness and nonfitness traits: empirical patterns and development of ideas the mouse universal genotyping array: from substrains to subspecies natural selection and the heritability of fitness components the founder strains of the collaborative cross express a complex combination of advantageous and deleterious traits for male reproduction inbred strain variant 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the optimal balance between size and number of offspring olfactory exposure to males, including men, causes stress and related analgesia in rodents general vs. specific combining ability in single crosses of corn genomes of the mouse collaborative cross selection, structure and the heritability of behaviour diallel analysis involving maternal and maternal interaction effects parent-offspring conflict reduced male fertility is common but highly variable in form and severity in a natural house mouse hybrid zone joint analysis of binomial and continuous traits with a recursive model: a case study using mortality and litter size of pigs variance stabilizing transformations of poisson, binomial and negative binomial distributions countreg: count data regression mixed model approaches for diallel analysis based on a bio-model key: cord- - lt h authors: jarvis, matthew c.; lam, ham ching; zhang, yan; wang, leyi; hesse, richard a.; hause, ben m.; vlasova, anastasia; wang, qiuhong; zhang, jianqiang; nelson, martha i.; murtaugh, michael p.; marthaler, douglas title: genomic and evolutionary inferences between american and global strains of porcine epidemic diarrhea virus date: - - journal: prev vet med doi: . /j.prevetmed. . . sha: doc_id: cord_uid: lt h porcine epidemic diarrhea virus (pedv) has caused severe economic losses both recently in the united states (us) and historically throughout europe and asia. traditionally, analysis of the spike gene has been used to determine phylogenetic relationships between pedv strains. we determined the complete genomes of pedv field samples from us swine and analyzed the data in conjunction with complete genome sequences available from genbank (n = ) to determine the most variable genomic areas. our results indicate high levels of variation within the orf and spike regions while the c-terminal domains of structural genes were highly conserved. analysis of the receptor binding domains in the spike gene revealed a limited number of amino acid substitutions in us strains compared to asian strains. phylogenetic analysis of the complete genome sequence data revealed high rates of recombination, resulting in differing evolutionary patterns in phylogenies inferred for the spike region versus whole genomes. these finding suggest that significant genetic events outside of the spike region have contributed to the evolution of pedv. porcine epidemic diarrhea virus (pedv) causes diarrhea, vomiting, and dehydration, leading to high mortality (up to %) in suckling piglets. pedv was first discovered in the united kingdom in , and later was found in belgium, hungary, france, italy, and the czech republic (chasey and cartwright, ; fan et al., ) . in , pedv was first reported in china, and proceeded to spread throughout asia (cui, ; song and park, ) . in late , a "variant" pedv strain with increased pathogenesis compared to the pedv is a single-stranded, positive sense rna virus belonging to the family coronaviridae, genus alphacoronavirus. the pedv genome is approximately kb in length and roughly two-thirds of the genome consists of open reading frame (orf) , which encodes non-structural proteins (nsps) (lai et al., ) . these nsps play important roles in viral replication, post-translational processing, and immune evasion (lai et al., ) . the virus produces various structural proteins, including spike, membrane, and nucleocapsid (lai et al., ) . the spike protein is crucial to cell attachment and infection, and the envelope is an integral membrane protein, aiding in membrane fusion while the nucleocapsid protein is necessary for genomic packaging (hagemeijer and de haan, ) . in addition, the pedv genome includes orf , located between the spike and membrane genes, that encodes an ion channel protein possibly associated with pedv pathogenesis (park et al., ; wang et al., ) . researchers have explored various regions of the coronavirus (cov) genome to link specific areas with virulence and host cell attachment. for example, the spike gene codes for a viral attachment protein that can be divided into the s ( - aa) and s ( - aa) regions (song and park, ) . comparative analysis of transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), and murine hepatitis virus (mhv) revealed two main antigenic sites in the s region: the n-terminal domain (ntd) and the c-terminal receptor binding domain (rbd) (li et al., ) . while both domains can influence virus infectivity, such as in tgev, one domain tends to be central to a cov's tropism: the ntd is important for mhv tropism, and the rbd is central to pedv infectivity and virulence (reguera et al., ) . the ntd can bind to various sialic acids on the host cell surface (reguera et al., ) . the rbd contains residues that bind to the porcine aminopeptidase-n (papn), the host receptor utilized by tgev and pedv (delmas et al., ) . since the last large-scale north american pedv outbreak ended in the spring of , the complete genomes of pedv strains from the us were sequenced and analyzed to further understand the origin and phylogenetic relationships among the american and global pedv strains. in-depth nucleotide and amino acid analysis was conducted to identify genes of high diversity. bayesian analysis was performed to understand the evolution of pedv and the emergence of different clades within us strains. in addition, the rbd was modeled to visualize the differences between american and asian strains to better understand how changes in the rbd might affect vaccine efficacy and development. samples were routinely submitted to the university of minnesota veterinary diagnostic laboratory (umvdl) for pathogen detection. between january and december samples were screened for pedv by real time rt-pcr . samples for complete genome sequencing were selected based on the criteria of a high viral concentration from the rt-pcr results and geographical diversity within the us. a total of samples, including fecal (n = ), intestinal homogenate (n = ), fecal swab (n = ), oral fluid (n = ), feedback (n = ), and environmental (n = ) samples were selected for complete genome sequencing using next generation sequencing (ngs) techniques as previously described (genbank numbers kr -kr , kr -kr ) (marthaler et al., ; marthaler et al., ) . whole genomic pedv sequences obtained using ngs techniques were also generously supplied from iowa state university (n = , genbank numbers km -km ) and the ohio department of agriculture (n = , genbank numbers kp -kp ), using previously described methods chen et al., ) . using the complete pedv genome sequences from this study (n = ) and the available pedv sequences from genbank (n = ), two nucleotide alignments were created and analyzed to determine the phylogenetic relationships between american and global pedv sequences: the concatenation of all orfs (orf , s, orf , envelope, membrane, and nucleocapsid), and a s alignment. vaccine and cell-passaged strains were excluded from the analysis (table s ). nucleotide and amino acid entropy analyses were performed using the matlab software (matlab v . and statistics toolbox v . , the mathworks, inc., natick, ma, usa). threshold values were determined using previously published methods (shannon, ; litwin and jores, ) . recombination analysis was performed using the recombination detection program (rdp) v , which uses multiple detection algorithms, including the rdp method, genecov, and maxchi, to check for the presence of recombinant sequences in the sequence dataset (martin et al., ) . window size was set to bp. breakpoints, the presence of major/minor donor sequences, and confidence intervals were used to determine regions that required excision from the alignment, or if entire sequences needed to be removed from the analysis due to multiple recombination events within the sequence. recombinant sequences were removed only prior to the bayesian analysis, but remained in the alignments for all entropy analysis and molecular modeling. bayesian markov chain monte carlo (mcmc) approach using beast v . . , with a relaxed molecular clock and bayesian skyline population (bsp) prior, with a general-time reversible nucleotide substitution and gamma distributed among-site rate variation was used to infer time-scaled phylogeny (drummond et al., , , drummond and rambaut, minin et al., ; drummond and suchard, ) . the mcmc chain was run for million generations, with sub-sampling every , iterations. a maximum clade credibility (mcc) tree was created by discarding the initial % of the chains and summarized in treeannotator (v. . . ). key nodes were identified using figtree (v. . . ) to determine time to most recent common ancestor (tmrca). the putative papn receptor-binding residues were analyzed to determine residue trends between classical and pandemic strains (reguera et al., ) . the c-terminal rbd within the s region of the spike gene was modeled using the open-source modeling server swiss-model provided by the swiss institute of bioinformatics (biasini et al., ) . predicted tertiary structure of the pedv papn rbd was modeled using prcv as a template since a pedv template was not available. spike monomer and trimer models were developed using a theoretical sars-cov model as a template (bernini et al., ) . illustrations were created using the open-source javabased molecular viewer jmol (herraez, ) and the python-based molecular viewer pymol (the pymol molecular graphics system, version . . schrödinger, llc.). , , , , , , , , , , , , , , , , , , , , , , , the pedv nucleotide sequences ranged from , to , bases in length. two pedv genomes from our study had insertions or deletions. ohio had a -nt insertion between positions , and , in the spike gene while minnesota with a -nt deletion from positions , to , in the utr compared to the original us strain, usa/colorado/ . entropy analysis was conducted with whole nucleotide and amino acid sequences, containing the concatenated orfs excluding and utrs. entropy values greater than . and . were considered highly variable for the nucleotide and amino acid alignments, respectively, based on the level of diversity in the dataset and previously determined entropy values (litwin and jores, ) . within the nucleotide alignment, of the pedv regions lacked positions with entropy levels above . (nsp , nsp -nsp , nsp , nsp , nsp , s , orf , envelope, membrane, and nucleocapsid) while regions had entropy levels above . (nsp , nsp , nsp , nsp , and s ) (fig. ) . the nsp and were the most divergent regions containing and diverse nucleotide positions, respectively (table ) . interestingly, the nsp gene contained diverse nucleotide positions, which were absent in the amino acid sequence (fig. a) . inversely, high amino acid diversity was observed in the nsp , nsp , nsp , and s genes, which were absent in the nucleotide alignment (fig. b) . higher entropy levels were present in the nsp , nsp , and s regions in both the nucleotide and amino acid alignments. overall, the orf a entropy levels were higher compared to orf b in the amino acid analysis. of the structural genes, the s gene had the highest entropy levels compared to the envelope, membrane, and nucleocapsid genes. recombination was detected in main areas of the concatenated full genome, including the nsp , nsp , nsp - , s domain, and nucleocapsid gene ( fig. a) . in these areas, recombination was present in the majority of the sequences, so the entire region was excised from the alignment prior to bayesian analysis. in addition, sequences ( from asia, from the americas) were omitted from the bayesian analysis due to evidence of widespread recombination throughout the genome (table s ). for example, the pandemic sequence minnesota contained a recombinant region with the characteristic s-indel deletions and insertions in the s domain, indicating a recombinant event occurred between an s-indel strain and a non s-indel pathogenic strain in the us (fig. b) . a maximum clade credibility (mcc) phylogeny was inferred for both the concatenated genomic sequences excluding the recombinant regions ( , nt) and the spike s gene ( nt). the analysis was run independently twice until convergence was reached, with high agreement between the two runs. in the concatenated alignment tree, the classical and pandemic asian strains were positioned as basal to the us strains, consistent with an asian origin for the us outbreak (fig. ) . importantly, the concatenated alignment tree suggests that the us epidemic may have resulted from two independent pedv introductions into the us, including minor and major clade of viruses. the minor clade contained the american and european s-indels, and a small subclade of non s-indel sequences from the ohio, including ohio / , pc a/ , and oh / . the major clade of us pedv strains was supported by high posterior probability ( %) and appears to have diverged further into two highly supported sublineages ( % and % posterior probability). the phylogeny is consistent with multiple incursions of the major clade of us pedv viruses into mexico, canada, and south korea. the minor clade includes sequences from late to early that are localized to the midwestern and eastern us regions. the estimated tmrca of the minor clade of us pedv strains is july - , and the estimated tmrca of the major clade of us pedv strains is september -august . the estimated evolutionary rate for the complete genome (excluding recombinant regions and sequences) is . × − substitutions/site/year ( . × − - . × − , % highest posterior density (hpd)). the rate estimate for the us strains is slightly higher, but not significantly: . × − substitutions/site/year ( . × − - . × − , % hpd). the spike tree illustrates the evolutionary relationship between the classical strains and the s-indels, which suggests a classical origin for the s-indel genotype (fig. ) . the pathogenic strains form a highly diverged major clade (fig. b) , which braches into large american clades. in addition, the bayesian analysis of the spike gene might suggest separate introductions of pedv into the us. the evolutionary rate for the s gene is . × − substitutions/site/year ( . × − - . × − , % hpd). considering the high entropy levels in the spike gene and the evolutionary rate determined from the s bayesian spike tree, the rbd within the s was further examined. the s-indel and classical pedv strains shared similar amino acid substitutions, specifically in the ntd of the s region (fig. s ) . furthermore, the pandemic pedv strains from china had an increased number of substitutions within the s domain when compared to the american strains due to the longer circulation time in china. compared to the attenuated vaccine strain dr , of ( %) american strains and of ( %) asian strains had at least one amino acid substitution in the papn rbd ( table ). the majority of the american strains (n = ) did not represent any amino acid differences in the papn rbd. in this region, positions in the american strains had amino acid differences compared to the vaccine strain dr (fig. a) . the most common substitutions were in the fourth region of the papn rbd at positions e d (n = ) and g d (n = ), which were substituted with aspartic acid. more substitutions (n = ) occurred in the papn rbd of the asian strains (table ) , with the most common substitutions at position h l (n = ). the rbd regions were three-dimensionally modeled to illustrate the and amino acid positions at which substitutions occurred in the north american and asian strains, respectively. the modeling of the spike protein suggests that the papn rbd residues cluster around the inner pore created by the trimer molecule, while the ntd is oriented around the outer surface of the s domain ( fig. b through f). genomic analysis depends critically on complete sequence data to conduct accurate research on phylogeny, evolution, and gene regulation. in the past, it was more economically and time effective to sequence smaller pieces of a genome and develop evolutionary conclusions from these relatively small genomic pieces. however, without full genomic sequences, it is impossible to compare variations within a genome to determine selective pressures on specific genes or regions. because of ngs technology, tools like site-specific entropy analysis can be used to examine variability throughout the genome of many pedv sequences. sun and collaborators reported four regions of diversity within the pedv genome, including v in the nsp and nsp , v in the s , v in the s and orf , and v in the nucleocapsid (sun et al., ) . while our results support the nucleotide variance in the nsp , nsp , and spike genes, high levels of diversity were not present in the s , orf , and the nucleocapsid. this could be due to the omission of pedv isolates in our analysis, as well as the comparatively large number of new us sequences in our dataset. our variance results may more accurately represent variance within american pedv strains, and underrepresent variance within chinese strains. the diversity in the s region is comprehensible since it is under strong immunological pressure while the s region is more conserved throughout covs (aydin et al., ) . the functions of nsp and nsp remain relatively ambiguous. despite being involved in viral growth and propagation, nsp is dispensable for viral replication because cov strains can replicate in absence of the nsp (graham et al., ) . the function of nsp may be related to innate immune evasion since it encodes proteases that facilitate proteasome degradation, changes in intracellular destination, signaling, protein interactivity, and host type i interferon (ifn) antagonist activities (xing et al., ) . due to the multifaceted nature of nsp , other nsp regions could produce proteins with novel effects not yet understood that mediate the virulence of cov species. acquired nucleotide differences throughout the nsp and nsp regions could contribute to the evasion of host immunity. thus, future research should focus on the functionality and importance of all pedv genes to further understand cov pathogenesis. recombination plays a pivotal role in the evolution of covs by creating new strains with altered virulence. the minnesota strain originated from a recombination even between an s-indel and a us pandemic strain, which has been associated with altered pathogenesis. while recombination may occur more often during an epidemic, recombination events occurred in most of the asian strains. recombination events can affect the phylogenetic analy-sis because different regions of the genome may have different evolutionary histories (spade et al., ) . our recombination analysis resulted in a significant portion of the complete genome being removed prior to more detailed phylogenetic analysis. at this time, the beast program cannot accommodate genetic data that includes recombined regions. our analysis supported an asian origin for the us outbreak while the inference is biased by the lack of background sequences from other regions. although over genomes were from the us, interpretation of the evolutionary and spatial his- tory of this data is limited by the lack of genomic pedv data from other regions, including europe and asia. us strains had a higher evolutionary rate compared to the global strains, but the bayesian skyline plot did not show any significant increase in evolutionary rate, possibly due to the lack of temporal sampling in the us dataset. the evolutionary rate for the spike gene was higher compared to the rest of the genome, reflecting greater selective pressure. the overall evolutionary rate of pedv ( . × − substitutions/site/year) is similar to that of tgev and wild animal covs ( . × − substitutions/site/year, . × − - . × − % hpd), but lower than that of sars-cov ( . × − nucleotide substitutions/site/year), except during the time of the us pedv epidemic ( . × − sub-stitutions/site/year) (song et al., ; vijaykrishna et al., ) . surprisingly, pandemic strains were positioned within the s-indel clade. possibly, a pandemic and s-indel strain were introduced into the americas, and a recombination event occurred in the ntd that removed the characteristic insertions and deletions of an s-indel strain, as indicated in the minnesota sequence. the relationship between the us minor clade and the recent pedv strains from europe is less clear. while these viral populations are closely related (posterior probability of %), the direction of transmission is unclear at this time. additional sequences from europe might help to resolve the origins of these recent european pedv cases. (e) a monomer model of the pedv spike protein, with the c-terminal rbd represented in green, dark blue represents the s region, light blue represents the s region, and yellow represents the n-terminal rbd. (f) a theoretical tertiary structure model of the pedv spike protein. blue represents the s region, with the specific n-and c-terminal rbds highlighted in yellow and green, respectively. the papn-rbd is shown in violet.(for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) examining the spike gene can reveal interesting conclusions about the papn rbd. while the ntd spans a larger region of the s domain, it has not been directly linked to pedv tropism and functionality, as in tgev and mhv (reguera et al., ) . the korean attenuated vaccine strain and the us strains share similar residues in the papn rbd, with numerous differences compared to the older classical strains, suggesting this vaccine may protect against the american strains. however, developing a consistently and longitudinally efficacious vaccine may prove challenging, considering the high evolutionary rate of the s region. failures in the development of an efficacious vaccine have been reported, further supporting the difficulty in generating vaccines for pedv . despite some uncertainties in vaccine efficacy, a recent study demonstrated that prior exposure of sows to the s-indel strain provided a level of protective immunity when their piglets were challenged with the more virulent original us pedv strain, which is probably due to conservation within the c-terminal region of the viral genome (goede et al., ) . while the exact functionality of all the genes of pedv and other covs is unknown, adding the complete genomes of diverse strains to the global database promotes better understanding of evolutionary and phylogenetic relationships. multiple regions within the genome are variable, and recombination is common between pedv strains. despite excising a large portion of the genome prior to analysis, the bayesian trees illustrate two distinct entries of pedv into the us and characterize the evolution of pedv compared to other covs. 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insights into the ecology of coronaviruses distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase this study was supported partially by the rapid agricultural response fund, established by the minnesota legislature and administered by the university of minnesota agricultural experiment station, and by boehringer ingelheim vetmedica, inc.the authors thank the faculty and personal at the umvdl for their technical services. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.prevetmed. . . . key: cord- -ttb o lv authors: choi, jeong-won; jung, ji-youl; lee, jae-il; lee, kyoung-ki; oem, jae-ku title: molecular characteristics of a novel strain of canine minute virus associated with hepatitis in a dog date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: ttb o lv a -year-old female yorkshire terrier dog died a few days following hernia and ovariohysterectomy surgeries. necropsy performed on the dog revealed that the surgeries were not the cause of death; however, degenerative viral hepatitis, showing intranuclear inclusion bodies in hepatic cells, was observed in histopathologic examination. several diagnostic methods were used to screen for the cause of disease, and minute virus of canines (mvc) was detected in all parenchymal organs, including the liver. other pathogens that may cause degenerative viral hepatitis were not found. infection with mvc was confirmed by in situ hybridization, which revealed the presence of mvc nucleic acid in the liver tissue of the dog. through sequencing and phylogenetic analysis of the nearly complete genome sequence, the strain was found to be distinct from other previously reported mvc strains. these results indicate that this novel mvc strain might be related to degenerative viral hepatitis in dogs. viruses the family parvoviridae are currently categorized into two subfamilies: densovirinae and parvovirinae, members of which infect in-vertebrate and vertebrate hosts, respectively [ , ] . the subfamily parvovirinae is further divided into eight genera: amdoparvovirus, aveparvovirus, bocaparvovirus, copiparvovirus, dependoparvovirus, erythroparvovirus, protoparvovirus, and tetraparvovirus [ ] . the genus bocaparvovirus contains small, non-enveloped, autonomously replicating, single-stranded dna viruses with an icosahedral capsid. bocaparvoviruses are unique among parvoviruses, as they contain a third open reading frame (orf) between the regions encoding the non-structural and structural proteins, and they have a genome length of approximately . kb. the genus was originally named according to its initial two members, bovine parvovirus (bpv) and minute virus of canines (mvc) [ , , , ] . minute virus of canines, also known as canine minute virus or canine parvovirus type , is an autonomous parvovirus of dogs that is genetically and antigenically unrelated to canine parvovirus type , which belongs to a separate genus in the family parvoviridae and is a common agent of canine hemorrhagic gastroenteritis [ ] . mvc was first discovered in germany in in feces samples of clinically healthy dogs [ , ] . initially, mvc was not thought to cause disease; however, several studies, including experimental infections, revealed its role as a pathogen in newborn pups and canine fetuses [ , ] . recently, mvc was reported to be associated with neurological disease in dogs of various ages [ ] and with severe gastroenteritis in an elderly dog [ ] . a seroprevalence study undertaken in showed an . % mvc-positive rate for korean canines [ ] , and an mvc strain was isolated from a korean dog in [ ] . since then, mvc has not been reported in korea for over years, but it has been reported infrequently in other areas of asia, including japan and china [ , ] . the present study describes a previously unreported mvc strain, isolated from a canine in korea, and we have analyzed its genetic relationship to other bocaparvoviruses. a five-year-old female yorkshire terrier dog underwent a hernia operation and ovariohysterectomy on dec , . five days after surgery, the dog began to experience loss of appetite and vomiting. the dog died on jan , , following symptoms of hypothermia, bloody vomiting, and bloody diarrhea. the dog was submitted to the animal and plant quarantine agency for diagnostic examination. a full necropsy was performed; brain, lung, kidney, liver, spleen, lymph node, heart, and intestine were among the tissues collected, which were subsequently fixed in % buffered formalin, dehydrated in graded ethanol solutions, and embedded in paraffin wax. four-micrometer serial sections from the paraffin-embedded organs were stained with hematoxylin and eosin for histological analysis. for the generation of an mvc-specific in situ probe, pcr labeling with a pcr dig probe synthesis kit (roche diagnostics gmbh, mannheim, germany) was performed according to the manufacturer's protocol. briefly, a specific region (nucleotides - ) of the ns gene of the d strain was amplified by pcr, using ns forward ( ' aacgcgatttgcaccttcat ') and ns reverse ( ' catcaaacatttctccggca ') primers. the pcr product was labeled with digoxigenin (dig) and subsequently used as a hybridization probe. using this probe, the tissues of parenchymal organs taken from the infected dogs were investigated for the presence of mvc nucleic acid. to detect mvc dna, additional paraffin-embedded tissues were sectioned at -lm thickness and hybridized in situ using a fully automated system (nexes ihc instrument; ventana medical systems, inc., tucson, az, usa) and a dab detection system (ventana medical systems inc.). the tissue sections were deparaffinized (standard xylene and industrial methanol) and fixed in % paraformaldehyde for min. tissues were then permeabilized by incubation in . m hcl containing mg of pepsin per ml for min at °c, followed by denaturation at °c for min before hybridization with the dig-labeled riboprobes at °c for h. after hybridization, samples were washed twice . ssc at °c for min, followed by pbs at room temperature for min. after hybridization, slides were incubated with qds conjugated anti-dig antibody (invitrogen, carlsbad, ca) for min and then rinsed twice in pbs for min. all tissue sections were mounted with coverslips using % (v/v) glycerol/pbs mounting solution. the dab detection system was then used to detect antibody binding. finally, sections were incubated at °c and then counterstained with hematoxylin and post-counterstained with bluing reagent. polymerase chain reaction (pcr) and reverse transcription (rt)-pcr was performed for genetic diagnosis. extraction of viral rna and dna from organ tissue was performed using an inclone mini extraction kit (inclone biotech co., yongin, gyeonggi, , korea), according to the manufacturer's instructions. canine parvovirus types and (cpv- , ), canine distemper virus (cdv), and canine influenza virus (civ), were examined using a virus detection kit (intron biotechnology, inc., sungnam, gyeonggi, , korea). canine herpes virus (chv) and canine adenovirus types and (cadv- and, - ) were amplified by pcr, using a hotstart pcr premix (imod, hanam, gyeonggi, , korea). canine corona virus (ccv) and canine parainfluenza virus (cpiv) were screened using an rt-pcr one-step kit (imod). pcr products ( ll) were subjected to electrophoresis in . % agarose gels at v for min to visualize the amplified dna. a molecular weight marker was run with the samples to determine the length of the amplified product. bands were visualized under ultraviolet illumination after ethidium bromide staining and photographed using an ultraviolet gel imaging system. for genome sequencing and phylogenetic analysis, sequence-specific oligonucleotides were designed based on the conserved regions of several published mvcs [ ] [ ] [ ] . the amplified pcr products were purified using an agarose gel dna extraction kit (intron biotechnology, korea) and cloned into the pgem-t vector (promega, madison, wi, usa). the cloned plasmids were purified and sequenced using t and sp primers, a bigdye terminator cycle sequencing kit (applied biosystems, foster city, ca, usa), and an automated dna sequencer (abi xl genetic analyzer, applied biosystems). all nucleotide sequences were confirmed by three or more independent, bi-directional sequencing runs. nucleotide and putative amino acid sequence alignments were generated using the bioedit sequence alignment editor (ibis biosciences, carlsbad, ca, usa). the nearly complete sequence of the mvc genome was deposited in the gen-bank database, under accession number kt . this sequence was compared to those of previously reported mvcs and bocaparvoviruses from other species at both the nucleotide and amino acid level, using orthologous sequences available in genbank. phylogenetic analysis was conducted using bioedit and molecular evolutionary genetics analysis (mega) . software, with bootstrap values calculated from , replicates. the neighborjoining phylogenetic algorithm was used to construct phylogenetic trees. from post-mortem examination, the immediate and surrounding areas of the surgical incisions showed no indication of surgical complications. the gastrointestinal tract was sparsely filled with fluid contents, and liver and spleen damage was evident. histopathologic examination revealed massive hepatic necrosis and basophilic intranuclear inclusion bodies in degenerated hepatic cells (fig. ). there were no other macroscopic or significant histological findings in the other organs. on analyzing the in situ hybridization images, hepatic cells surrounding the damaged regions and intranuclear inclusion bodies were found positive for mvc nucleic acid (fig. ) . in situ hybridization in other tissues also revealed cells that were positive for mvc nucleic acid; however, with the exclusion of the liver, there was no evidence of lesions in any other organs. therefore, it was difficult to establish a correlation between the results of in situ hybridization for these organs and the presence of disease in the present case. by using pcr and rt-pcr, cpv- , cadv- , , cdv, civ, chv, ccv, and cpiv were not detected; however, positive pcr products of the expected size of bp, corresponding to mvc, were detected in samples of the brain, lung, kidney, liver, spleen, lymph node, heart, and intestinal tissue. the isolated mvc strain, designated d , was sequenced and aligned with the other mvc strains obtained from genbank, using bioedit. the mvc d strain has a , -nucleotide-long genome. the non-coding region of the d strain on the left-hand side (lhs) terminus, located at the -end of positive-sense ssdna genomes, was nucleotides in length. the right-hand side (rhs) non-coding region of the d strain, found at the end of positive-sense ssdna genomes, was nucleotides in length. however, the very end of the and termini of this genome could not be definitively sequenced, presumably because of its peculiar hairpin structures, which are also present in other parvovirus genomes [ ] . orf of the d strain encodes a -aa non-structural (ns) protein, which is - aa longer than the ns protein of other mvc strains. orf and orf of the d strain encode a -aa protein that overlaps (table ) , which is also lower than the mean nucleotide and amino acid sequence identity values for the vp /vp region of other mvc strains (approximately . % and . %, respectively). phylogenetic analysis based on these ns and vp /vp regions also showed that the d strain formed a distinct branch that was separate from the other previous mvc strains. this indicates that this strain is distinct from these previously isolated strains (fig. ) . however, the np region of the d strain showed greater nucleotide and amino acid sequence similarity to that of the hm- strain (ab ), which was isolated from a korean dog in ( . % and . %, respectively), compared to those of the other mvc strains (mean similarities of . - . % and . %, respectively) ( table ). phylogenetic analysis based on the np region of the mvc strains also showed that the d and hm- strains fell into the same cluster, which is distinct from other mvc strains (fig. ) . the high level of nucleotide and amino acid sequence similarity in the np region between these two korean strains implies that the d strain could be a mutated strain of hm- or an endemic strain from korea that has not been isolated previously. in addition, the coding region of d shared . - . % nucleotide sequence identity to the other mvc strains (table ) , and this value is lower than the mean nucleotide sequence identity of the coding region of the other mvc strains (approximately . %). phylogenetic analysis based on the complete sequence of the d strain indicated that this strain could be correctly classified as an mvc strain, although it forms a distinct branch on phylogenetic trees when analyzed with respect to the ns and vp /vp regions. interestingly, the mvc strains, including the d strain, are more closely related to feline bocaviruses than the novel canine bocaviruses (fig. ) . this observation supports the possibility of a common origin and crossspecies transmission between mvc and feline bocavirus. in conclusion, our results indicate that the mvc d strain is a possible cause of degenerative viral hepatitis in dogs. although liver degeneration in canines has also been described at post-mortem examination of a pup infected with mvc [ ] , and a novel bocavirus (kc ) that was highly divergent from known mvc strains has been isolated from canine liver [ ] , hepatitis with evidence of intranuclear inclusion bodies that correspond to mvc infection has not yet been reported. therefore, the present study suggests two possible mechanisms for the observation of hepatic intranuclear inclusion bodies following infection with the novel mvc d strain. first, there is the possibility of pathogenic variation between the d and other mvc strains, based on differences in nucleotide and amino acid sequences. the d strain showed comparatively low nucleotide and amino acid sequence similarity to other mvc strains, except in the np region of the hm- strain, and the region related to pathogenicity might be altered, resulting in an acquired ability to cause hepatitis. the second possibility is that an immunocompromised state of the host animal might affect virulence. generally, the pathogenic potential of mvc for adult dogs has been considered minimal [ , , , ] . however, the host animal in the present study had surgery a few days before presenting with symptoms, and this procedure may have weakened the host immune system, which can be influenced by factors such as stress, anesthetic effects, or the effects of other drugs. this weakened immunity might explain the occurrence of hepatitis; in fact, simultaneous human bocavirus infection and hepatitis has been reported in a boy with severe t-cell immunodeficiency [ ] . further studies, such as isolation of the d strain and a subsequent animal inoculation tests, are required to clarify the exact relationship between this strain and hepatitis and to determine the distribution, diversity, and pathogenesis of mvc in dogs. recovery and characterization of a minute virus of canines novel canine bocavirus strain associated with severe enteritis in a dog litter the expanding range of parvoviruses which infect humans pathogenicity of minute virus of canines (mvc) for the canine fetus minute virus of canines (mvc, canine parvovirus type- ): pathogenicity for pups and seroprevalence estimate molecular characterization of canine minute virus associated with neonatal mortality in a litter of jack russell terrier dogs minute virus as a possible cause of neurological problems in dogs virus taxonomy: eighth report of the international committee on taxonomy of viruses seroprevalence of canine calicivirus and canine minute virus in the republic of korea hepatitis and human bocavirus primary infection in a child with t-cell deficiency a novel bocavirus in canine liver animal bocaviruses: a brief review virologic and serologic identification of minute virus of canines (canine parvovirus type ) from dogs in japan sequence analysis of an asian isolate of minute virus of canines (canine parvovirus type ) a minute virus of canines (mvc: canine bocavirus) isolated from an elderly dog with severe gastroenteritis, and phylogenetic analysis of mvc strains the canine minute virus (minute virus of canines) is a distinct parvovirus that is most similar to bovine parvovirus sequence analysis of an isolate of minute virus of canines in china reveals the closed association with bocavirus experimental infection of calves with hemadsorbing enteric (haden) virus parvoviruses associated with diarrhea in calves acknowledgments this research was supported by a grant from the animal and plant quarantine agency, anyang, gyeonggi, republic of korea. conflict of interest the authors declare that they have no conflict of interest.ethical approval this article does not contain any animal studies that were performed by any of the authors. key: cord- -osf t cw authors: cercenado, emilia; garau, javier; almirante, benito; ramón azanza, josé; cantón, rafael; cisterna, ramón; maría eiros, josé; fariñas, carmen; fortún, jesús; gudiol, francisco; mensa, josé; pachón, jerónimo; pascual, Álvaro; luis pérez, josé; rodríguez, alejandro; sánchez, miguel; vila, jordi title: update on bacterial pathogens: virulence and resistance date: - - journal: enfermedades infecciosas y microbiología clínica doi: . /s - x( ) -x sha: doc_id: cord_uid: osf t cw the present article is an update of the literature on bacterial pathogens. recognizing the interest and scientific and public health importance of infections produced by bacterial pathogens with new virulence mechanisms and/or new mechanisms of resistance to antimicrobial agents, a multidisciplinary group of spanish physicians and microbiologists organized a joint session and revised the most important papers produced in the field during . each article was analyzed and discussed by one of the members of the panel. this paper focus on a variety of diseases that pose major clinical and public health challenges today; and include infections produced by community-acquired methicillin-resistant staphylococcus aureus and s. aureus small colony variants, infections produced by multiply resistant coagulase-negative staphylococci, pneumococcal infections, human listeriosis, meningococcal disease, haemophilus influenzae, pertussis, escherichia coli, esbl-producing organisms, and infections due to non-fermenters. after a review of the state of the art, papers selected in this field are discussed. although predictions during the th century indicated that the incidence of infectious diseases would diminish as a result of improvements in sanitation and by the introduction of many vaccines and antibiotics, at the beginning of the st century the rates of infections produced by new pathogens or by reemerging microorganisms possessing new virulence or resistance phenotypes is increasing, threatening the overall human health [ ] [ ] [ ] [ ] [ ] . over the last years we have moved from the pre-antibiotic era through the antibiotic era into the era of emerging infectious diseases. the discovery of new infectious diseases and the steady increase in the number of microorganisms that are resistant to multiple antimicrobial agents have altered the practice of medicine within the hospital, and are affecting the management of infections in the ambulatory care setting , . it is in this scenario where community-acquired methicillin-resistant staphylococcus aureus (ca-mrsa) has emerged as the most common pathogen isolated from patients with skin and soft-tissue infections attending to the emergency departments in many united states and australian cities , , and at present, its incidence is increasing in other parts of the world , . despite the growing prevalence of mrsa in hospitals, these strains have been uncommon in the community. in many circumstances mrsa escape into the community when patients still harbouring the organisms are discharged or when hospital personnel go from work to home. these strains can be associated with infections that begin in the community, but the isolates are hospital-associated. however, there is now an increasing number of reports of mrsa infections in the community that have not escaped from the hospitals, and no longer can mrsa be considered as an exclusively nosocomial pathogen. there is molecular evidence that strains of mrsa have also evolved in the community, are well adapted to survive there, and are causing an epidemic outside the hospitals , . ca-mrsa infections refer to mrsa infections in patients lacking established mrsa risk factors and without a previous history of mrsa infection or colonization: they do not have a medical history in the past year of hospitalization, healthcare-related admission (to a nursing home, skilled nursing facility or hospice), dialysis, surgery, or implantation of a permanent indwelling catheter or other medical devices , . ca-mrsa tends to cause infections that occur in clusters or small outbreaks that affect otherwise healthy and unique populations such as children and young adults, australian aborigines, native americans, alaskan natives, prisoners, military recruits, men who have sex with men, college athletes and players competing in contact sports. recent antimicrobial exposure is less likely to be reported as a risk for mrsa infections in the community that in health care settings. most infections are mild and limited to skin and soft tissues, though rapidly fatal invasive infections such as necrotizing fasciitis and overwhelming pneumonia and sepsis accompanied by the waterhouse-friderichsen syndrome do occur and may serve as "sentinels" that first bring a more widespread community problem to the attention of public health personnel [ ] [ ] [ ] [ ] [ ] [ ] . because skin infections caused by these organisms often have necrotic centers, the disease can be misdiagnosed as a "spider bite". similar to the epidemiology of methicillinsusceptible s. aureus, ca-mrsa infections occur in children in different regions of the united states and throughout the world , [ ] [ ] [ ] [ ] . although minor skin and soft-tissue infections predominate, life-threatening invasive disease and death can result. in children, ca-mrsa can cause septic thrombophlebitis of the extremities and a "pelvic syndrome" consisting of septic arthritis of the hips, osteomyelitis of the pelvic bones, pelvic abscesses, and septic thrombophlebitis . unlike hospital strains, which typically are resistant to multiple antimicrobial agents and share a common genotype with other hospital isolates, community acquired strains tend to be susceptible to non-betalactams and have genotypes distinct from hospital isolates in the same community . two main clones (usa and usa , belonging to st and st by mlst, respectively) have been described as responsible for the majority of infections caused by ca-mrsa throughout the united states , . in other continents, the most frequent sts of ca-mrsa are the st for the southwest pacific clone, and the st for the european clone, although in europe the st and st clones have also been described . the meca gene, the genetic determinant necessary for the expression of oxacillin resistance, resides in these strains on genetic elements (usually sccmec types iv and v) that are different from those encoding methicillin resistance in hospital-acquired mrsa, although in recent years sc-cmec type iv has been frequently found in nosocomial mrsa isolates mainly in europe , , . another key difference is that almost all ca-mrsa isolates contain genes encoding the panton-valentine leukocidin, which is a cytotoxin that causes leukocyte destruction and tissue necrosis. although the exact role of panton-valentine leukocidin in the serious infections caused by these organisms is unclear, it is in general a marker for ca-mrsa , , . a hypothesis in order to explain the origins of ca-mrsa is that the meca gene or the sccmec have been transferred horizontally to one or more previously oxacillin-susceptible s. aureus strains that occupy traditional community niches. this possibility does account for the distinct phenotypic and genotypic characteristics of ca-mrsa. the relatively long time lag from the appearance of mrsa in hospitals to its emergence in the community may in part be due to the low frequency of horizontal chromosomal gene transfer . at present, the knowledge of ca-mrsa epidemiology is incomplete which adds to the challenge of controlling the infection. in addition, nasal colonization may not precede ca-mrsa infections and since nasal carriage is not the prime predictor of subsequent infection it is more difficult to identify and control populations that are at risk. colonization of the gastrointestinal tract and of household pets may act as additional reservoirs for ca-mrsa , . moreover, ca-mrsa are now being introduced into hospitals, thus blurring the borders between community-acquired and hospital-acquired strains . therapy for infections due to ca-mrsa includes appropriate drainage of skin and soft-tissue lesions, since milder infections often respond to local incision and drainage alone . the clinician should know when to use drugs with activity against ca-mrsa (clindamycin, minocycline, doxycycline, trimethoprim-sulfamethoxazole or vancomycin), although optimal antimicrobial agent therapy is unknown. for severe and invasive infections therapy with vancomycin or linezolid should immediately start since antimicrobial agents may be ineffective when the patient receives treatment too late , . the recognition that partial vancomycin resistance may result in some vancomycin treatment failures for serious mrsa infections, portends the near-term loss of this firstline drug as the treatment of choice. vancomycin-intermediate s. aureus (visa) and heterogenous visa (hvisa) have become a significant problem in many parts of the world [ ] [ ] [ ] . visa/hvisa isolates can arise from fully vancomycin-susceptible s. aureus during persistent infection that fails to respond to glycopeptide therapy and are associated with significant phenotypic changes . resistance may develop by different pathways, but these strains have a thickened cell wall with reduced peptidoglycan crosslinking leading to cell wall "clogging" with vancomycin. moreover, a marked reduction in autolytic activity and reduced cell wall turnover have been found in visa /hvisa strains . several studies have demonstrated a number of metabolic pathways and regulatory genes that may contribute to resistance . in particular, the agr twocomponent regulatory system has been linked to low-level vancomycin resistance in s. aureus, with reports suggesting that agr type ii strains and loss of agr function are associated with visa . it has also been noted that many reported visa infections have involved biomedical devices, and biofilm formation on these devices could be an important initial step in the pathway to vancomycin resistance . an "emerging" pathogen can be seen as a well-known pathogen for which newly discovered subpopulations of the parent strain are able to produce disease. staphylococcus aureus small-colony variants (scvs) fall into this category. this variant subpopulation is defective in electron transport, grow slowly, and produce colonies < % the size of the parent strain grown on the same medium for the same amount of time. most of these clinical isolates cannot synthesize menadione or hemin, and these results in a block of electron transport at the level of menaquinone or the cytochromes . scvs might seem to be less virulent because of their slow growth and decreased production of coagulase and alpha-toxin. however, the intracellular location of scvs shields them from host defenses, their slow growth reduces the efficacy of cell wall-active antibiotics, and a decreased membrane potential protects them from positively charged antimicrobials . scvs represent a subpopulation of s. aureus that can cause persistent and recurrent infections. while their overall prevalence has not been firmly established, problems in discovering and identifying these organisms may cause an underestimation or their prevalence . coagulase-negative staphylococci can also be seen as an "emerging" pathogen since it has acquired new virulence factors and resistance to new antimicrobials . these microorganisms can colonize indwelling catheters (including central venous catheters) and form biofilms, that can result in bloodstream infections. the organisms embedded in a matrix of extracellular polymeric substances that they have produced exhibit an altered phenotype with respect to growth rate and gene transcription. treatment of these infections with conventional antimicrobial agents alone is frequently unsuccessful due to the high tolerance of these agents in microorganisms comprising the biofilm . several preventative and treatment approaches have been investigated for catheter-related infections including a recent renewed interest in the use of bacteriophages for mitigating biofilm formation on indwelling catheters . recently, another cause of concern among coagulase-negative staphylococci is their resistance to new antimicrobial agents such as linezolid. although resistance to linezolid is very infrequent among staphylococci, these organisms are able to acquire de novo resistance among patients who have a long duration of hospitalization and that have received high amounts of linezolid. moreover, linezolid-resistant strains can be transmitted from patientto-patient with their establishment as part of the skin flora and may be a precursor of linezolid-resistant mrsa . streptococcus pneumoniae is a cause of serious infections that are a major source of morbidity and mortality among all age groups in both the developed and the developing worlds. in addition, the emergence of strains with high-level resistance to penicillin and to other antimicrobial agents has raised concerns about the current and future effectiveness of antibiotic regimens. nevetheless, there is now evidence of decreasing resistance to some antimicrobials in some regions of the world. reduction in antimicrobial use in some community settings has been associated with a decline in resistance of pneumococci to some classes of antimicrobials, mainly beta-lactams. however, we are still witnessing a relentless increase in resistance to the macrolide class of antimicrobials . fortunately, control of pneumococcal disease through vaccination with the -valent polysaccharide vaccine in adults has demonstrated a reduction in risk of bacteremic pneumococcal disease after vaccination, as well as decreased risk of death and complications of hospitalization, which reinforces the need to improve compliance with existing pneumococcal vaccination recommendations for adults. moreover, the introduction of the -valent pneumococcal conjugated vaccine not only prevents invasive disease in children but also appears to have resulted in a decline in the prevalence of resistant serotypes included in the vaccine, resulting in an overall decrease in the prevalence of pneumococcal resistance . however, penicillin-nonsusceptible pneumococcal clones of nonvaccine serotypes have been reported as a cause of bacteremia and other infections. these clones may have been derived from capsular transformation of vaccine-related serotypes . in recent years an upsurge in the incidence of human listeriosis seems to have occurred in some geographical areas. although most cases are foodborne, the epidemiology is complex. prevention of the disease must include dietary advice on avoiding high-risk foods routinely to pregnant women, to the elderly and to immunocompromised patients. listeriosis manifests primarily as abortion, septicemia, or central nervous system infections with a high case-fatality rate in all patient groups. listeria monocytogenes is the third most common cause of bacterial meningitis that occurs among immunocompromised patients and elderly individuals . symptoms and signs of patients presenting with l. monocytogenes meningitis are not different from those found in the general population of patients with community-acquired bacterial meningitis, albeit with a longer prodromal phase; however typical cerebrospinal fluid findings predictive for bacterial meningitis might be absent, and gram stain has a low yield. in patients aged > years old or with risk factors for l. monocytogenes meningitis, an amoxicillin-based empirical antimicrobial regimen is mandatory, since this bacterium is resistant to cephalosporins. despite the availability, for decades, of meningococcal vaccines, neisseria meningitidis remains a leading cause of meningitis, sepsis, and other serious infections in both industrialized nations and the developing world. group c meningococcal conjugate-vaccine effectiveness in the united kingdom declines from ~ % in the first year to % between and years after immunization in infants immunized at , , and months of age and to % in toddlers given a single dose. a recent study did not find evidence of lower immunity in children immunized as infants than as toddlers. on the basis of serum bactericidal activity and/or passive protection, - % of both age groups are protected at - years after immunization, which is significantly greater than in unimmunized historical controls (< %). the study of snape et al on antibody responses to either a reduced dose of meningococcal c polysaccharide vaccine, which is meant to simulate exposure to n. meningitidis, or meningococcal c conjugate vaccine in healthy - -year-olds in the united kingdom who had been primed with a serogroup c conjugate vaccine - years previously, adds new data on the number of days required for antibody levels to increase after boosting with either of the vaccines. in addition to susceptibility associated with flawed complement pathway function, several other pathways have also been convincingly linked to altered host susceptibility to meningococcal disease. the article by jack et al breaks new ground in increasing the understanding of the human genetic basis for some host defence failures in meningococcal disease. surfactant protein (sp)-a and sp-d are pattern-recognition molecules of the respiratory tract that activate inflammatory and phagocytic defences after binding to microbial sugars. variation in the genes of the surfactant proteins affects the expression and function of these molecules. routine use of haemophilus influenzae type b (hib) conjugate vaccines has dramatically decreased the incidence of invasive hib disease in the western world. despite the effectiveness of the vaccine, an increase in the incidence of hib disease has recently been observed in some european countries. careful analysis of circulating hib strains is essential for prompt detection of any change in the properties of bacteria, enabling particular clones to overcome the host's immune response. in a recent italian study , contrary to previous reports, neither increased genetic diversity of hib strains isolated from children nor the disappearance of individual clones was observed after the rountine immunization of infants against hib was established; however, an upward temporal trend in proportion of strains possessing multiple copies of the capsulation b locus was detected. the results of this study suggest that vaccine pressure may be positively selecting for strains that harbour amplified cap b sequences. interactions of nontypeable haemophilus influenzae (nthi) with human alveolar macrophages are implicated in the persistence of nthi in chronic obstructive pulmonary disease (copd). a recent study by sethi's group confirmed the hypothesis that immunologic responses of alveolar macrophages to nthi are impaired in copd. the same group has described a phenotypic variant of nthi, haemophilus haemolyticus that frequently colonizes the airway of copd patients and is not found in normally sterile sites. these findings substantially strengthen the association of true h. influenzae with clinical infection and exacerbation of chronic bronchitis . the control of pertussis infection is not optimal, because neither vaccination nor natural infection induces long-lived immunity. increasing proportions of reports of pertussis cases involve adolescents and adults. the aim of a recent us study was to define, among unimmunized control subjects, a yearly infection rate and to characterize the proportion of those infections that include prolonged coughs . a similar analysis was attempted for acellular pertussis (ap)-vaccinated subjects. pertussis infections in older persons was found to be largely asymptomatic; ap boosters confer protection for adolescents and adults against symptomatic pertussis.the diagnosis of pertussis in older individuals is problematic because of the lack of specific clinical criteria, insensitivity of culture and pcr, and the limited availability of standardized serologic tests and criteria for diagnosis. a spanish study suggests that real time-pcr is the most sensitive and specific test available for the diagnosis of pertussis and that the direct fluorescent assay should be abandoned . three recent studies in escherichia coli infections deserve comment. the first two deal with fluoroquinolone resistance in this species. the first shows the increasing prevalence of strains with reduced sensitivity to the quinolones in the gut of hospitalized patients , and the second analyses the differences and similarities of fluoroquinolone-resistant e. coli from humans and from chicken. the similarities among the strains studied strengthen the hypothesis of an animal origin of the human strains of fluoroquinolone-resistant e. coli . a third study addressed the progressive importance of infections caused by esbl-producing e. coli . bacteremia caused by these organisms is increasing and given the difficulties encountered in its treatment,-frequent multiresistance to other traditionally used agents,-a reappraisal of current empirical regimens is in order. a very good example of the difficulties found treating esbl-producing organisms, is the case reported from israel, a patient with an esbl-producing klebsiella pneumoniae endocarditis that developed resistance to ciprofloxacin and piperacillin/tazobactam while on therapy . the presence of esbl-producing enterobacteriaceae in the stools of humans, sewage and animals has been found to be high in areas where these studies have been done. from to % of the farm animals examined in a recent spanish study harbour esbl-producing enteric bacteria . the potential for dissemination of multi-resistance isolates is illustrated by two recent reports: the emergence of proteus mirabilis carrying the bla metallo-beta-lactamase gene, the first instance in this species , and bla vim- , and bla vim- carbapenemase-producing pseudomonas aeruginosa . among the latter, the emergence of resistance to carbapenems can be very rapid; when associated with resistance to polimixins the problem at clinical level can become insoluble . acinetobacter baumannii has recently emerged as a major cause of hospital-acquired infection, because of its propensity to accumulate mechanisms of antimicrobial resistance that lead to pan-drug resistance and cause large nosocomial outbreaks that often involve multiple facilities. the problem is particularly serious in intensive care settings. an ex-cellent review has been published recently on the epidemiology and control of a. baumannii infections in health care facilities . finally, although rare, communityacquired pneumonia due to a. baumannii is known to occur. a retrospective case-control study (cases: community-acquired pneumonia; controls: hospital acquired pneumonia) from hong-kong has been published . below, a group of spanish physicians and microbiologists with an interest in the field of bacterial pathogens expand on the most remarkable papers published in this area during . the following are the publications selected for discussion. these studies are an example of the ca-mrsa epidemic in the united states at present time. a population study conducted in three united states communities during - established the annual incidence of ca-mrsa to be - per . , and most isolates were associated with clinically relevant infections. the current relevance of mrsa as a cause of skin and soft-tissue infections has been demonstrated in a prospective prevalence study involving patients presenting to emergency departments in u.s. cities (moran gj et ) . according to these observations, the distinction between healthcare and community-associated mrsa is rapidly blurring. these studies demonstrate: ) an increase in the incidence of skin and soft-tissue infections as well as in the incidence of invasive infections due to ca-mrsa. ) the current predominance of the genotype usa . ) the migration of ca-mrsa strains into the healthcare settings. stapylococcus aureus is an infrequent cause of community-acquired pneumonia (cap), but it is a recognized cause of influenza-associated cap. mrsa commonly causes nosocomial pneumonia, but relatively few cases of mrsa cap have been reported. during the - influenza season, cases of cap due to s. aureus were reported to the cdc from states; ( %) were caused by mrsa, and all isolates available for microbiological study presented genes for the panton-valentine leukocidine. thirteen were admitted to the icu, and death occurred in patients. authors suggest that empiric therapy of severe cap during periods of high influenza activity should include coverage for mrsa. in a country with high incidence of ca-mrsa, as it is in the united states, empiric therapy of severe cap during periods of high influenza activity should include coverage for mrsa, even in patients without recognized risk factors for mrsa since they can be infected by a ca-mrsa strain. pyomiositis is an acute bacterial infection of skeletal muscle with localized abscess formation caused in - % of cases by s. aureus. acute bacterial myositis is a less common muscle infection, but inflammation extends to more than one muscle group without distinct abscesses. although commonly caused by streptococcus pyogenes, myositis caused by s. aureus have been described. cases of pyomiositis and myositis have increased in pediatric patients since at texas children´s hospital, and this increase appears to correlate with the emergence of ca-mrsa. this study reviews the medical records of all patients admitted in this hospital from to with infective pyomyositis and myositis. forty-five cases were analyzed. forty percent had negative culture but . % were caused by s. aureus. the number of cases increased from to as a result of an increase in the prevalence of ca-mrsa. the thigh and pelvis were the most commonly affected sites. fifteen out of available s. aureus isolates were ca-mrsa and were community-acquired methicillin-susceptible s. aureus (ca-mssa). by pfge, isolates were found to be usa , and carried the panton-valentine leukocidin (pvl) genes. patients with ca-mrsa, usa and/or pvl-positive strains required more drainage procedures that did those with ca-mssa, non-usa and/or pvl-negative strains ( vs. %, vs. %, and vs. %, respectively). the authors strongly consider coverage for mrsa in the empirical treatment regimen (vancomycin or clindamycin) of these infections in children. ca-mrsa is an increasing cause of pyomyositis and myositis in children. although in this study the infections caused by ca-mrsa, usa , and pvl-positive isolates were more severe than those caused by ca-mssa, non-usa , and pvl-negative strains, additional studies are needed to find out the virulence factors associated with muscle infection caused by ca-mrsa. the epidemiology of ca-mrsa among healthy children has been recently described. however, little is known about ca-mrsa in children with underlying medical conditions. the authors compare in this study the clinical and molecular epidemiology of ca-mrsa in children with and without risk factors for health care-associated infections (rf-hai). a -year retrospective cohort study of children with ca-mrsa infection was conducted. rf-hai, including hospitalization within the past year, indwelling medical devices or chronic medical condition, were identified by chart review. the authors identified episodes of community-acquired s. aureus infections, of which ( %) were caused by mrsa. during the -year study period, the proportion of s. aureus infections caused by mrsa rose from % ( of ) to % ( of ) (p < . ) with the increase noted predominately in children with skin and soft tissue infections. rf-hai were identified in ( %) patients with ca-mrsa. among subjects with ca-mrsa, children with rf-hai were more likely to have had an invasive infection than healthy children ( versus %; p < . ). ca-mrsa isolates from children with rf-hai were similar to those without rf-hai; all laboratory-retained ca-mrsa isolates harbored the sccmec type iv cassette, and almost all isolates were susceptible to cotrimoxazole and clindamycin. pfge revealed greater molecular diversity among ca-mrsa isolates recovered from children with rf-hai compared with those from otherwise healthy children (p = . ). additionally, ca-mrsa isolates from children with rf-hai were less likely to be pvl-positive (p < . ) and more likely to be resistant to or more classes of antibiotics (p = . ). this study demonstrates that the borders between community-acquired and hospital-acquired mrsa strains are blurring, and suggests that ca-mrsa strains might have become endemic not only in adult health care facilities but also within paediatric facilities. ca-mrsa was first reported in western australia (wa) in the early s from indigenous people living in remote areas. a statewide policy of screening all hospital patients and staff who have lived outside the state for mrsa has prevented the establishment of multidrug-resistant epidemic mrsa (memrsa), however, this study demonstrates that this policy has not prevented sccmec type iv and type v mrsa clones (community clones) from becoming established in wa. all mrsa isolated in wa from july to december were included in this study. isolates were recovered from clinical and infection control screenings. of the , mrsa isolates analysed (those sent to a reference center), . % were ca-mrsa. using different molecular methods, a total of different ca-mrsa clones were characterized. of these isolates, . % were resistant to ≥ non-beta-lactam antimicrobial drug. five pvl-positive ca-mrsa clones were identified. the australian policy that prevented the establishment of memrsa, has not prevented sccmec type iv and v mrsa clones, including non-memrsa and ca-mrsa, from becoming established in wa. the emergence of multidrug-resistant ca-mrsa clones and the detection of pvl toxin genes in clones previously reported as pvl-negative is a major public health concern. molecular typing is very important in tracing the origin of isolates and in designing antimicrobial drug prescribing policies for their control, particularly in the community. some studies have indicated that ca-mrsa strains are generally more virulent than hospital-acquired mrsa, a finding consisting with the ability of ca-mrsa to cause disease in individuals without predisposing risk factors. although the molecular basis for the enhanced virulence is not known, there is strong association between ca-mrsa infections and the presence of pvl. however, the role that pvl plays in the pathogenesis of ca-mrsa has not been tested directly. in this study the authors evaluated, in a mouse infection model, the role of pvl in the virulence of ca-mrsa as well as the lytic activity and the intracellular survival in human polymorphonuclear leukocytes (pmns) of isogenic strains of ca-mrsa, expressing or not pvl. they compared the virulence of pvl-positive with that of pvl-negative ca-mrsa representing the leading disease-causing strains. unexpectedly, strains lacking pvl were as virulent in mouse sepsis and abscess models as those containing the leukotoxin. isogenic pvlnegative (luks/f-pv knockout) strains of usa and usa were as lethal as wild-type strains in a sepsis model, and they caused comparable skin disease. moreover, lysis of pmns and pathogen survival after phagocytosis were similar between wild-type and mutant strains. although the toxin may be a highly linked epidemiological marker for ca-mrsa strains, the authors conclude that pvl is not the major virulence determinant of ca-mrsa. it may be that pvl contributes to specific pathologic conditions such as necrotizing pneumonia, a disease not tested in this study. probably future studies using other different animal models will help to understand the pathogenesis of ca-mrsa or to identify other factors responsible for the type and severity of disease caused by these emerging pathogens. the main limitation of this study is its retrospective nature, which cannot exclude bias. unrecognized common factors leading to fluoroquinolone therapy and mrsa acquisition cannot be ruled out. in addition, adverse events associated with increased use of those antimicrobials substituting the fluoroquinolones need to be considered. the authors investigate the association between the use of fluoroquinolones and the recovery of mrsa in hospitalized patients. four hospitals located in the same region of france with a similar pre-study incidence of mrsa are selected. the study manoeuvre consists of discontinuing the use of fluoroquinolones in one hospital for the study period, while the three others serve as controls without changing their antibiotic policies. as a consequence, the study hospital reduces the use of fluoroquinolones from to ddd per bed-days and increases the consumption of amoxicillin-clavulanate by %, ceftriaxone by %, gentamicin by %, amikacin by %, erythromycin by % and cotrimoxazole by %. during the intervention period the incidence of mrsa tended to be lower in the study hospital (odds ratio . ; % ci . - . ). no reduction in the rate of fluoroquinolone resistance occurred in gram-negative bacilli and the study centre suffered a nosocomial esbl-producing klebsiella pneumoniae outbreak during the intervention period. the observed reduction in mrsa rate is significant, although small, in this interestingly designed study. unfortunately, a spontaneous decrease of the incidence of mrsa, rather than due to the absence of the use of fluoroquinolones, may be responsible for the observed % reduction. in addition, a potential causal relation between the changes in antibiotic policy and the occurrence of an esblproducing k. pneumoniae outbreak is a matter of concern. howden bp, johnson pd, ward pb, stinear tp, davies jk. isolates with low-level vancomycin resistance associated with persistent methicillin-resistant staphylococcus aureus bacteremia. antimicrob agents chemother. ; : - . low-level vancomycin-resistant staphylococcus aureus (vancomycin-intermediate s. aureus -visa-and heterogenous visa -hvisa-) leads to glycopeptide treatment failure. the genetic changes leading to hvisa and visa have not been clearly determined. this study characterizes five clinical pairs of fully vancomycin-susceptible s. aureus (vssa) and hvisa/visa (vancomycin mics, to mg/l) isolates obtained before and after failed vancomycin therapy, in patients with bacteremia due to mrsa, in order to better understand the changes associated with this type of resistance. the hvisa/visa phenotype was associated with increased cell wall thickness, re-duced autolytic activity in four of five hvisa/visa strains, and a striking reduction in biofilm formation compared to the parent strains in all pairs. all five pairs were isogenic, and genomic dna microarray comparison suggested that major genetic changes are not required for the development of the resistant phenotype in these strains. a marked reduction in rnaiii expression of the agr gene was found in four pairs. this study performs a detailed analysis of the different mechanisms that could explain the conversion of a vssa into a visa strain. many of the phenotypic changes are consistent with previous reports; however, reduced autolytic activity is not essential for the expression of low-level vancomycin resistance in s. aureus. the use of pairs of clinically derived, isogenic strains of vssa and hvisa/visa will be a valuable resource to elucidate the genetic mechanism of low level glycopeptide resistance in the latter. staphylococcus aureus small-colony variants (scvs) are able to persist inside of host cells and to resist antibiotics. this characteristic is especially important when involved in implant-associated infections. the authors analyze cases of hip-prosthesis-associated infections due to scvs, including their course prior to identification of the pathogen. the patients' mean age was . years. all patients experienced treatment failures prior to isolation of scvs, despite as many as surgical revisions and up to months of antibiotics. transmission electron microscopy performed on biopsy specimens from periprosthetic tissue revealed intracellular cocci in fibroblasts. all prostheses were removed without implanting a spacer, antimicrobial agents were administered for . - weeks, and reimplantation of the prostheses was performed for patients. this -stage exchange was associated with successful outcome, with a mean follow-up of months. slow growth, atypical colony morphology, and unusual biochemical profile make clinical laboratory personnel likely to miss or misidentify scvs. in the case of a poor response to adequate antimicrobial and surgical treatment in implant-associated staphylococcal infections, the clinician and the clinical laboratory should consider special efforts to search for scvs. this study investigates an unusually high incidence of % linezolid resistance in coagulase-negative staphylococci (lr-cns) at their centre in the usa. they perform an analysis of linezolid use and a retrospective case-control study to identify the risk factors and epidemiological profile of patients in whom lr-cns had been identified. each of the case patients were first matched with randomly selected concomitant patients at the same hospital ward. in a second analysis, each case patient was matched with patients with linezolid-susceptible cns isolated from clinical samples. the authors report that the use of linezolid had increased from . ddd per bed-days in to . in . in of ( %) patients with lr-cns they detected prior use of linezolid. lr-cns was identified in blood cultures of patients, times it was considered to be a contaminant and in patients it was identified in of blood samples. all except one isolate (staphylococcus lugdunensis) were identified as staphylococcus epidermidis. mics were higher than mg/l and genotyping revealed relatedness of strains from ( %) patients, of whom had been admitted to the same icu. compared to random controls, prior treatment with linezolid was significantly associated with acquisition of lr-cns (odds ratio . , . - ), as was admission to ward "c" (odds ratio . , . - . ). patients with linezolid-susceptible cns had not been given linezolid (odds ratio . , . - . ). this paper describes a clonal outbreak due to lr-cns associated with extensive use of linezolid. although the information provided is of interest, it is based on experience from only one centre, and it is a short series of strains and only clinical infections. this in vitro study investigates the effect of a bacteriophage on biofilm formation by staphylococcus epidermidis on hydrogel-coated foley catheters. they demonstrate that pre-treatment of catheters with the lytic s. epidermidis bacteriophage prevents formation of biofilm. while new technologies, such us pre-treatment of catheter surfaces with antiseptic or antibiotic agents show promise as prophylaxis/treatment of catheter-related infections, the results of this study highlight the potential of phages for the reduction of biofilm development on biomedical implant surfaces and suggest future developments in catheter-associated infection prevention. in spite of the antibiotic treatment and intensive care, the mortality of community-acquired pneumonia (cap) remains high. on the other hand, pneumococcal vaccination reduces the incidence of pneumococcal bacteremia, which itself has a high fatality rate. the objective of this study was to evaluate the impact of prior -valent pneumococcal vaccination (pv ) on the early mortality and complications in hospitalized adults with cap. the study was carried out in us teaching and community hospitals that share a common database. a total of , individuals with cap, identified by the incd th rev. codes . - . entered the study during a five-year period ( ) ( ) ( ) ( ) ( ) . data were obtained systematically using standardized definitions by trained nurses, concurrently with patient care, and validated monthly. variables included those necessary for calculation of the fine score, vaccination status (yes, %; no, %; unknown, %) and other comorbidities. no data on microbial etiology were recorded. statistical analysis was done with multivariable logistic regression models. vaccine recipients were less likely to die during hospitalization that were unvaccinated patients (adjusted or . ; % ci . - . ). this trend remained under varying assumptions about missing vaccination data. vaccination also had a positive effect on the risk of respiratory failure (or . ; % ci . - . ). length of stay was also reduced in vaccinated patients (p < . ). in conclusion, prior vaccination with the pv is associated with a % mortality reduction and a lower risk of complications, then reinforcing the efforts towards improving vaccination coverage in adults. the absence of microbiological data, and the high proportion of individuals with unknown vaccination status were the main weaknesses of this observational study. this is a case-control study on the effectiveness of the -valent pneumococcal conjugate vaccine (pcv ) to prevent invasive pneumococcal disease in children - months old, including those vaccinated with incomplete schedules. the study was carried out in several urban areas and two entire states of the usa. cases were identified by an active surveillance program operated by the cdc during - . a total of cases were included, and controls per case, matched by age and zip code, were selected. a total of cases were excluded, mostly those who had no isolate available for serotyping, or those whose parents refused to participate. serotyping (danish scheme), and susceptibility tests were done at cdc or at some state laboratories. matched odds ratio (or) was calculated by using conditional regression models controlled by underlying disorders, race, sex, and access to scheduled vaccinations, among other variables. effectiveness was defined as one minus the adjusted matched orx %. a total of % of cases were caused by serotypes included in the pcv (vt), mostly and f, of which % were in children who had received al least one dose of the vaccine, and % were in fully vaccinated. effectiveness in healthy children was % for vt strains, and % for vaccine-related serotypes [same serogroup but different type (vrt)]. lower effectiveness was observed in children with underlying diseases ( % and % for vt and vrt, respectively. the level of protection was also lower for strains with decrease susceptibility to penicillin. the pcv did not prevent serotype a infection. several schedules were more protective than no vaccination, being doses plus a booster more protective than doses alone. in summary, the pcv prevents invasive disease in both healthy and chronically ill children, even with various no standard schedules. this study opens new doors to prospective studies for investigating simpler vaccination schedules. the vaccine fails with some serotypes and is less efficient with penicillin non-susceptible strains. in this observational study from the children's hospital of philadelphia, the authors aimed to evaluate the effect of the pcv vaccine on the pneumococcal bacteremia (pnb) caused by vrt or by pneumococci non susceptible to penicillin (pnsp) in the post-licensure period of this vaccine. from january to may , all episodes of pnb attended at the pediatric emergence unit were revised retrospectively, as well as bacteremias caused by other respiratory bacterial pathogens (orbp; haemophilus influenzae, neisseria meningitidis, and moraxella catarrhalis), that were used as a non-equivalent dependent variable with the purpose of statistical analysis. blood cultures were done according with the physician indication. serotyping was performed with the staten seruminstitut antisera collection, and mics to penicillin by using e-test strips (ab biodisk). pcv vaccination status was no recorded among variables entering the study. an overall % decrease per year in the incidence of pnb was observed, mainly due to the reduction of cases caused by vaccine type strains, although a shift in this trend was noted in - . the incidence of pnb caused by non-vaccine types and orbp remains stable, but that of vrt increased by % along the study period, being serotype a responsible for most cases. the percentage of pnsp strains also increased from to % (p < . ). this study calls attention on the possibility of serotype replacement after vaccination with the pcv , and on the emergence of pnb caused by pnsp strains, possibly related with selection of some serotypes ( a) for which the pcv has poor immugenicity. in addition to its retrospective nature, the study has other limitations. the absence of records on the previous pcv vaccination in the study population, the use of a non-equivalent dependent variable for control of biases, and changing criteria in performing blood cultures along the study period (confound-ing by indication) were the main weaknesses of the study, although the authors believe that some of these variables would be controlled in a certain extent. therefore, prospective multicenter studies on this subject would be welcome. after large-scale introduction of the pcv vaccine, two effects have become apparent. first, increasing pharyngeal colonization by serotypes not covered by the pcv (serotype replacement), and second, acquisition of a new nonvaccine capsule through recombination in naturally transformable clones (serotype swiching). authors from taiwan investigated the in vitro capacity of clinical strains of s. pneumoniae isolated in an area with low pcv coverage to become transformed (competence) in vitro. a total of strains belonging to serotypes were prospectively collected from taiwanese hospitals from to . with the exception of serotype , all strains were included in the pcv . competence studies were done with the use of two variants of the competence-stimulating peptide (csp and ) and the pdl plasmid as a vector. genetic diversity were also studied by pulse field gel electrophoresis (pfge). serotype b had the greatest competence, followed by types , f, v, f, , and c. serotype b also showed the highest genetic diversity and higher proportion of penicillin nonsusceptibility rates. conversely, strains belonging to types and c showed the highest clustering, were mostly incompetent for transformation, and were % susceptible to penicillin. in spite of this qualitative association between serotype, competence and resistance, no correlation was observed between the level of competence and the degree of resistance to penicillin (or . ; % ci . - . ). in this way, serotype f was relatively incompetent, but had a clear trend to higher proportion of resistant strains, and with higher penicillin mics. this study shows how certain serotypes with lower capacity to be transformed are genetically more stable, and why resistance to penicillin is rare in strains belonging to incompetent serotypes all over the world. competence would be a necessary condition, but no sufficient, for explaining the genetic diversity and, secondarily the acquisition of penicillin resistance determinants in s. pneumoniae. streptococcus pneumoniae is the leading cause of cap in adults. bacteremic pneumococcal pneumonia (bpp) is among the most serious forms of pneumococcal disease. this population-based case-control study identifies clini-cal and demographic factors associated with macrolideresistant bpp in adults from acute-care hospitals at the pennsylvania region. from december , , to april , patients included in the study were individuals who ) were years or more; ) had at least one blood culture that grew s pneumoniae drawn within hours of hospital admission; ) resided in one of the five counties surrounding pennsylvania, and ) had a bacterial isolate confirmed in the laboratory as s pneumoniae. seventy-six patients had erythromycin-resistant infections and were selected as the case-patients for this study. the authors found that exposure to macrolides in the six months preceding infection, a history of influenza vaccination in the previous year, and hispanic ethnicity were all independently associated with an increased probability of erythromycin -resistant s. pneumoniae infection. among patients who reported taking antimicrobial agents in the months preceding infection, failure to complete the course of prescribed drugs was associated with an increased probability of macrolide resistance ([or = . ]; % ci . - . ). however, most patients with macrolide-resistant infections did not report any prior antimicrobial drug exposures. the authors assume in the discussion potential biases that may have affected the assessment of different risk factors. future studies correlating duration of therapy with risk for colonization with macrolide-resistant pneumococci would be useful to further explore this phenomenon. the incidence of invasive pneumococcal disease (ipd) in children and adults increases in winter, however possible underlying causes have not been carefully studied. this ecological study correlated population-based data on ipd and respiratory virus activity in the year in metropolitan new south wales, australia, with climatic parameters. seven hospitals participated in the study from may to october. during a year with good separation of respiratory syncytial virus (rsv) and influenza virus activity, it was demonstrated in children a significant correlation with rsv activity and ipd activity, but no significant correlation between influenza virus activity and ipd. in adults the epidemic curves of rsv and influenza virus activity corresponded with peaks of ipd, but it was only possible to demonstrate a statistically significant correlation when the activity of both viruses was combined. of the climatic parameters there was a clear inverse relationship between weekly mean minimum and maximum temperatures and ipd activity in adults and children. data from other temperature geographic areas and data obtained after the introduction of vaccines are required to confirm these findings. probably a case-control study comparing the isolation of respiratory viruses in patients with ipd matched with age, sex, location and time with control subjects without ipd would be required to specifically examine the role that respiratory viruses play in predisposing to ipd. the effect of falling temperatures on the pathogenicity of s penumoniae has not been extensively studied and warrants further investigation. human metapneumovirus (hmpv) has been found to be associated with lower respiratory tract infections although the pathogenesis remains to be elucidated. there are few reports of respiratory tract infections due to bacterial coinfection with hmpv. this hypothesis-generating study, performed from march to october , involved a cohort of , children in south africa randomized to receive the -valent pneumococcal polysaccharide-protein conjugate vaccine (pcv ) or placebo. these that were hospitalised ( . ) for lower respiratory tract infection (lrti) were tested for hmpv infection. by use of a nested reverse-transcription pcr assay targeted at amplifying a fragment of the hmpv fusion protein gene, such infections were identified among episodes of lrti in children. in fully vaccinated children, the incidence of hospitalization for a least one episode of hmpvassociated lrti was reduced by % (p = . ) overall, by % (p = . ) in human immunodeficiency virus (hiv)-uninfected children, and by % (p = . ) in hiv-infected children. there was a significant reduction in the incidence of clinical pneumonia among vaccine recipients overall ( %; p = . ), in hiv-uninfected children ( %; p = . ), and in hiv-infected children ( %; p = . ). there was no significant reduction in the incidence of hospitalization for hmpv-associated bronchiolitis among vaccine recipients in the entire study population. the absence of sensitive tools to diagnose bacterial pneumonia has been a major obstacle in order to define the role of bacterial-virus coinfection in humans. it has been documented that approximately one-third of children with respiratory syncytial virus associated pneumonia may have pneumococcal coinfections. the results of the present study suggest that bacterial coinfections, particularly pneumococcal infections, are an essential part of the pathogenesis of most severe hmpv infections progressing to pneumonia. an implication of this observation is that children hospitalized with the diagnosis of hmpv-associated pneumonia should be treated with antibiotics, and a significant proportion of these hospitalisations may be prevented by vaccination with pneumococcal vaccine. nevertheless more information is needed in order to clarify the pathogenic mechanisms of the mixed infections. in this study, the authors describe the clinical features, complications, treatment and outcome of episodes of community-acquired listeria monocytogenes meningitis in adults. these cases were included in a prospective nationwide observational cohort study of bacterial meningitis with a positive lcr culture, performed in the netherlands between october and april . over the period of study, cases of bacterial meningitis were diagnosed and these caused by l. monocytogenes represented %. the annual incidence l. monocytogenes meningitis was . cases per , adults. all patients were immunocompromised ( %) or > years old. in ( %) of patients, symptoms were present for > h, and the clinical presentation was subacute in patients ( % had symptoms for ≥ days). the classical triad of stiff neck, fever and alterations in mental status was present in % of the cases. gram staining of cerebrospinal fluid (csf) samples revealed the causative organism in ( %) of cases and a biochemical csf indicator of bacterial meningitis was present in ( %). the initial antimicrobial therapy was amoxicillin based for ( %) of patients. the coverage of initial antimicrobial therapy was microbiologically inadequate for ( %) of the patients. the mortality rate was % ( patients), and ( %) experienced an unfavourable outcome. inadequate initial antimicrobial therapy was not related to outcome. the study have limitations due to the selection of patients with a positive l. monocytogenes csf culture. the most severe patients (in which antimicrobial treatment is started before the csf is obtained or these with important neurological alterations) might have been excluded. nevertheless, this prospective study confirms that typical csf findings predictive for bacterial meningitis might be absent (biochemistry and gram stain), but in contrast with previous reports, patients with meningitis due to l. monocytogenes do not present with atypical clinical features. it is interesting to note the high rate of patients ( %) with hyponatremia and the development of a subarachnoidal hemorrhage in one patient, complications more frequently related with tuberculous meningitis. the authors review the microbiologic and epidemiologic data of , cases of human listeriosis reported in england and wales from to . ten common-source outbreaks affecting patients were excluded from the study. a significant increase was observed during the period - compared to - . the majority of the cases were sporadic ( , ) and not pregnancy-related ( , ) . deaths were more frequent in the group of nonpregnancy-related cases ( vs. %). the majority of the sporadic cases diagnosed between and occurred in patients > years of age with bacteriemia without cns affectation and without relation to sex, race, season of the year, socioeconomic differences or underlying conditions. serotypes b and / a were the most frequent throughout the entire study. the increase of cases of listeriosis shown in the second period analysed in this study could be due to an increased interest in reporting this illness. nonetheless, a change in the pathogenicity of l. monocytogenes could also explain the results. the advice to avoid high-risk foods for people of advanced age and for those that are immunocompromised, and not only for pregnant women, is a plausible message of this study. the immunized toddlers have a higher meningococcal antibody in serum samples than immunized infants. the authors determined meningococcal antibodies in serum samples obtained from children who were immunized with group c meningococcal vaccines - year earlier as infants or toddlers.the geometric mean serum antibody concentrations were higher in immunized infants or immunized toddlers than in un-immunized historic control ( . and . mcg/ml vs. . mcg/ml, respectively; p < . ). the proportion of immunized infants who had bactericidal titers ≥ : (considered to be protective when measured with human complement was higher than of immunized toddlers ( vs %; p < . ). even if threshold for protection is considered to be a serum bactericidal titer ≥ : then the respective percentages of serum samples with protective titers is % for immunized infants, compared with % for the immunized toddlers (p = . ). when passive protective was tested, % of serum samples from immunized infants and % of serum samples from immunized toddlers conferred protection against group c bacteremia, compared with % of serum samples from unimmunized historic controls (p < . ). there was no evidence of lower immunity in children immunized as infants than as toddlers. on the basis of serum bactericidal activity and/or passive protection, - % of both age groups are protected at - years after immunization, which is significantly greater than in unimmunised historical controls (< %). the aims of this study were to determine hsba (human serum bactericidal assays) gmts (geometric mean titers) and the percentage of participants with an hsba titer ≥ at - days after vaccination with menps. secondary endpoits were to determine the hsba gmt and geometric mean antibody concentration measured by elisa for the following populations: ) all vaccine recipients (at day ); ) all recipients of menps and mencv (analyzed separately for values at both day and day after vaccinations), and groups - (analyzed separately for all days on which blood samples were obtained) this was a phase iv, open-label randomized comparative trail. healthy - year olds who were vaccinated with a single dose of menjugate (chiron vaccines), a meningococcal serogroup c-crm glycoconjugate vaccine (mencv) in the - . previous immunization status was determined by reference to the centralized immunization records of the relevant child health computer departments. subject numbers were prospectively randomized in blocks of according to computer-generated blocked randomization scheme that allocated equal numbers of subjects to the groups. participants randomized to groups , , , or received the plain polysaccharide serogroup a and c meningococcal vaccine (menps), where persons randomized to groups or received mencv. a one-fifth dose of menps was used. blood samples of up to ml were obtained prior vaccination, twice more in the week after vaccination and at day - after vaccination. serum samples were analyzed for menc sba titer using hsba. an hsba titer ≥ was used to indicate a very conservative measure of protection. a total of participants were randomized to of groups. one hundred seventy-one of these had been randomized to group - (received menps) and randomized to groups ot (received mencv). an hsba titer ≥ was measured in the serum samples of . % of participants. no increase in hsba gmt was detected until days after administration of menps or mencv. all participants demonstrated an hsba titer ≥ at day after vaccination. the hsba gmts observed days following vaccination with mencv ( . ) were higher than those observed following vaccination with menps ( . ). elisa gmcs were similarly higher days after vaccination with men c or menps ( . vs. . , respectively) . this study shows persistence of sustained levels of bactericidal antibodies for at least years after vaccination of adolescent with mencv. after challenge of immunized adolescents with menps, there was no increase in sba observed until days after vaccination, indicating that immunological memory may be too slow to generate protection against this potentially rapidly invasive organism. the distribution of polymorphisms in three genes (sp-a ; sp-a and sp-d) in a cohort of patients with microbiologically proven meningococcal disease was studied. this study was realized in a cohort of patients with proven meningococcal disease. from july through november , all whole-blood samples obtained from patients were stored. the serogroup of the infecting organism, the disease outcome and the age of patients were recorded. the control group comprised healthy volunteers. samples were chosen at random from the cohort for sp-a/sp-d genotyping. in total, patients were genotyped for polymorphisms at all loci in sp-a , and for sp-a . data for sp-d polymorphisms were available for patients. in the control group, and individuals were typed successfully for sp-a and sp-a respectively, and were typed for sp-d. two alleles of sp-a had a significant association with susceptibility to meningococcal disease. homozygosity for a was associated with an increased risk of meningococcal disease (or . ; % ci . - . ), with a . % of the patient population homocygous for a compared with only . % of the control population. homozygosity for the rare snp (sp-a at codon ) was associated with an increased risk of meningococcal disease (or . ; % ci . - . ) and increased risk of death (or corrected for age . ; % ci . - . ). haplotype a/ a was higher in patients ( . %) than in control subjects ( . %; or . % ci . - . ). gene polymorphism of the binding domain of surfactant protein a- , sp-a at codon , increases susceptibility to meningococcal disease, as well as the risk of death. in the era of universal vaccination of children with the h. influenzae conjugate vaccine, it is critical to characterize the currently circulating strains for detection of any genetic change that could render the current vaccine no longer protective. in italy, this vaccine is included in the vaccine calendar since , ant the uptake of the population exposed has been > % since . the aims of this study were to evaluate the genetic and capsular structures of the available strains from patients with invasive disease from to , to assess the influence of vaccination in the changes observed in these structures.. the genetic diversity of the strains was analysed by pfge and capsular changes by southern blot. the strains isolated pre licensure ( - ) was compared with the strains isolated post licensure ( ) ( ) ( ) ( ) ( ) . the strains had by pfge a total of restriction patterns, with a clear predominante of of them. it should be stressed that almost half of the strains had a restriction pattern indistinguishable from f, the dominant and endemic clone in italy since . four strains were fromchildren that had received at least one dose of the vaccine. a high genetic homology was detected in . % of the isolates. the amplification of the locus of capsulation b showed that there is a significant difference between the two analysed periods. thus, the strains from the vaccine period, including those from children previously vaccinated, had a number of copies higher than in , % of the cases, compared to , % in the prevaccine period (p = , ). the results of this study indicate the importance of monitoring invasive strains. the use of molecular genetics applied to this end, has shown that vaccination has not conditioned the occurrence of genetic changes nor the disappearance of dominant clones; it has been followed, however, by relevant changes in the expression of the capsulation genes, that could explain, at least in part, the lack of immune response in some vaccinated children. non-typable haemophilus influenzae (nthi) is a frequent pathogen in patients with copd. when acute exacerbations are due to bacterial infection, nthi are the most frequent isolated organisms. in this report, nthi isolates from a variety of respiratory sources, were studied. also, the microbiological results of copd patients of monthly sputa collected prospectively or in case of exacerbation, collected during a period of years, were evaluated. identification and typing were done by standard methods. phenotypic variants were identified as h. haemolyticus with independent methods: análisis of rdna sequence, mlst, hybridization dna-dna y sequencing the gene encoding protein p . of a total of strains studied, . % of sputum isolates and . of nasopharingeal isolates had genotypic characteristics of h. haemolyticus. none of the invasive isolates studied had the genotypic characteristics of h. haemolyticus. molecular typing of sputum isolates made possible to demonstrate a statistically significant difference in the acquisition of a new strain of h. influenzae as a causal agent of an exacerbation in patients with copd ( . vs . %; p < . ; rr . ic % . - . ). the differences in the case of h. haemolyticus were not significant ( . vs. . %; p = . ; rr . ic % . - . ). from the results of this study, it can be inferred that h. haemolyticus is a frequent inhabitant of the respiratory tract of children and adults with copd, as a commensal, without pathogenic capacity. unfortunately, with the standard methodology used for identification of h. influenzae it is not possible the identification of the phenotypic variant h. haemolyticus, a fact that might have important clinical implications in the use of antibiotic treatment in many patients. the persistance of non-typable haemophilus influenzae (nthi) in patients with copd is thought to be related with interactions between this bacterium and human alveolar macrophages. the immune mechanisms that mediate this macrophage response are poorly known. the aims of this study were to demonstrate the importance of the alteration of the immune response of alveolar macrophages in the persistence of nthi in the lower airways in patients with copd. three groups of patients were included: esmokers with copd (n = ), esmokers without copd (n = ) and non-smokers (n = ). respiratory and blood samples were used for separation of purified macrophages. phagocytosis of adherent cells and survival of intracellular nthi, three different strains of nthi from patients with copd were used. alveolar macrophages of copd patients had diminished phagocytosis as compared with with macrophages from the other two groups in front of the three strains. however, phagocytosis of nthi by blood macrophages was similar in all three groups studied. finally, intracellular survival of nthi was not altered in the alveolar macrophages of the patients with copd. the results of the study demonstrate the existence of an alteration in the immune response of alveolar macrophages of patients with copd vs. nthi, as sown by a diminution of phagocytosis, that explains the persistence of this organism in the airways. the absence of alterations in blood macrophages indicates the existence of a compartmentalised immune response in this population. however, intracellular lysis of nthi is not altered in alveolar macrophages of copd patients the control of pertussis infection is not optimal, because neither vaccination nor natural infection induces longlived immunity. the estimated annual incidence of clinical pertussis infection in usa in adolescents and adults is about - cases per , person-years. the lack of specific clinical criteria and the limited availability of standardized serologic criteria make difficult the diagnosis of pertussis infection. a study to determine the impact of vaccination in adolescents and adults, as a part of a national institutes of health-sponsored multicenter, prospective, randomized acellular pertussis vaccine (ap) efficacy trial in the us, was recently reported. usa. the study was performed in adults ( - yearsold) in which one-half of the subjects received ap vaccine and one-half received hepatitis a vaccine (control subjects). all patients were observed for years for clinical illness (telephone calls every days) and serological studies obtained at baseline, + month and + months after vaccine application. for serological evaluation iga and igg antibodies to pertusis toxin (pt), filamentous hemagglutinin (fha), pertactin (prn) (antigens all included in vaccine), and fimbriae / (fim) (not included in vaccine), were quantitated by elisa. seroconversion rates for each antigen (pt was the most specific) ranged from . % to . % in unvaccinated pa-tients. authors estimate that the infection rate among unvaccinated adults is close to % over an -month period. in vaccinated patients the infection rate and the efficacy of vaccine are difficult to obtain. only . % of vaccinated patients had titers at year that were equal to or higher than the titres month after immunization and only . % of patients a -fold cut-off. fim antibodies (antigen not included in vaccine) were significantly less common among vaccinated subjects than among controls (p < . ) suggesting that infection was less frequent (although marginally significant) in vaccinated patients. in vaccinated and unvaccinated patients symptoms probably related to pertussis infection were more common in patients who had serologic evidence of infection. the incidence of b. pertussis infection in adults is about % per year and is usually asymptomatic. b. pertussis boosters may confer protection for adolescents and adults against symptomatic pertussis infection and may reduce transmission to others. the diagnosis of pertussis infection is problematic because of the lack of specific clinical criteria, insensitivity of culture and pcr and the limited availability of standardized serology tests. the aim of this study was to determine the usefulness of several procedures, including real-time pcr, for the laboratory diagnosis of pertussis, and to investigate clonal relationships among clinical isolates of bordetella pertussis. the study was performed in one tertiary hospital in madrid, spain. during months (august to october ) nasopharyngeal swabs were collected from paediatric and adult patients with symptoms of pertussis, and contact cases. the samples were processed by culture (regan-lowe medium), direct fluorescence assay (with polyclonal antibodies against wall antigens), and real-time pcr (light-cycler, primers bp , region is ). most of the isolates were further characterized by pulsed-field gel electrophoresis. among clinical samples corresponding to patients, b. pertussis was detected in samples by culture ( . %), samples ( . %) by dfa and samples ( . %) by real-time pcr. real-time pcr diagnosed and more cases than culture and dfa, respectively. in relation to culture, sensitivity and specificity of pcr were . % and %, respectively. authors consider that a probable partial immunity in patients may justify better results for pcr in relation to culture. seventeen clinical isolates were available for pfge analysis and different genotypes were identified. fourteen isolates were included in two genotypes (genotype c and genotype e). real-time pcr applied to the diagnosis of pertussis provides more positive results than dfa and culture. on the basis of these results, dfa should no longer be used for the diagnosis of b. pertussis infection. a to years cycle is observed in relation to b. pertussis infection, independently of vaccine programmes. some clones are dominant and one of them has circulated at least since . the aims of this study were to determine the prevalence of clinical isolates of e. coli resistant to the fluoroquinolones in hospitalizad patients, as well as the underlying mechanisms of resistance (mutations in genes gyra and parc), and tolerance to organic solvents as markers of of overexpression of the active efflux system acrab. e. coli with a levofloxacin mic > , mg/l were studied. of the fecal samples analysed, ( . %) e. coli with diminished fluoroquinolone susceptibility were isolated ( . %). of these isolates, had a levofloxacin mic > mg/l, these strains had a mean of mutations in genes gyra and parc and presented tolerance to organic solvents more frequently than the isolates with a levofloxacin mic < mg/l that exhibited a mean of one mutation in gyra. the prevalence of isolates with tolerance to organic solvents varied during the period of study. no clonal dissemination was detected. the main conclusions of this study are that colonization of the gut by escherichia coli with reduced susceptibility to quinolones in hospitalizad patients is common, and that the resistant determinants to the quinolones vary over time. resistance to nalidixic acid can be used in the identification of strains of e. coli with mutations in gyra. comparative study of the molecular epidemiology of escherichia coli of humans ( blood isolates and faecal isolates) and chicken ( faecal isolates) that were susceptible (n = ) o resistant (n = ) to ciprofloxacin, where an analysis of the filogenetic group, virulence genotype and the epidemiologic relationship with rapd and pfge were carried out. among human isolates, those resistant to ciprofloxacin were different of the ciprofloxacinresistant strains, while the sensitive and resistant isolates of chicken were indistinguishable. susceptible human isolates had more genes associated with virulence factors than resistant human isolates or susceptible or resistant chicken isolates. some resistant human isolates were very similar to some chicken isolates by rapd and pfge. this is another study that increases the degree of evidence to the hypothesis that ciprofloxacin resistant isolates can appear de novo (after selection pressure by enrofloxacin and other quinolones used in animal husbandry), in susceptible progenitors in the animal's gut, then be transmitted to humans via the food chain and, finally cause infections in humans with ciprofloxacin-resistant strains. this is a retrospective study of the predisposing factors, clinical presentation and evolution of episodes of esbl-producing escherichia coli bacteraemia seen from january to march in a single institution; it represents a , % of the total cases of e. coli bacteraemia seen during the same period. % of esbl-harbouring e. coli produced ctx-m types and the great majority was not clonally related; % were of nosocomial origin, % were from geriatric centers, and % from the community. the number of cases increased from in to in . the mean age of the infected patients was , % were male, % had a foley catheter, % had urinary tract obstruction and patients ( %) had received antimicrobials in the recent past (aminopenicillins, third generation cephalosporins, and fluoroquinolones).crude mortality was %. patients treated with a combination of β-lactam/β-lactamase inhibitor or a carbapenem had a mortality of % as compared with a mortality rate of % in those treated with cephalosporins or fluoroquinolones (p = . ). the failure rate of those treated with a cephalosporin was independent of the mic values; however the number of patients studied was small and this finding has o be interpreted with caution. in areas where the prevalence of esbl-producing e. coli is increasingly represented, therapeutic guidelines should be revised. given the characteristics of the population described, this suggerence is particularly important when sepsis is diagnosed in old males, carriers of a urinary catheter and those that had been treated with antimicrobials in the recent past. the auhors decribe the case of a years old woman with a prosthetic mitral valve that had a klebsiella pneumoniae bacteraemia secondary to an infected intravenous catheter. the initial isolate produced an esbl, susceptible to ciprofloxacin (mic, . mg/l) and piperacillin/tazobactam (pt). patient was with a combination of ciprofloxacin and amikacin, and had recurrent bacteraemia days later. this time the blood isolate was resistant to ciprofloxacin (mic, mg/l) and an echocardiogram showed vegetations in the prosthetic valve. ciprofloxacin was replaced by piperacillin/tazobactam. however, after two weeks treatment bacteraemia persisted and repeat blood cultures grew pt resistant e. coli. finally, the patient underwent prosthetic valve replacement and was treated with meropenem. the three isolates were genotypically identical. the resistance to ciprofloxacin was associated to a mutation in gyra. the activity of pt against inolcula of and ufc/ml showed that with low inocula, the first two isolates were sensitive to pt, while the third isolate had an mic > mg/l. using high inocula, all three isolates had an mic > mg/l. no changes in activity were seen with meropenem in relation to the bacterial inoculum used. the three isolates were susceptible to cefoxitin and amikacin (mic, mg/l). this case exemplifies two important points: first, the possibility of therapeutic failure with pt in case of infection by esbl-producing k pneumoniae and, second, the combination with amikacin does not avoid the emergence of resistance to the companion antibiotic, in this instance, ciprofloxacin. the mechanism of resistance to pt was not characterized; no mutations were identified in the gen bla that could explain the resistance to tazobactam, on the other hand, the susceptibility to cefoxitin ruled out the possibility of resistance due to a loss of a porin. this study analysed the presence of esbl-producing enterobacteriaceae in clinical samples, in faecal samples of the population seen in the emergancy department, in sewage waters, in faeces of farm animals, in faeces of patients with gastroenteritis, and in samples from food. faecal samples were cultured in a mg cefotaxime containing medium. the study was carried out in and during the same period the estimated comsumption of antimicrobials in ambulatory care was measured. the prevalence of esbl-producing enterobacteriaceae was . % in clinical isolates, . % in faecal samples, and % in samples obtained in patients with gastroenteritis. their prevalence in samples from farm animals ranged from and %, and in food was . %. of note, % of food samples analysed had been previously cooked. the prevalence of faecal carriers was significantly higher in july to november as compared to the period of february to may. the lowest prevalence rates corresponded to the periods with highest antibiotic consumption. given the fact that amoxicilin-clavulanate was the most frequently used antimicrobial, the authors speculate on the possibility that its use could be a reason for a lower detection of esbl-producing strains. however, the impact of other variables such as the difference in alimentary habits (increased comsumption of of uncooked foods during the summer) should be considered. the prevalence of esbl-producing enterobacteriaceae in this area of spain is considerable in all different environments studied. these findings suggest that the wide dissemination of these strains in the community could be transmitted to humans via the food chain. metalo-beta-lactamase (mlb) producing enterobacteriaceae are increasingly recognized in some european countries, particularly vim derivatives. in greece, they have been identified in escherichia coli, enterobacter cloacae and in klebsiella pneumoniae. the last species is considered endemic in this country. vourli et al detected (between june and march ) in a single institution in greece seven proteus mirabilis isolates resistant to cefotaxime, ceftazidime and imipenem, but susceptible to aztreonam. the use of the double disk diffusion assay with edta and imipenem was useful to ascertain the production of a mlb, unlike the e-test. pulsed-field electrophoresis analysis revealed four related clones representing all these isolates indicating persistence of this strain in the hospital setting. in all cases, vim- metallo-beta-lactamase was identified. the corresponding bla vim- gene was detected in a chromosomally encoded integron platform widely disperse in greece and commonly associated with a plasmid. all p. mirabilis isolates were recovered form patients hospitalized during a prolonged period of time in three different wards, which indicate persistence over time and dispersion through the single institution. this is the first description of a p. mirabilis producing vim- that also illustrates the potential for dissemination of multi-resistance isolates. this article should also be considered as an alert of potential failure of the etest strips in the detection of mlb in certain enterobacteriaceae. metalo-beta-lactamases (mbls) in pseudomonas aeruginosa have been mainly described in asia and europe and remain scarce in the usa. aboufaycal et al. detected three multi-resistant p. aeruginosa isolates with a positive metallo-beta-lactamase test during a continuous surveillance study (the mystic study) performed in a single institution in usa (anderson cancer center) over a year period ( to ). isolates were recovered from unrelated patients and displayed different pulsed-field electrophoresis patterns as well as different ribotypes suggesting an independent emergence, possibly under carbapenem usage. two of these isolates produced the vim- enzyme, in one of them simultaneously with oxa- , whereas vim- enzyme was identified in the remaining one. in both vim- producing isolates, bla vim- gene was associated with an identical integron platform, which may suggest horizontal gene transfer processes. both patients were treated with carbapenems. interestingly, the vim- producing isolate was recovered form a patient previously treated with carbapenems in jordan. this study shows a well documented example of selection and spread of multi-resistant isolates from distant geographic areas, in this case, a vim- producing pseudomonas aeruginosa. an interesting point addressed by the authors was the phenotypic detection of mlbs in these p. aeruginosa isolates. in one them the double disc synergy approximation test with edta was negative with imipenem and in another one with meropenem. in addition, in two isolates this double disk diffusion assay was negative with ceftazidime. the use of -mercaptopropionic acid did not enhanced detection of mlb producing p. aeruginosa isolates. these results also illustrate the laboratory difficulties to detect the presence of mlbs in p. aeruginosa. pseudomonas aeruginosa is a nosocomial pathogen with resistance to antimicrobials, both intrinsic and acquired. the rise of resistance has evolved to the appearance of epidemic caused by strains only susceptible to polymyxins, but the risk factors associated to this fact are not well known. the present study was designed to know the risk factors associated to the development of infections caused by strains of p. aeruginosa only susceptible to polymyxins. a retrospective case-control study was carried-out in a tertiary hospital in athens, greece, including all the patients with a episode of p. aeruginosa bacteraemia (n = ) between january and august . cases were those with bacteraemia caused by strains only susceptible to polymyxins (n = ) and controls those with bacteraemia caused by strains susceptible at least to polymyxins and carbapenems (n = ). those patients with bacteraemia caused by p. aeruginosa strains susceptible to polymyxins and other antimicrobials, but resistant to carbapenems, were excluded (n = ). the most common sources of bacteraemia were pneumonia, unknown, urinary tract infections, and intra-abdominal infections. different factors were evaluated, including the use of antimicrobials, considering only those that the patients had received during the hospitalization and at least for three days. mortality rates were . % and . % in cases and controls, respectively. a multivariable logistic regression model revealed that the only factor associated to the development of infec-tions caused by strains of p. aeruginosa only susceptible to polymyxins was the previous use of carbapenems (or ; p = . ). this study proved the rapid emergence of resistance to carbapenems among patients infected by p. aeruginosa receiving this antimicrobial class. the exclusion of patients infected by strains susceptible to polymyxins but resistant to carbapenems is a major limitation of the study because the over-estimation of the use of carbapenems as a risk factor, prompted by the criteria to select the control group and to exclude patients of the study. also, the authors do not comment if the occurrence of infections by strains only susceptible to polymyxins was related to outbreaks nor they made molecular analysis of these strains, to be sure that there was not horizontal transmission of them. the objective of this manuscript was to review the non resolved aspects of the infections caused by acinetobacter baumannii. the genus acinetobacter consists of strictly aerobic, gram-negative cocobacillary rods. different studies have resulted in the description of genomic species, of the have been validated. in relation with the habitat, acinetobacter colonize skin of human beings (< % a. baumannii); recent studies have shown a. baumannii in unsuspected reservoirs as foods (fruits and vegetables) and arthropods (body lice); a. baumannii has been the cause of infections in traumatic injuries in iraq, kuwait, afghanistan, and vietnam, suggesting environmental contamination of wounds, although the source remains unknown; also, a. baumannii shows tolerance to the desiccation. a. baumannii develops rapidly antimicrobial resistance, related to the use of antimicrobials in hospitals, through multiple mechanisms: plasmids, integrons, transposons, and natural transformation; it results in the frequent appearance of strains resistant to almost all available antimicrobials. the most frequent infections caused by a. baumannii affect respiratory tract, surgical wounds, urinary tract, and bacteraemia. the crude mortality is high in some infections, as bacteraemia and ventilator-associated pneumonia, although they seem have non-attributable mortality in case-control studies. the frequency of infections and colonisations by a. baumannii differs among hospitals, and they are mostly nosocomial; the incidence in paediatric wards is low. the outbreaks of infections by a. baumannii are facilitated by its resistance to the desiccation and antimicrobial resistance; they occurred in all hospital areas, being more frequent in intensive care units; multiple risks factors have been identified, including wards with high density of colonized/infected patients, environmental contamination, and carriage by hands of staff members; the adherence to strict infection-control measures is useful. the complexity of the epidemiology of a. baumannii infections is highlighted by the existence of centres with endemic/epidemic situations in which multiple clones are responsible of colonisations/infections; also, there is coexistence of outbreaks with sporadic cases of infection. there has been transmission of strains among different sanitary centres in several countries. respect to the treatment, different clinical studies have shown the efficacy of sulbactam and colistin in the treatment of cases of infections caused by multi-drug resistant strains, although there have been contradictory results in experimental models in the case of colistin. in depth review of some selected aspects of acinetobacter baumannii infections. the authors identify several fields in which we need more knowledge: virulence of a. baumannii, identification of reservoirs, strategies for the control of multi-drug resistant strains, and the treatment of infections caused by these strains. acinetobacter baumannii is a nosocomial pathogen causing, among other infections, hospital-acquired pneumonia (hap). previously, there were some reports of communityacquired pneumonia (cap) caused by a. baumannii. the objective of this manuscript was to analyze the clinical and prognostic characteristics of cap by a. baumannii and to compare them with hap by this bacterium. the method was a retrospective case-control study (cases: cap; controls: hap) carried-out in a regional hospital in hong kong between july and december . pneumonia was defined by clinical and radiographic criteria from the centers for disease control and prevention. a. baumannii was considered "definite pathogen" if isolated from blood or pleural fluid, and "probable pathogen" if isolated from sputum or tracheal aspirate cultures. nineteen cases (cap) and controls (hap) were analyzed. compared with the hap, cap had more smokers ( vs. %; p = . ) and more patients with chronic obstructive pulmonary disease ( vs. %; p = . ). also, from a clinical point of view, cap had bacteraemia more frequently ( vs. %; p < . ), as were the presence of acute respiratory distress syndrome ( vs. %; p < . ), and disseminated intravascular coagulation ( vs. %; p = . ). finally, the mortality rate at days, was higher in cap than in hap ( vs. %; p < . ). factors associated to mortality in cap were: bacteraemia, low platelet count, acidosis, and disseminated intravascular coagulation. these data are consistent with that from previous studies. one limitation of the results is that the number of polymicrobial pneumonias was high: % and % in cap and hap, respectively, resting specificity to 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staphylococcal infections: diagnostic and therapeutic implications staphylococcus epidermidis: why is it so successful? biofilms: survival mechanisms of clinically relevant microorganisms susceptibility of staphylococcus epidermidis biofilm in csf shunts to bacteriophage attack key: cord- - b rvr authors: abrantes, joana; droillard, clément; lopes, ana m.; lemaitre, evelyne; lucas, pierrick; blanchard, yannick; marchandeau, stéphane; esteves, pedro j.; le gall-reculé, ghislaine title: recombination at the emergence of the pathogenic rabbit haemorrhagic disease virus lagovirus europaeus/gi. date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: b rvr rabbit haemorrhagic disease is a viral disease that emerged in the s and causes high mortality and morbidity in the european rabbit (oryctolagus cuniculus). in , a new genotype of the rabbit haemorrhagic disease virus emerged and replaced the former circulating lagovirus europaeus/gi. strains. several recombination events have been reported for the new genotype lagovirus europaeus/gi. , with pathogenic (variants gi. a and gi. b) and benign (genotype gi. ) strains that served as donors for the non-structural part while gi. composed the structural part; another recombination event has also been described at the p /p junction involving gi. strains. in this study, we analysed new complete coding sequences of four benign gi. strains and four gi. strains. phylogenetic and recombination detection analyses revealed that the first gi. strains, considered as non-recombinant, resulted from a recombination event between gi. and gi. , with gi. as the major donor for the non-structural part and gi. for the structural part. our results indicate that recombination contributed to the emergence, persistence and dissemination of gi. as a pathogenic form and that all described gi. strains so far are the product of recombination. this highlights the need to study full-genomic sequences of lagoviruses to understand their emergence and evolution. in this study, we obtained eight new genome sequences: four for gi. and four for gi. . these included four strains previously identified based on their capsid sequences as gi. (ja / - , bo / - , ja / - and cha / - ; genbank accession numbers: lt , lt , lt and lt , respectively) and two strains as gi. ( - and - ; genbank accession numbers: he and he , respectively) , . we obtained the full-length genomic sequences of strains - and - , the complete coding sequences (cds) of strains - and - , and the cds along with the ′ untranslated region (utr) of the strains ja / - , bo / - , ja / - and cha / - . the genomes of the earliest gi. strains - and - were , nucleotides (nt) long and shared . % nucleotide identity. the length of the ′ utr sequences is nt and that of the ′ utr is nt. the closest strains were gi. strains with an average nucleotide identity of . % ( . % for the non-structural part of the genomes), while the average identity with gi. strains was % ( . % for the non-structural part of the genomes). the genomes of the gi. strains were , nt long (excluding the ′ utr), except the cha / - strain that was , nt long. the ′ utr sequences had the same length ( nt) . when including the gi. - sequence (genbank accession number mn ), the average nucleotide identity between the five gi. strains was . %. the cds of the four gi. showed between . % and . % of nucleotide identity ( . % between - and - strains) , and when compared to gi. cds presented no deletions or insertions ( , nt long), while the gi. cds presented a deletion of six consecutive nucleotides within the capsid gene ( , nt long instead of , nt). this deletion is present in the benign strains - , ashington and the italian rcv (genbank accession numbers: ef and x ) , , and comprises a codon putatively under positive selection in gi. strains . the strain cha / - had a further three nucleotide deletion in the minor structural protein vp (positions - , amino acid ). the complete dataset ( sequences; , nucleotides) was screened for recombination with rdp (recombination detection program) . the different methods available in rdp detected as recombinants, with strong statistical support (p-values < . ; table ), all the strains from the early gi. outbreaks (strains - , - and n ), all the gi. strains previously identified as non-recombinants (strains cbval , zar - , rij - , tar - , zar - and seg - ) and gi. strains more recently detected ( plm , nl , canada , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a single recombination breakpoint was determined at the non-structural/structural parts boundary (table ) . for most of these strains, the gi. strain - was identified as the most likely donor for the non-structural part, with the exception of strain - for which the most likely donor is cha / - . a gi. strain ( - _barrancos_portugal_ or sos _portugal_ , genbank accession numbers kf and mg , respectively) was the most likely donor for the structural part. thus, according to rdp all these sequences (gi. strains from the early outbreaks and gi. non-recombinant strains) are gi. /gi. recombinants. the tanglegram based on the alignments for the non-structural and the structural genome partitions also evidenced as recombinants the strains identified by rdp (fig. ) . the phylogenetic analysis was performed taking into account the location of the recombination breakpoint detected by rdp at the junction of the non-structural and structural parts. in the ml tree of the structural part, which includes vp and vp (fig. i) , the gi. strains formed a strongly supported group (bootstrap value ), that was a sister group to gi. (bootstrap value ). the mrcv strain, a gi. -like/gi. -like recombinant , also grouped with the gi. strains (bootstrap value ). according to the nomenclature recently proposed for lagoviruses, mrcv does not meet the criteria to be considered as a member of the gi. group and is currently unclassified . indeed, the genetic distance between mrcv and gi. vp gene sequences is > % (data not shown) and no other similar strains (from independent outbreaks) had been identified. as for the four gi. sequences obtained in this study, they clustered within a highly supported group (bootstrap value ) formed , gi. strains previously considered as non-recombinants (marked with an *), and the strains from canada (canada ) and the netherlands (nl ). the ml tree for the non-structural protein p (see supplementary information), recapitulated most of the results observed in the ml tree for the non-structural part with the exception of the positioning of the portuguese strains sos , sos , sos , sos , sos , sos , sos and sos , that appear clustered with gi. . strains sos and sos were previously shown to be triple recombinants between gi. , gi. b and gi. (gi. for p , gi. b for the remaining non-structural proteins and gi. for the structural proteins) which is in agreement with the results obtained in this study. as for strains sos , sos , sos , sos , sos and sos , the tree suggests that their genome also originated from three different strains: gi. for p , gi. for the remaining non-structural proteins and gi. for the structural proteins. in this case, it most likely involved recombination between a gi. strain and a gi. /gi. recombinant strain. a tanglegram analysis and rdp further confirmed these results (see supplementary information). gi. was identified as a novel pathogenic form of lagovirus in france in . field observations and further characterisation of the strains revealed unique characteristics in comparison with former strains such as the ability to cross the species boundaries [ ] [ ] [ ] [ ] [ ] and to kill young rabbits , . previous studies also revealed the occurrence of recombination of gi. strains with non-pathogenic (gi. ) and pathogenic (gi. a and gi. b) strains - , showing an important role of recombination in generating diversity in gi. and confirming the high capacity of recombination within lagoviruses. our results show that the strains circulating at the time of the first noticed gi. outbreaks were already recombinants between a european non-pathogenic strain (gi. ), that was donor for the non-structural part, and the new lagovirus genotype, gi. , that formed the structural part. indeed, in our ml tree for the non-structural part, the strains from the first gi. outbreaks ( - and - ) grouped with the non-pathogenic gi. group, while for the structural part these strains clustered with all the strains of the newly emerged gi. genotype. strains from the earliest outbreaks in spain and portugal in - , and strains collected in the canary islands (spain) , france, the netherlands and canada in are also gi. /gi. recombinants. these results, that were confirmed by the rdp, the ml trees and the tanglegram analyses, demonstrate that all the gi. strains described so far, including those that were considered non-recombinants and that in some instances co-circulated with other recombinant gi. strains , resulted from recombination. several recombination events are thus associated with the evolution of this new genotype: with gi. , gi. , gi. b and gi. a, with a recombination breakpoint at the non-structural/structural boundary , . an additional recombination event was further previously reported with a breakpoint at the p /p boundary and involved gi. as the donor of p , while gi. /gi. (see also supplementary information) or gi. b/gi. recombinants were the donors for the remaining viral genome . notably, the pattern described here for lagoviruses, with a recombination hotspot at the start of the major structural gene that encodes the capsid, is typical of caliciviruses [ ] [ ] [ ] . indeed, the combination of low sequence divergence , presence of complex rna secondary structures that may promote template switching by the virus polymerase, and the existence of a subgenomic rna that may act as a secondary template when rna replication cbcoruche - _portugal_ www.nature.com/scientificreports/ is resumed upon template switching, seem to contribute for the high rates of recombination observed in this region in the genome of caliciviruses and that likely constitutes an important survival strategy in the evolution of this family . this strategy, that resembles antigenic shift in influenza virus , allows caliciviruses to rapidly generate diversity by producing new genomic combinations, which might be beneficial for the adaptation to new hosts and environments, and to overcome selective pressures. recombination is important in shaping the evolution of rna viruses, including the closely related picornaviruses and coronaviruses and, more importantly, is often associated with the emergence of new viruses and even families, e.g. the hepeviridae family . as for the recombination involving the breakpoint at the p /p boundary, it may have originated from a non-replicative recombination mechanism where rna strands are randomly self-ligated or joined by cellular ligases . atypical recombination breakpoints such as this have been also observed for other caliciviruses and a similar mechanism proposed . estimation of the time to the most recent common ancestor (tmrca) of gi. by using capsid sequences pointed to an emergence in july . although the estimated substitution rates may be inaccurate due to limited sampling, reduced sequence variation and low temporal spread, they tend to underestimate rather than overestimate the real tmrca , . thus, it is possible that despite surveillance efforts, gi. circulated unnoticed in wildlife prior to its detection . the virus that recombined with gi. to produce this new genotype is currently unknown and undetected, even with the molecular surveys of lagoviruses performed in european leporids and our improved knowledge of the complete coding sequences of both pathogenic and benign lagoviruses. the same occurs for some older recombinant iberian strains where the virus that originated the non-structural part has never been detected . both viruses, the one originating gi. and the one of these iberian strains, could have either circulated harmlessly prior to their detection, thus making difficult to detect them, or have become extinct due to their lower fitness as a non-recombinant form, as suggested for noroviruses that recombined and whose partial sequences were never found . the evidence for recombination with gi. a , gi. b , gi. and gi. , (see also supplementary information) reveals that gi. successfully recombines with a great diversity of pathogenic and non-pathogenic lagoviruses. this, together with the tmrca estimates and the hypothesis of circulating harmlessly before its emergence, supports an evolution from a non-pathogenic form that acquired pathogenicity . we suggest that evolution of www.nature.com/scientificreports/ pathogenicity was not driven solely by point mutations, but was aided by recombination events. similarly to other rna viruses, lagoviruses genomes may function as interchangeable modules rather than as strict genomes, which leads to the appearance of mosaic-like genomes through recombination, resulting in a semi-independent evolution of structural and non-structural genes , and with low-fitness regions being eliminated by recombination . this might help to explain how a non-pathogenic strain (gi. ), combined with a potentially benign strain (gi. ), gave rise to a highly lethal, pathogenic strain. non-pathogenic forms are thus likely to be involved in the evolution of pathogenic forms and recombination might have precipitated the emergence of lagovirus europaeus/ gi. . the hypothesis of a species jump may not be discarded. indeed, reservoir species could have acted as source for the original virus that jumped to the final host, the european rabbit . whether recombination occurred in the reservoir species or in the final host is unknown. nonetheless, reservoir species have yet to be identified in which lagoviruses could have evolved and replicated without being detected before "jumping" into the european rabbit (le gall-reculé, personal communication) [ ] [ ] [ ] [ ] . genetic mechanisms such as recombination require coinfection of a host cell by two (or more) viruses. the high frequency of recombination detected in lagoviruses also implies a high frequency of co-infection, either in the host population or in the reservoir species. leeks and colleagues established a link between co-infection and viral diversity, as more diverse populations can produce more virulent infections and better adapt to new hosts . this might explain the array of hare species infected by gi. [ ] [ ] [ ] [ ] [ ] , which constitutes a novelty in relation to gi. . our results add complexity to the diversity of lagoviruses, but help to elucidate our current understanding on the emergence of pathogenic forms. in addition, they show that full genomic sequences of lagoviruses are crucial to understand their evolutionary history and genetic relationships. future studies should focus on unravelling the role of the structural and the non-structural parts in the emergence of pathogenicity in lagoviruses. virus samples and full-length genome sequencing. no animals were captured, handled or killed specifically for the purpose of this study. duodenum samples were collected from four hunted french rabbits by the national hunting and wildlife agency (oncfs) during the hunting seasons in - and - . presence of lagoviruses had previously been detected in these samples and phylogenetic relationships of the obtained capsid vp gene sequences had been determined ; these were identified as gi. strains. four liver samples were further collected in france from dead rabbits collected in three rabbitries affected by rhd, two on november ( - and - gi. strains ) and one in june (strain [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and in the field on june (strain . the presence of gi. in the - sample was diagnosed by labovet conseil (les herbiers, france). we characterised this strain as well as the - one based on their complete vp gene sequences, confirming that they belong to the gi. genotype. for the sequencing of the complete coding genome of gi. strains, ~ mg of duodenal tissue were homogenized in a mixer-mill disruptor (tissuelysed, qiagen, hidden, germany). rnas were first extracted with trizol ® (trizol ls reagent, ambion, thermo fisher scientific, villebon-sur-yvette, france) before using the rneasy mini kit (qiagen, hilden, germany) to increase sensitivity . for gi. strains, rna was extracted from µl of liver exudate using the rneasy mini kit. except for two gi. strains ( - and - ) for which the coding sequence was obtained using ngs (see below) from purified and quantified rna using the rneasy minelute cleanup kit (qiagen, hilden, germany) and the qubit rna hs assay kit with the qubit . fluorometer (thermo fisher scientific, villebon-sur-yvette, france), respectively, rnas were reverse-transcribed using oligo-dt as a primer and maxima reverse transcriptase (thermo fisher scientific, villebon-sur-yvette, france). protocols were performed as recommended by the manufacturers. according to the strains, different pcr strategies were attempted for genome sequencing either by using combinations of newly designed primers (primer sequences available upon request) or primers previously published , , . thus, cdnas of ja / - , bo / - and cha / - gi. strains were amplified using several overlapping pcrs, including one that covered the recombinant breakpoint between the non-structural and structural encoding genes, using expand high-fidelity pcr system (roche, sigma-aldrich, saint-quentin-fallavier, france). for ja / - gi. strain, due to lower viral loads, after two first pcrs that amplified two overlapping fragments of about , and , bp, respectively, several nested or semi-nested overlapping pcrs were performed. the complete coding sequences of the four gi. genomes were obtained following either one pcr amplification of about , bp using the primers u and l followed by smaller overlapping pcrs for sequence confirmation ( - and - ; primer sequences available upon request), or ngs ( - and - , see below). for these two last strains, the ′ end sequence of the cds ( bp) was confirmed using the pcr primers u and l as described in le gall-reculé et al. ( ) . pcrs were performed using expand high fidelity enzyme (roche-applied-science). the different pcr products were analysed by electrophoresis, purified using the minelute pcr purification kit (qiagen, hilden, germany) and quantified using the qubit dsdna hs assay kit with the qubit . fluorometer (thermo fisher scientific, villebon-sur-yvette, france). dna sequences were determined with an abi prism genetic or a series genetic analysers in both directions (applied biosystems, foster city, ca, usa), using the pcr and several inner primers and big dye terminator v . (life technologies) as recommended by the manufacturer. the ′ and ′ ends of the - and - strains were obtained using the rapid amplification of cdna ends (race) method following the protocol developed in lemaitre et al. ( ) . pcr products were purified and sequenced as described above. consensus sequences were compiled using vector nti advance software (life technologies, thermo fisher scientific, villebon-sur-yvette, france). for ngs, cdna libraries were prepared using the ion total rna-seq kit (life technologies, carlsbad, ca, usa) according to a protocol adapted from the supplier's instructions (available upon request from the authors). sequencing was performed using ion proton sequencer (life technologies). the reads were cleaned with the the aligned reads of this third alignment were collected and then down-sampled to fit a global coverage depth estimation of x. these cleaned reads were submitted to the spades (version . . ) de novo assembler and their raw related reads to the mira (version . . ) de novo assembler. the de novo contigs were then submitted to megablast on a local copy of nucleotide databank. the best lagovirus reference identified with megablast was used in a last bwa alignment of cleaned reads. the genomic sequences produced in this study have been deposited in genbank under the accession numbers: mn , mn , mn , mn (gi. strains ja / - , bo / - , ja / - and cha / - , respectively); mn , mn , mn , mn (gi. strains - , - , - and - , respectively). complete coding sequences obtained in this study were aligned with publicly available complete coding sequences of lagovirus europaeus representing genotypes gi. , gi. , gi. and gi. in bioedit software version . . . the final dataset included sequences, , nucleotides in length (see supplementary information for the list of the sequences used). the dataset was screened for recombination by seven methods available in the rdp software version . (rdp, geneconv, bootscan, maxchi, chimaera, siscan and seq) with the following parameters: sequences were set to linear, bonferroni correction, highest acceptable p-value of . and permutations. only recombination events detected by three or more methods were considered. recombinant strains were visualized by plotting a tanglegram using the "dendextend package" in the rstudio software (version . . ) . phylogenetic analysis. pairwise nucleotide distance comparison (p-distance model) was computed using mega . following the results obtained with rdp, phylogenetic analyses were carried out separately for the following genome partitions: nucleotide positions (i) - (p ; supplementary information), (ii) - , (p , p , p , vpg, protease and rdrp), and (iii) , - , (vp and vp ). maximum-likelihood phylogenetic trees were inferred in mega using the best model of nucleotide substitution determined in the same software for the different genome partitions, according to the lowest aicc value (akaike 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and g.l.g.r. performed the analyses, discussed the data, wrote and revised the first draft of the manuscript. c.d. obtained the molecular data, performed the analyses, discussed the data and revised the first draft. e.l. obtained the molecular data, discussed the data and revised the first draft. p.l. and y.b. analysed the ngs data. s.m. was responsible for the gi. strains sample collection, discussed the data and revised the first draft. p.j.e. discussed the data and revised the first draft. all authors contributed to the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to j.a.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - qcoxl authors: nicklas, werner; bleich, andré; mähler, michael title: viral infections of laboratory mice date: - - journal: the laboratory mouse doi: . /b - - - - . - sha: doc_id: cord_uid: qcoxl viral infections of laboratory mice have considerable impact on research results, and prevention of such infections is therefore of crucial importance. this chapter covers infections of mice with the following viruses: herpesviruses, mousepox virus, murine adenoviruses, polyomaviruses, parvoviruses, lactate dehydrogenase-elevating virus, lymphocytic choriomeningitis virus, mammalian orthoreovirus serotype , murine hepatitis virus, murine norovirus, murine pneumonia virus, murine rotavirus, sendai virus, and theiler’s murine encephalomyelitis virus. for each virus, there is a description of the agent, epizootiology, clinical symptoms, pathology, methods of diagnosis and control, and its impact on research. in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but clinically inapparent infections may also have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system, or both at the same time but on different parts of the the laboratory mouse Ó elsevier ltd. all rights reserved. isbn - - - - doi: . /b - - - - . - immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase interindividual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells or tumours [ , ] . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals [ ] . mouse antibody production (map) testing or polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to find. to address this problem, several meetings have been held on viral complications on research. the knowledge available is summarized in conference proceedings [ , ] and has later repeatedly been reviewed [ ] [ ] [ ] . viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. the nomenclature and taxonomy of viruses is described based on recent nomenclature rules by the international union of microbiological societies [ ] and the universal virus database of the international committee on the taxonomy of viruses (http://www. ictvdb.org). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with the methods presently available. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via the germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with a virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of the virus. an animal may have been infected, mounted an effective antibody response and cleared the virus, but remains seropositive for weeks or months or for ever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct detection methods such as virus isolation, electron microscopy or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and, depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary, including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpesviruses and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices [ ] [ ] [ ] [ ] [ ] [ ] . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, these animals are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can cause natural infections in mice (mus musculus). mouse cytomegalovirus (mcmv- ) or murid herpesvirus (muhv- ) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus (muhv- ) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both are enveloped, double-stranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar [ ] . mcmv- is very uncommon in european and american colonies of laboratory mice and is found at a very low rate [ ] or reported as not found [ , ] . seropositivity has, however, been reported from asian countries [ , ] . testing for mtv is not frequently reported, and no sample tested positive in recent studies [ ] . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv- and mtv are frequently found in wild mice, which may be coinfected with both viruses [ , [ ] [ ] [ ] . mouse cytomegalovirus (mcmv- ) or murid herpesvirus (muhv- ) natural infection with mcmv- causes subclinical salivary gland infection in mice. like other cytomegaloviruses, mcmv- is strictly hostspecific. it persists in the salivary glands (particularly in the submaxillary glands) and also in other organs [ ] [ ] [ ] . the virus can be cultured in mouse fibroblast lines like t cells, but primary mouse embryo fibroblasts are more sensitive to infection and produce higher virus titres. however, passage in cell culture results in its attenuation. to maintain virulence, the virus is best propagated by salivary gland passages of sublethal virus doses in weanling mice of a susceptible strain (e.g. balb/c) [ ] . most information concerning the pathogenesis of mcmv- infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history [ ] , virus dose and route of inoculation [ ] . in general, newborn mice are most susceptible to clinical disease and to lethal infection and develop higher levels of resistance with increasing age. infection of neonates leads to abnormal brain development [ , ] . virus replication is observed in newborn mice in many tissues and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days and of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains [ ] . mice of the h- b (e.g. c bl/ ) and h- d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h- k haplotype (e.g. c h), which are approximately -fold more resistant to mortality than those of the b or d haplotype [ ] . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone) or critical illness such as sepsis [ ] . reactivation of mcmv- also occurs after implantation of latently infected salivary glands into prkdc scid mice [ ] . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn nu and lyst bg mice are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver and other organs, and multinucleate syncythia with inclusion bodies in the liver [ ] . similarly to aids patients infected with human cytomegalovirus, athymic foxn nu mice experimentally infected with mcmv- also develop adrenal necrosis [ ] . the virus also replicates in the lungs, leading to pneumonitis, whereas replication and disease are not seen in heterozygous (foxn nu/þ ) littermates [ ] . the pathogenesis of mcmv- infection in immunocompetent and in immunocompromised mice, as well as the role of the immune system, have been reviewed by krmpotic et al. [ ] . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and are found also in other organs such as liver, spleen, ovary and pancreas [ ] . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis or splenic necrosis in susceptible strains [ , , ] . virus is transmitted by the oronasal route, by direct contact and is excreted in saliva, tears and urine for several months. the virus is ubiquitous in wild house mice worldwide. they serve as a natural reservoir for infection and can even be infected with different virus strains [ ] . the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. sexual transmission and transmission with tissues or organs is also possible. the virus does not cross the placenta in immunocompetent mice, although infection of pregnant females results in fetal death or resorption and wasting of borne pups. however, fetal infection is possible by direct injection of mcmv- into the placenta [ ] and also occurs by transplacental transmission in mice with severe immunodeficiency [ ] . vertical transmission is also possible by milk during lactation [ ] . it is generally assumed that mcmv- has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, different serological tests, including multiplex fluorescent immunoassay (mfia) [ ] , are used for health surveillance of rodent colonies. as the virus persists, direct demonstration of mcmv- in infected mice is possible by pcr [ ] [ ] [ ] or by virus isolation using mouse embryo fibroblasts ( t cells). although mcmv- does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection [ ] . these aspects have been discussed in detail by shellam et al. [ ] . mcmv- has also been used as a vaccine vector aiming at a disseminating mouse control agent by inducing immunocontraception in mice [ ] . the virus is known to influence immune reactions in infected mice [ , ] and may therefore have an impact on immunological research [ , ] . mtv was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, mtv is difficult to culture in vitro and is usually propagated by intraperitoneal infection of newborn mice. the thymus is removed - days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason, and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice [ , ] . further, mtv obviously represents a significant source of contamination of mcmv- (and vice versa) if virus is prepared from salivary glands, since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about days. infection results in nuclear inclusions in thymocytes and their almost complete destruction within weeks. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. the virus persists here and is shed via saliva for months. mtv also establishes a persistent infection in athymic foxn nu mice, but virus shedding is reduced compared to euthymic mice, with virus recovery possible only in a lower percentage of mice [ ] . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion [ ] . lymphoid necrosis may also occur in lymph nodes and spleen [ ] , with necrosis and recovery similar to that in the thymus. in mice infected during the first days after birth, necrosis of thymus becomes evident within - days, and the animals' size and weight are markedly reduced at day - . intranuclear inclusions may be present in thymocytes between days - after infection. the thymus and the affected peripheral tissues regenerate within weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t-cell mitogens) returns to normal as the histological repair progresses [ ] . selective depletion of cd þ t cells by mtv results in autoimmune disease [ , ] . information about additional influences on the immune system is given in textbooks [ , ] . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion and virus shedding, suggesting that the oral-nasal route is likely to be involved in natural transmission [ ] . the virus spreads to cagemates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mtv is not transmitted to fetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion [ ] . mtv and mcmv- do not cross-react serologically [ ] . serological monitoring of mouse populations for antibodies to mtv is possible by indirect immunofluorescent assay (ifa) testing, which is commercially available; enzyme-linked immunosorbent assays (elisa) tests have also been established [ , ] . elisa and complement fixation yield similar results [ ] . serological tests based on recombinant proteins and direct detection of virus by pcr are currently not possible because the genome of the virus has not yet been sequenced. it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns, whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes and spleens for lymphoid necrosis [ ] . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa [ ] and map testing, although this is slightly less sensitive than infectivity assays [ ] . there is very little experience of eradication methods for mtv because of its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses will likely eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction of the virus into laboratory animal facilities. murid herpesvirus (muhv- ) or rat cytomegalovirus infects rats and is also a member of the genus muromegalovirus. murid herpesvirus (muhv- ) is a member of the genus rhadinovirus in the subfamily gammaherpesvirinae and is also known as mouse herpesvirus strain (mhv- ). other murid herpesviruses are not yet assigned to a subfamily within the family herpesviridae. among these is murid herpesvirus (mouse thymic herpesvirus), but also murid herpesvirus (field mouse herpesvirus) which infects voles (microtus pennsylvanicus), murid herpesvirus (sand rat nuclear inclusion agent), and murid herpesvirus [ ] . furthermore, a gammaherpesvirus of house mice (mus musculus) has been described recently which is clearly distinct from mhv- [ ] . experimental infection of laboratory mice with mhv- is a frequently used model system for the study of human gammaherpesvirus pathogenesis, e.g. of kaposi's sarcoma-associated herpesvirus or epstein-barr virus (ebv) [ , ] which are members of the same subfamily. they are also important models to study viral latency and immune mechanisms controlling latency [ ] [ ] [ ] . mus musculus is not the natural host for this virus; it was first isolated in slovakia from bank voles (myodes glareolus). additional closely related strains (mhv- , mhv- ) exist from the same host species, and similar strains (mhv- , mhv- ) were isolated from wood mice (apodemus flavicollis and apodemus sylvaticus). apodemus sp. seem to be the major hosts for mhv- in great britain [ ] . different virus strains exhibit different genetic and biological properties and also differ in their pathogenicity, e.g. for prkdc scid mice [ ] . infections in laboratory mice take the same course as in their natural hosts [ ] . there are, however, some differences as, e.g. higher virus levels are reached in the lungs of balb/c mice, and wood mice develop higher titres of neutralizing antibodies [ ] . house mice develop an acute infection in the lungs after intranasal infection. a latent infection develops within weeks and the virus persists lifelong in epithelial cells in the lungs and also spreads to the spleen and other organs (e.g. bone marrow, peritoneal cells) where it persists in different cells of the immune system. it behaves like a natural pathogen in inbred strains of mice and persists without causing disease. the mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses, with a diameter of nm and a length of - nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of nearly nucleotides [ ] . it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to days of age) is possible [ ] . unlike various other orthopoxviruses, ectromelia virus does not infect humans [ ] . the virus is resistant to desiccation, dry heat and many disinfectants. it is not consistently inactivated in serum heated for min at c [ , ] and remains active for months when maintained at c in fetal bovine serum [ ] . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite and iodophores [ , ] . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between and almost individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in - and has been described in great detail [ ] . severe outbreaks were also described in various european countries [ ] [ ] [ ] . a more recent outbreak in the usa, which resulted in the eradication of almost mice in one institution, was described by dick et al. [ ] . another recent and well-documented case of mousepox was published by lipman et al. [ ] . few additional but unpublished cases of ectromelia have been observed since then; the latest report of an outbreak was published in [ ] . in general, positive serological reactions are occasionally reported from routine health surveillance studies [ ] but the virus is extremely rare in european and american colonies of laboratory mice [ ] [ ] [ ] . natural infections manifest differently, depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs and may therefore be very difficult to diagnose [ ] . the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form) or subacute to chronic infection (cutaneous form) [ , [ ] [ ] [ ] . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent [ ] [ ] [ ] . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/ , dba/ , balb/c, a and c h/hej. immunodeficient mice may also be very susceptible [ ] . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (> %). clinical disease may not be evident in resistant strains such as c bl/ and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice [ , ] . killer cells are necessary to control mousepox infections [ ] . mice that are resistant to mousepox may lose their resistance with increasing age, most likely due to the decreased number and activity of nk cells [ ] . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h d b (termed rmp , resistance to mousepox), on chromosome [ ] ; the c genes (rmp , on chromosome ); rmp , localized to a region on chromosome encoding the nk cell receptor nkr-p alloantigens [ ] ; the nitric oxide synthase locus on chromosome [ ] ; and the signal transducer and activator of transcription locus on chromosome [ ] . mousepox infections are controlled for several days during the initial course of infection by the complement system until the adaptive immune system can react. loss of the complement system results in lethal infection [ ] . clearance of the virus by the immune system is dependent upon the effector functions of cd þ t cells while nk cells, cd þ t cells and macrophages are necessary for the generation of an optimal response [ , ] . t-and b-cell interactions and antibodies play a central role during recovery from a secondary infection [ ] . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes [ , , ] . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages, resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis and skin lesions (papules, erosions or encrustations mainly on ears, feet and tail; figure . . ). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen [ , , , ] . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure . . ), lymph nodes, peyer's patches, thymus and liver [ , , , , ] . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viraemia ( natural transmission of ectv mainly occurs by direct contact and fomites [ , , , ] . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved [ ] . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection [ ] . intrauterine transmission is possible at least under experimental conditions [ ] . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about - weeks, even though the virus can persist for months in the spleen of an occasional mouse [ , ] . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding [ ] . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays [ ] . in the s, diagnosis relied on clinical signs, histopathology and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also used. serology is currently the primary means of routine health surveillance for testing mouse colonies for exposure to ectv. the methods of choice are mfia, elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay [ , , ] . serological tests based on virus particles detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus or other orthopoxviruses, respectively. vaccinia virus is commonly used as an antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding [ ] . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. [ ] depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical signs suggestive of mousepox are observed. pcr assays to detect different genes of poxviruses in infected tissues have been used [ , , ] . other pcr tests which were developed to detect smallpox virus have also been shown to detect ectromelia virus and can be used as well [ , ] . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines [ ] , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last three published outbreaks of ectromelia were introduced into the facilities by mouse serum [ , , ] . lipman et al. [ ] found that the contaminated serum originated from a pooled lot of l that had been imported from china, but in both other cases, serum was obtained from animals in the usa. because mouse serum is commonly sold to the end user in small aliquots (a few millilitres), it has to be expected that aliquots of the contaminated lot may still be stored in freezers. these published cases of ectromelia outbreaks provide excellent examples of why testing should be performed on all biological materials to be inoculated into mice. in the case of ectromelia virus it was shown that pcr is more sensitive, and map testing failed to detect contamination [ ] . eradication of mousepox has usually been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method because of the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious [ , , ] and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring, with removal of clinically sick and seropositive animals, are a potential alternative. the period from the last births until the first matings after cessation of breeding should be at least weeks [ ] . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population [ , ] . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs and thymus [ ] . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals and financial resources can be substantial. experimental mousepox infections are frequently used as a model to study various aspects of smallpox infections of humans [ ] [ ] [ ] . mousepox shares many aspects of virus biology and pathology, and models the course of human smallpox. experimental mousepox infections are used to study vaccination procedures [ , ] or anti-poxvirus therapies [ ] . murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae. two distinct strains have been isolated from mice. the fl strain (madv- ) was first isolated in the usa as a contaminant of a friend leukaemia [ ] and has been classified as a member of the genus mastadenovirus. the k strain (madv- ) was isolated in japan from the faeces of a healthy mouse [ ] and has not yet been assigned to a genus. both strains are considered to represent different species [ ] [ ] [ ] . they are host species specific and are not infectious for infant rats [ ] . madv- can be cultured in vitro in mouse fibroblasts (e.g. t or l cells), madv- is usually cultured in vitro in a mouse rectum carcinoma cell line (cmt- ). in laboratory mice, seropositivity to adenoviruses was reported to be very low [ , , , , , ] or negative [ , ] . antibodies to madv are also found in wild mice [ , ] and in rats [ , ] . neither virus is known to cause clinical disease in naturally infected, immunocompetent mice. however, madv- can cause a fatal systemic disease in suckling mice after experimental inoculation [ , , ] . disease is characterized by scruffiness, lethargy, stunted growth and often death within days. experimental infection of adult mice with madv- is most often subclinical and persistent but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia and paralysis in susceptible c bl/ and dba/ j mice [ ] . balb/c mice are relatively resistant to this condition. athymic foxn nu mice experimentally infected with madv- develop a lethal wasting disease [ ] . similarly, prkdc scid mice succumb to experimental infection with madv- [ ] . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv- include small surface haemorrhages in the brain and spinal cord of c bl/ and dba/ j mice [ ] , duodenal haemorrhage in foxn nu mice [ ] and pale yellow livers in prkdc scid mice [ ] . histologically, experimental madv- infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas and brown fat [ , , , ] . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns) [ ] . histopathological manifestations in madv- -infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes [ ] . in contrast to madv- , the tissue tropism of madv- is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum [ , , ] . transmission of madv primarily occurs by ingestion. madv- is excreted in the urine and may be shed for up to years [ ] . madv- infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice [ ] ; immunodeficient mice may shed the virus for longer periods [ ] . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross-reactivity of madv- with madv- [ ] . serum from mice experimentally infected with madv- yielded positive reactions in serological tests with both viruses, while serum from mice infected with madv- reacted only with the homologous antigen [ ] . smith et al. [ ] reported that sera might react with madv- or madv- or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv- ) indicate that the infection may not be detected if only one virus is used as an antigen [ ] . it is therefore usual to test sera for antibodies to both madv- and madv- . the commonly used methods are ifa, elisa and mfia. the low prevalence in colonies of laboratory mice indicates that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance, which is also indicated by the fact that recent publications about murine adenoviruses are very rare. however, the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only a few influences on research attributable to madv have been published. for example, it has been shown that madv- significantly aggravates the clinical course of scrapie disease in mice [ ] . natural infections with madv could also interfere with studies using adenovirus as a gene vector. a novel murine adenovirus classified as a mastadenovirus has recently been isolated from a striped field mouse (apodemus agrarius) [ ] . it was cultured in vero e cells and named madv type (madv- ). it revealed the highest similarity to madv- but it represents a separate serotype. however, there is some cross-reactivity between madv- and both other mouse viruses [ ] . in addition to serological and antigenic differences it also shows a unique organotropism and infects predominantly the heart tissue of c bl/ n mice after experimental infection. experimentally infected mice show no clinical signs. the virus is not easily transmitted from experimentally infected mice to contact sentinels [ ] . polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) was formerly known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related, but antigenically distinct, from each other [ ] , and also viruslike particles from the major capsid protein (vp ) do not cross-react [ ] . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible [ ] . seropositivity to these viruses was not reported in a recent survey conducted in the usa [ ] , and other publications also indicate that these viruses do not presently play a significant role in laboratory mice [ , , ] . because of their low prevalence, neither virus is included in the list of agents for which testing is recommended on a regular basis by felasa [ ] . although polyomavirus genes, especially those of sv , are widely used in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last - years. the reader is therefore referred to previous review articles for details [ , ] . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence [ , ] . clinical signs are observed only after infection of infant mice less than - days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium (figure . . ). endothelial cells in other organs are also involved in virus replication [ , ] (figure . . ). in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn nu mice, experimental infection of adults is clinically asymptomatic, although virus is detectable for a period of several months [ ] . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays [ ] . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available [ ] . furthermore, a pcr test for demonstration of mptv in biological samples has also been published [ ] . mpyv was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours [ ] . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs when mice are experimentally infected at a young age or when inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage [ ] . parotid, salivary gland and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels and thyroid also occur. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c bl and c br/cd lineage are considered to be the most resistant strains; athymic foxn nu mice are considered to be most susceptible; c h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn nu ) also supports the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses [ ] . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney and colon become infected [ ] . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection [ ] . mpyv persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about weeks [ ] . however, virus may persist and can be reactivated by prolonged immunosuppression [ ] or during pregnancy, at least in young mice [ ] . it has been shown that interferon-gamma is an important factor of the host defence against tumour formation and mpyv infection [ ] . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice, emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa, mfia and ifa; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing [ ] . while mpyv infections are of low importance for laboratory animal medicine, the virus is used in models of persistent virus infection [ , ] . virus-like particles from both murine polyomaviruses have been used as a vector for gene therapy or vaccines [ , ] . parvoviruses are non-enveloped small viruses (approximately nm in diameter) with a singlestranded dna genome of approximately nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. up to date, two distinct species that infect laboratory mice are officially listed: the minute virus of mice (mvm), previously named mice minute virus (mmv), and the mouse parvovirus (mpv). non-structural proteins (ns- and ns- ) are highly conserved among both viruses whereas the capsid proteins (vp- , vp- , vp- ) are more divergent and determine the serogroup [ ] . both viruses require mitotically active cells for replication. severe clinical signs are therefore not found in mature animals because of the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed [ , , [ ] [ ] [ ] [ ] [ ] . already in the mid- s mouse colonies were identified that gave positive reactions for mvm by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses [ ] and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and subsequently characterized on a molecular basis [ , ] . on the basis of epidemiological analyses, further parvoviruses were recently identified in mice, sequenced, and tentatively named serially mpv- and mpv- [ ] , mpv- (genbank fj ) and mpv- (genbank fj ). in addition, several variants are published for mpv- [ , , ] . at present, mpv is among the most common viruses found in colonies of laboratory mice. the prevalence of sera positive for parvoviruses ranged from % to nearly % in western europe and north america, with the majority of sera being positive for mpv in studies differentiating between the two parvovirus species [ , , , ] . these prevalence data are based on testing at commercial laboratories and do not reflect that, despite highly specific and sensitive test methods, enzootic parvovirus infections are difficult to detect due to virus-associated characteristics [ , ] . a recent survey conducted in the usa showed that during a - month period mouse parvoviruses were detected at almost all facilities that responded to a questionnaire, with mpv being more often diagnosed than mvm [ ] . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals [ , ] . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice [ , ] . humoral immunity to mpv does not protect against mvm infections, and vice versa [ ] . serological surveys have indicated that mpv naturally infects only mice, with the exception that mpv- shows genetic similarity to hamster parvovirus, suggesting that a cross-species transmission has occurred, where the mouse probably served as the natural host [ , ] . differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection with mpv- b, seroconversion occurred in all c h/hen mice, fewer balb/c, dba/ and icr mice, and seroconversion could not be detected in c bl/ mice [ ] . upon mpv- f inoculation, antibody response was absent in balb/carc mice [ ] . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/ even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than months [ ] , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for - weeks [ ] , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice might not transmit the virus under similar experimental conditions [ ] . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi is a practical screening test for mpv because they require large quantities of purified mpv, which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mvm hi-negative result coupled with an mvm ifa-positive result. ifa provides the opportunity to detect both serogroup-specific vp proteins as well as ns proteins that are conserved among mouse parvoviruses. a generic rodent parvovirus elisa using a recombinant ns- protein as antigen has been developed [ ] , but mpv ifa and mpv hi assays are more sensitive techniques than the ns- elisa and the mvm ifa [ ] . in contrast, elisa tests that use recombinant vp- provide sensitive and serogroup-specific assays for the diagnosis of mpv infections in mice [ , ] , although considerable cross-reactivity with heterologous capsid antigens exists [ ] . nevertheless, when using the elisa technique, one needs to consider that mpv- may not consistently be detected by mpv- vp- elisa [ , ] , especially when antibody titres are low (own observations). therefore, elisas using mpv- vp- and mpv- vp- antigens are also used for diagnostics. as parvovirus diagnostics using recombinant assays should be based on a combination of antigens, bead-based mulitplex assays are a convenient extension of traditional elisa, allowing the use of multiple antigens simultaneously. in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv [ , ] and other parvoviruses. mpv has been shown to persist for at least weeks in the mesenteric lymph nodes [ ] . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success [ ] . for antemortem detection, shedding of parvoviruses can also be detected by pcr of faecal samples [ ] . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigen-based serological assays. the pcr is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours [ ] . in addition, the map test has been reported as a sensitive tool to detect mpv [ ] . given the high environmental stability of the virus and the potential fomite transmission, together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations by embryo transfer or by hysterectomy. it should be noted that recent studies suggest a risk of virus transmission by embryo transfer, though successful sanitation of immunodeficient mice was achieved despite antibody response in recipients and progeny after embryo transfer [ , ] . although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts [ ] . mvm is the type species of the genus parvovirus. the virus was intermediately named mice minute virus (mmv). it was originally isolated by crawford [ ] from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma [ ] and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. mvmp infects fibroblast cell lines and does not cause clinical disease [ , ] . both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mvmp. a third strain, the cutter strain mvmc, was isolated from bhk- cells [ ] . in contrast to these three strains detected as cell culture contaminants, an isolate was obtained from naturally infected mice with a b-cell maturational defect maintained at the university of missouri and therefore denominated mvmm [ ] . serological surveys show that the mouse is the primary natural host [ , , ] , but the virus is also infective for rats, hamsters [ , ] , and mastomys [ ] during fetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis [ ] observed mild growth retardation a few days after experimental infection of neonatal mice with mvmp. studies of transplacental infection yielded no pathological findings in mice [ ] . the immunosuppressive variant, but not the prototype strain, is able to produce a runting syndrome after experimental infection of newborn mice [ ] . depending on the host genotype, experimental infections of fetal and neonatal mice with mvmi produce various clinical presentations and lesions. infection in c bl/ mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/ mice. infection of strains such as balb/c, cba, c h/he and sjl is also lethal and mice have renal papillary haemorrhage [ ] . the mvmi also infects haematopoietic stem cells and mediates an acute myelosuppression [ , ] . because of its dependence on mitotically active tissues, the fetus is at particular risk for damage by parvoviruses. mvm and other parvoviruses may have severe teratogenic effects and cause fetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in fetal death. adult prkdc scid mice develop an acute leukopenia month after experimental infection with mvmi and die within months. the virus persists lifelong in the bone marrow of these mice [ ] . during a natural concurrent outbreak of mvmm and mpv, a runting syndrome with lymphohistiocytic renal inflammation and inclusion bodies in cells resembling splenic haematopoietic progenitor cells was reported in b-cell (ighm)deficient mice [ ] . mmv is shed in faeces and urine. in faecal samples, mvm was detected for up to - weeks by pcr [ , ] , although shorter periods ( - days) have been observed [ ] . notably, shedding re-occurred after immunosuppression by irradiation [ ] . contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mvm can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. hi is a highly specific diagnostic test whereas ifa always exhibits some degree of cross-reactivity with mpv and other closely related parvoviruses. elisa is probably the most frequently used test, but depending on the purity of the antigen preparation, cross-reactions with mpv may occur due to contamination with non-structural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp- antigen [ ] . by using serological methods, one needs to consider that the mouse strain has a considerable effect on seroconversion so that an antibody response might not be detectable despite infection; while c bl/ j mice showed good antibody response, seroconversion was observed only in some balb/c, akr/n, dba/ j, fvb/n and c h/hen, but not in nmri and icr mice upon contact exposure to mvmi-inoculated mice [ ] . viral detection is also possible by pcr in biological materials, organs (intestine, mesenteric lymph node, kidney, spleen) and faeces from infected animals [ , , , , ] . although mvm was not thought to cause persistent infection in immunocompetent mice, recent data show that it can be detected in spleens for up to weeks after exposure in some mouse strains [ ] . therefore, pcr may be considered as a confirmatory method for serology. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. however, certain precautions such as careful washing and accompanying testing need to be minded, as mvm has been detected in reproductive organs and gametes and this virus firmly attaches to the zona pellucida or might even cross it [ , ] . in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found to be free from viral contamination. both allotropic variants of mvm have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published [ , , , , ] . because of their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect [ ] . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mvm is a common contaminant of transplantable tumours, murine leukaemias and other cell lines [ , , ] . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. the genome organization and replication of ldv and other arteriviruses, their cell biology and other molecular aspects have been reviewed by snijder and meulenberg [ ] . ldv has repeatedly been detected in wild mice (mus musculus), which are considered to be a virus reservoir [ , ] . after infection of mice, virus titres of - particles per ml serum are found within - h after infection. the virus titre drops to particles per ml within - weeks and remains constant at this level for life. it persists in infected mice for the whole lifetime although it stimulates various immune mechanisms [ ] [ ] [ ] [ ] . the virus can be stored in undiluted mouse plasma at À c without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. only mice and primary mouse cells are susceptible to infection with ldv. it replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues [ ] . as suitable cell systems have not been available for virus production, routine serology has not been easily possible so that testing for ldv was not included in serological health monitoring programs. the prevalence of ldv in contemporary colonies of laboratory rodents is likely to be very low but detailed information about its prevalence comparable to most other agents is not available. ldv was first detected during a study of methods that could be used in the early diagnosis of tumours [ ] . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host [ ]. ldv has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours or matrigel prepared from such materials [ , , , ] , monoclonal antibodies or ascitic fluids [ ] , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after serial passage in an infected and viraemic mouse. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials [ , ] . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is poliomyelitis with flaccid paralysis of hindlimbs developing in c and akr mice when they are immunosuppressed either naturally with ageing or experimentally. it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis [ ] . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes [ , ] , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only h after infection and peaks - days after infection at - -fold normal levels, or can even be up to -fold in sjl/j mice. the enzyme activity declines during the next weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli [ ] . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brainstem. ldv is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cagemates, most likely via blood and saliva. infected females transmit the virus to their fetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection [ , ] . maternal immunity protects fetuses from intrauterine infection. immunodeficient prkdc scid mice also transmit virus to their offspring during chronic infection [ ] . an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principle, serological methods such as ifa may be used for detecting ldv infection [ ] but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests, and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle [ ] . however, it is meanwhile possible to use recombinant viral proteins of ldv as antigens [ ] in elisa and mfia tests so that routine testing by serology is possible. in the past, diagnosis of ldv infection has primarily been based on increased ldh activity in serum or plasma of mice. ldv activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within - days is measured. an - -fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear non-haemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. this assay was usually included in a 'map test', but antibody detection similar to other viruses was not involved for reasons mentioned earlier. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates [ , ] . however, reports exist that pcr may produce false-negative results and should be used cautiously [ ] . just as important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogenfree mice [ , ] or by pcr [ , , [ ] [ ] [ ] . ldv spreads slowly in a population because direct contact is necessary. therefore, ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations may be indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages [ ] . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats [ ] or other animal species. another method to remove ldv from contaminated cells, which is based on cell sorting, has recently been described [ ] . ldv is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. ldv may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice); [ , , , [ ] [ ] [ ] [ ] [ ] and enhance or suppress tumour growth [ , , ] . interaction with other viruses has also been described [ ] . lymphocytic choriomeningitis virus (lcmv) is an enveloped, segmented single-stranded rna virus of the genus arenavirus family, arenaviridae. it can easily be propagated in several commonly used cell lines like bhk- cells. however, cells are not lysed and a cytopathic effect (cpe) is not visible. the virus name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir [ ] , but syrian hamsters are also important hosts [ ] . additional species such as rabbits, guinea-pigs, squirrels, monkeys and humans are susceptible to natural or experimental infection [ ] . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' [ , ] . antibodies to lcmv have been found in wild mice in europe [ , ] , africa [ ] , asia [ ] , australia [ ] and america [ ] . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice [ ] . seropositivity to lcmv in laboratory mice was reported to be low during the last decade [ , , , ] or negative [ ] [ ] [ ] . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters [ , ] . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lcmv is frequently transmitted to humans from wild mice and is also endemic to a varying degree in the human population [ ] [ ] [ ] [ ] [ ] due to contact with wild mice. it has also been transmitted to humans by infected laboratory mice [ ] and by pet and laboratory syrian hamsters [ ] [ ] [ ] [ ] . in addition, contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours [ , ] . transmission of lcmv to humans also occurred repeatedly by organ transplantation and was most likely transmitted to organ donors by close contact with infected pets [ , ] . lcmv can cause mild-to-serious or fatal disease in humans [ , , ] . congenital infection in humans may result in hydrocephalus, or fetal or neonatal death [ ] . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation [ , , ] . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) non-tolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and viral shedding. mice may show growth retardation, especially during the first - weeks, but they appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at - months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites and occasional deaths. this immunopathologic phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs [ ] . the non-tolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung and kidney) of mice for at least weeks after infection as determined by sensitive assays such as nested reverse transcriptase (rt)-pcr or immunohistochemistry [ ] . such non-lethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg a antibodies [ ] . in experimentally infected mice, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease [ ] . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day after inoculation, and animals die within - days after the onset of symptoms, or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmvinduced disease by immunosuppression [ ] . lcmv disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis, clinical and pathological features of lcmv infection, the reader is referred to review articles [ , , ] . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection [ , ] . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv, indicating that oral infection is possible, e.g. by ingestion of contaminated food or by cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). the virus is not easily transmitted to dirty-bedding sentinels, and it is important that colony animals or animals having had direct contact with a population are tested to exclude lcmv infection [ ] . lcmv is most commonly diagnosed by serological methods such as mfia, ifa and elisa [ ] . all strains show a broad cross-reactivity and are serologically uniform. however, subclinical persistent infections may be difficult to detect because they may be associated with minimal or undetectable levels of circulating antibody. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals [ ] [ ] [ ] . both map test and pcr can also be used to detect contamination of biological materials [ , ] . specifically for exclusion of contamination by lcmv, it was requested by different authorities that virus is inoculated intracerebrally at a lethal dose - weeks after administration of the material to be tested. in case of contamination by lcmv and subsequent seroconversion, animals survive the challenge infection. vertical transmission of lcmv by transuterine infection is efficient so that this virus cannot reliably be eliminated by caesarean rederivation [ ] . caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (non-tolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting non-viraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals, or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn nu and prkdc scid mice may pose a special risk because infections are silent and chronic [ ] . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, hydrogen peroxide or other effective disinfectants. prevention of introduction into an animal facility requires that wild mice cannot get access to the facility. similarly important is screening of biological materials originating from mice and hamsters because these can be contaminated by lcmv. finally, it has been shown that the virus can also be introduced into a population by mice with an undetected infection [ ] . appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) will be considered to be sufficient in most cases. bsl practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching or aerosol formation from the bedding. animal biosafety level (absl) practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or absls by cdc [ ] . lcmv is frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro [ ] [ ] [ ] . accidental transmission may have a severe impact on various kinds of experiments [ , , , ] and also affect infection with other agents [ ] . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes, numbered - . mammalian orthoreovirus- (synonyms: hepatoencephalomyelitis virus; echo virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice [ , , ] . a study in france reported antibodies to mrv- in % of mouse colonies examined [ ] . in more recent studies in north america and western europe, such antibodies were detected in . - . % of mice monitored [ , , ] . schoondermark-van de ven et al. [ ] found antibodies to mrv- in . % of mouse samplings from western european institutions; and in a survey conducted by carty [ ] , about % of responding institutions in the usa reported mrv- infection in their mouse colonies. in addition, contamination of mouse origin tumours and cell lines by mrv- has been reported many times [ , , ] . experimentally, mrv- infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus and lymphoma [ , ] . the literature on mrv- infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice [ ] [ ] [ ] . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented [ , ] . the mechanisms of viral pathogenesis and their interactions with the host cell as well as the host's immune response are reviewed in detail by tyler et al. [ ] , schiff et al. [ ] and ward et al. [ ] . natural infection by mrv- in a mouse colony is usually subclinical, although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted [ , , [ ] [ ] [ ] . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia) and neurological signs such as incoordination, tremors or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv (murine hepatitis virus) or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns and intestine. the outcome of mrv- infection depends on age and immunological status of mouse, dose of virus and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease [ , ] . experimental infection of adult prkdc scid mice is lethal [ ] . depending on the route of inoculation, experimental infection of adult foxn nu mice is subclinical or results in liver disease [ , ] . histological findings reported to occur after experimental mrv- infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia [ , [ ] [ ] [ ] ] . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route [ , ] . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with mfia, elisa or ifa is in widespread use for detection of antibodies to mrv- in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice [ ] , although a recent report indicates that some ifa-positive mrv infections in mice may not be detected by commonly used elisas [ ] . the hi test does not detect such cross-reacting antibodies but is prone to give false-positive results due to nonspecific inhibitors of haemagglutination [ , ] . rt-pcr methods for the detection of mrv- rna [ , ] or mrv rna [ , ] are also available. reports on contamination of mouse origin tumours and cell lines by mrv- and its interference with transplantable tumour studies [ , ] emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv- without clinical disease is also observed in laboratory rats, hamsters and guinea-pigs [ , ] . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv- infection [ , ] . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, single-stranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). mhv is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens, by use of monoclonal antibodies, or by sequencing [ ] . the beststudied strains are the prototype strains mhv- , mhv- , mhv- , jhm (mhv- ), a , and s, of which mhv- is regarded as the most virulent. like other coronaviruses mhv mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism and cell tropism [ ] . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) also exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating ( c for min) and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity [ ] . given the environmental conditions present in mouse rooms, mhv might remain infective for several days, at low humidity ( % relative humidity) or low temperatures ( c) even for weeks on surfaces [ ] . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. monitoring results for research institutions across north america and europe indicate that the prevalence of mhv has decreased in the past, though it seems to have remained quite stable since the s [ , ] . recently . % of north american laboratory mouse serum samples tested positive [ ] . in europe, prevalence rates ranged from . % to % [ , , ] . a retrospective study in france covering the period from to reported antibodies to mhv in % of mouse colonies examined [ ] , and a survey performed in revealed that almost half of north american research institutions detected mhv in their mouse populations [ ] . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease [ ] . similarly, deer mice seroconverted but showed no clinical disease after experimental infection [ ] . mhv is also a common contaminant of transplantable tumours [ , ] and cell lines [ , ] . the pathogenesis and outcome of mhv infections depend on interactions between numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status) [ , , , , , ] . mhv strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv- , mhv- , mhv- , a , s and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than weeks of age, infection of genetically susceptible strains of mice or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of the brain, liver, lymphoid organs, bone marrow and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant [ ] . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells [ ] , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages [ ] . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain [ ] . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are self-limiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within - weeks [ , , ] . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement [ ] [ ] [ ] [ ] . therefore, immunodeficient mice such as foxn nu and prkdc scid mice cannot clear the virus [ , ] . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection [ ] . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv [ ] [ ] [ ] . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains [ , ] . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine [ ] . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice [ , ] . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach % in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hindlimbs, convulsions and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn nu and prkdc scid ) mice, infection with virulent polytropic mhv strains is often rapidly fatal while less virulent strains cause chronic wasting disease [ ] . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease [ , ] . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by coinfections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed in [ , ] ). in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach [ , , ] . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver [ , , ] and thymus involution [ , ] may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis [ ] . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow [ , , , ] (figure . . ) . recently, pulmonary inflammation has been observed in susceptible mouse strains (c h/hej and a/j) after intranasal inoculation with polytropic mhv- [ , ] . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brainstem and periependymal areas. lesions in peritoneum, bone marrow, thymus and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t-cell mediated [ ] . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytium arising from endothelium, parenchyma or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferongamma-deficient mice infected with mhv [ ] . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues [ , , , ] . the most common sites are terminal ileum, caecum and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells) and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within - h, but are usually more severe at - days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity [ ] , but other agents such as helicobacter spp. may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. in t-cell deficient mice, multisystemic lethal infection was observed after experimental infection with the enterotropic strain mhv-y [ ] . mhv is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol and fomites [ , ] . vertical (in utero) transmission has been demonstrated in experimental infections [ ] but does not seem to be of practical importance under natural conditions. mhv was transmitted by ovarian transplantation after reproductive organs became infected [ ] . however, risk of mhv transmission by sperm or oocytes (ivf) or by embryo transfer seems to be low, though thorough washing of gametes and embryos is required [ , [ ] [ ] [ ] . diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry [ ] or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc or dbt [ ] . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn nu mouse inoculation [ , ] . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay [ ] and a number of rt-pcr assays [ ] [ ] [ ] [ ] . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. multiplex fluorescent immunoassay, elisa and ifa are well established and sensitive, and all known mhv strains cross-react in these tests [ , , ] . the magnitude of antibody response depends on mhv strain and mouse genotype [ , ] . dba/ mice are poor antibody responders whereas c bl/ mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least months [ , ] . infected mice may not develop detectable antibodies for up to days after initial exposure [ ] . in such cases, a direct diagnostic method, as discussed above, may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are weeks of age [ ] . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas [ ] and embryonic stem cells [ , ] these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test and rt-pcr can be used for this purpose. therefore, surveillance programmes should combine careful evaluation of clinically ill animals, testing of biological materials and routine health monitoring. soiled-bedding sentinel mice, which are frequently used for routine monitoring, are likely well suited for detecting enterotropic strains of mhv, but might not indicate the presence of less contagious respiratory strains of mhv [ ] equally well. the mouse strain used as sentinel should be considered as a critical factor. furthermore, duration of mhv shedding and stability of the virus, which seems to be lower in static microisolator cages than in ivc cages, might interfere with detection. the amount of bedding transferred seems not to be as critical as for, e.g. parvoviruses, at least for enterotropic strains [ ] . use of contact and exhaust air sentinels and testing of exhaust filters by pcr was also shown to be effective at detecting mhv [ ] . the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchasing mice from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing has also been reported to be effective [ ] . quarantine of an affected colony with no breeding and no introduction of new animals for approximately months has been effective in immunocompetent mice [ ] . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains [ ] . it may be practical to select a few future breeders from the infected population and quarantine them for approximately weeks [ ] . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the -week interval should permit recovery from active infection, and the additional -week gestation period effectively extends the total quarantine to weeks. it is advisable to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion [ ] . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation is likely to be the most cost-effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters, as well as stringent personal sanitation, are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic foxn rnu rats [ ] . mhv is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed in [ - , , , ] ). noroviruses are non-enveloped, single-stranded rna viruses with high environmental resistance and belong to the family caliciviridae, genus norovirus. they were first identified after an outbreak of acute gastroenteritis at a school in norwalk (ohio, usa) in and cause about % of non-bacterial epidemic gastroenteritis in humans. noroviruses found in animals include bovine, porcine and murine noroviruses. noroviruses are not known to cross species. murine norovirus (mnv) is endemic in many research mouse colonies and currently the most commonly detected viral agent in laboratory mice [ , , ] . in the hitherto largest survey [ ] , about % of mouse serum samples examined had antibodies against mnv. the first norovirus to infect mice was described in [ ] . experimental inoculation studies with this murine norovirus (mnv- ) show that duration of infection and disease manifestation vary depending on the mouse strain [ ] [ ] [ ] . in immunocompetent strains, mnv infection is variable in length (e.g. ! - days in s mice, ! weeks in hsd:icr mice) and does not induce clinical signs. infection is associated with mild histopathological alterations in the small intestine (increase in inflammatory cells) and spleen (red pulp hypertrophy and white pulp activation) of s mice. in certain immunodeficient strains, however, infection can cause lethal systemic disease (encephalitis, vasculitis, meningitis, hepatitis and pneumonia in interferon-alpha-beta-gamma-receptor-deficient and stat tm mice) or persist without symptoms (! days in rag À/À and rag À/À mice). these findings indicate that components of the innate immune system are critical for resistance to mnv- induced disease. consistent with this hypothesis, it was demonstrated that mnv- replicates in macrophages and dendritic cells [ ] . meanwhile, many additional strains of mnv with diverse biological properties were isolated [ , ] . an analysis of mnv isolates revealed distinct mnv strains that comprise a single genogroup and serotype [ ] . experimental inoculation studies show that several mnv strains are able to persist in various tissues (small intestine, caecum, mesenteric lymph node, spleen) of immunocompetent (c bl/ j, hsd:icr, jcl:icr) and immunodeficient (cb -prkdc scid ) mice with viral shedding in faeces for the duration of at least - days [ ] [ ] [ ] . murine norovirus is transmitted via the faecaloral route and is efficiently transferred to sentinel mice by soiled bedding [ , ] . mnv infection can be detected directly by rt-pcr on faecal pellets or tissue specimens (see above) and indirectly by serology (mfia, elisa, ifa) [ , , ] . detection is facilitated by high stability of mnv rna in faeces (at least weeks at room temperature) [ ] and by broad serological cross-reactivity among different strains of mnv [ , ] . embryo transfer [ ] and hysterectomy [ ] are most likely effective means of eliminating mnv from mouse colonies. since -to -day-old pups are resistant to infection, elimination of mnv may also be achieved by transferring neonates from infected dams to uninfected foster dams ('cross-fostering') [ ] . this transfer should ideally be performed within h after birth. mnv is used as a surrogate to evaluate resistance of human noroviruses to disinfectants. the impact of mnv on animal experiments remains to be evaluated. recent studies show that mnv is immunmodulatory and may alter disease phenotypes in mouse models of inflammatory bowel disease [ ] [ ] [ ] and other experimental mouse models [ , ] . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies [ ] ; however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus (mpv). pvm infection remains prevalent in contemporary colonies of mice and rats throughout the world. a serological survey in france demonstrated antibodies to pvm in % of mouse colonies examined [ ] . in more recent studies in north america and western europe, the prevalence of pvm-specific antibodies in mice ranged between % and . % [ , , ] . schoondermark-van de ven et al. [ ] found antibodies to pvm in . % of mouse samplings from western european institutions. antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea-pigs and rabbits [ , , ] . experimentally, pvm infection of mice is used as a model for hrsv infection and has therefore been extensively studied (reviewed by rosenberg and domachowske [ ] ). in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings [ , , ] . however, natural disease and persistent infection may occur in immunodeficient mice [ ] [ ] [ ] . in particular, athymic foxn nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation and death due to pneumonia [ , ] . similar clinical signs have been reported for experimentally infected immunocompetent mice [ ] . necropsy findings in naturally infected foxn nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation [ ] . pulmonary consolidation (dark red or grey in color) has also been found after experimental infection of immunocompetent mice [ ] . histologically, natural infection of foxn nu mice with pvm presents as interstitial pneumonia [ , ] . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia [ , ] . hydrocephalus may result from intracerebral inoculation of neonatal mice [ ] . susceptibility to infection is influenced by age and strain of mouse, dose of virus, and a variety of local and systemic stressors [ , , ] . in terms of the extent of the alveolar inflammatory response, /sv and dba/ mice are susceptible to pvm infection, while balb/c and c bl/ mice are relatively resistant [ ] . in terms of the control of viral replication, mice of strains /sv, dba/ , balb/c and c bl/ are susceptible to pvm infection, while sjl mice are relatively resistant. pvm is labile in the environment and rapidly inactivated at room temperature [ , ] . the virus is tropic for the respiratory epithelium [ , ] , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol [ , ] . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (mfia, elisa, ifa or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections [ , ] ; however, proper sampling (see chapter . , 'health management and monitoring') is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported [ ] . however, the use of direct methods requires good timing because the virus is present for only up to about days in immunocompetent mice [ ] . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus [ , ] . pvm could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease [ , ] or become more susceptible to the deleterious effects of other agents such as p. murina [ ] . murv-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a non-enveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis [ ] . murv-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. large commercial laboratories found . % to % of mouse sera from north american and european facilities to be positive for antibodies against murv-a/edim [ , , , ] , and up to % of mouse colonies in the usa were identified as affected in a survey performed in [ ] . experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies [ ] . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first weeks of life [ , , , , ] . diarrhoea usually begins around h after infection and persists for about week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region (figure . . ) . in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrhoeic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea (figure . . ). they are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells; the large intestinal surface mucosa may also be affected. though inflammation is minimal, the lamina propria may be oedematous, lymphatics may be dilated and mild leukocytic infiltration in the large intestinal mucosa and submucosa has been observed in a recent outbreak of disease [ , ] . hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system [ ] and through the effects of a viral enterotoxin called nsp (for non-structural protein ) [ ] . it is hypothesized that nsp is released from virus-infected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between and days of age [ , , , , ] . mice older than about weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus and diarrhoea does not occur. the exact reason for this agerelated resistance to disease is unknown. pups suckling from immune dams are protected against edim during their period of disease susceptibility [ ] . in general, the infection is self-limiting and resolves within days. successful viral control and clearance is promoted by an intact immune response [ ] [ ] [ ] [ ] , and some immunodeficient mice (e.g. prkdc scid and rag tm fwa mice) may shed virus for extended periods or become persistently infected [ , ] . protection against murv-a/edim reinfection is primarily mediated by antibodies [ , ] . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route [ , , ] . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols and is facilitated by the general property of rotaviruses that they remain infectious outside the body, show resistance to inactivation (e.g. low ph, non-ionic detergents, hydrophobic organic liquids, proteolytic enzymes), and are shed in high quantities (> particles/g faeces) [ ] . murv-a/edim is stable at À c but otherwise tends to be susceptible to extreme environmental conditions, detergents and disinfectants containing phenols, chlorine or ethanol [ ] . mfia, elisa and ifa are in widespread use for detection of serum antibodies to murv-a/ edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also used [ ] . as murv-a/edim shares the vp protein determined group a antigen, for example, with human, simian or bovine rotavirus strains, commercially available elisa assays utilizing polyclonal or monoclonal antibodies have been used to detect rotavirus antigen in mice; however, great care must be taken in interpreting the results because some feeds have been reported to cause false-positive reactions with certain elisa kits [ ] . electron microscopy of faeces of diarrhoeic pups should reveal typical wheel-shaped rotavirus particles, - nm in diameter. rt-pcr also can be used to detect rotavirus rna in faecal samples [ ] . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks [ ] . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy for - weeks combined with excellent sanitation, filter tops and conscientious serological testing of offspring and sentinel mice has also been reported to be effective, and prolongation of breeding cessation up to weeks resolved infection even in immunocompromised mice [ ] . murv-a/edim has the potential to interfere with any research using suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice [ ] . a disease-induced stress-related thymic necrosis may occur and alter immunology experiments [ ] . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus . the virus was named after sendai, japan, where it was first isolated from mice. historically, infections were relatively common in mouse and rat colonies worldwide. in addition, there is evidence that hamsters, guinea-pigs and rabbits are susceptible to infection with sev [ , , , ] ; however, some apparently seropositive guinea-pigs may in fact be seropositive to other parainfluenza viruses instead of sev. a study in france reported antibodies to sev in % of mouse colonies examined [ ] . a low rate of seropositive mice ( . %) was found in a survey in north america [ ] . schoondermark-van de ven et al. [ ] also found antibodies to sev in . % of mouse samplings from western european institutions. in more recent surveys in north america and western europe, sev infection was not detected [ ] [ ] [ ] , indicating that sev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. sev can contaminate biological materials [ ] . sev is pneumotropic and can cause significant respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein [ ] . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type [ , , ] . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after - months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high, resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings and runting in young mice. breeding colonies may return to normal productivity within months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport and copathogens are important in precipitating overt disease. dba and strains of mice are very susceptible to sev pneumonia, whereas sjl/j and c bl/ /j and several outbred stocks are relatively resistant. resistance to sev infection is under multigenic control with epistatic involvement [ ] . there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia [ , ] . clearance of a primary sev infection is mediated by cd þ and cd þ t-cell mechanisms [ , ] . heavier than normal, consolidated, plumcolored or grey lungs are a characteristic gross finding in severe sev pneumonia [ , , , ] . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative and resolution phases [ , ] . lesions of the acute phase, which lasts - days, are primarily attributed to the cellmediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils and mononuclear cells. atelectasis, bronchiectasis and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week after infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial, rather than alveolar, as they are in the acute phase. the resolution phase may be complete by the fourth week after infection and lesions may be difficult to subsequently identify. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present [ ] . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens [ ] . sev has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice [ ] . sev is extremely contagious. infectious virus is shed during the first weeks of infection and appears to be transmitted by direct contact, contaminated fomites and respiratory aerosol [ , ] . serology (mfia, elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only - weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry [ ] and rt-pcr [ , ] . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently [ , ] . map testing and rt-pcr can be used to detect sev in contaminated biological materials. sev infection in mouse colonies has proved to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies is probably the most appropriate means of eliminating the virus in most situations. embryo transfer, or caesarean derivation, followed by barrier maintenance, can also be used to eliminate the virus [ , ] . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding and prevent addition of other susceptible animals for approximately months until the infection is extinguished, and then breeding and other normal activities are resumed. vaccines against the virus have been developed [ , , ] , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sev has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and fetal growth; alterations of macrophage, nk-cell, and t-and b-cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed in [ ] [ ] [ ] ). pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev), or murine poliovirus, is a member of the genus cardiovirus in the family picornaviridae. members of this genus are non-enveloped viruses with single-stranded rna. the virus is rapidly destroyed at temperatures above c. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev [ ] . the best-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates, this virus is not pathogenic for rats and mice after intracerebral inoculation [ ] . recently, another rat isolate has been characterized and shown to be most closely related to, but quite distinct from, other tmev viruses [ ] . antibodies to tmev (strain gdvii) have been detected in guinea-pigs and are considered to indicate infection with another closely related cardiovirus [ ] . seropositivity to tmev was reported in approximately % of french mouse colonies in a retrospective study [ ] . in more recent studies, the prevalence of tmev infections was found to be lower. schoondermark-van de ven et al. [ ] detected antibodies to tmev in . % of mouse samplings from western european institutions. in a survey conducted by carty [ ] , about % of responding institutions in the usa reported tmev infection in their mouse colonies. further surveys in north america and western europe revealed antibodies in . - . % of mice monitored [ , , ] . tmev is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to weeks, sometimes intermittently [ ] , and transmission under natural conditions is via the faecal-oral route, by direct contact between mice, as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only in - infected immunocompetent animals [ ] . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher [ ] . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hindlimbs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virusinduced demyelinating disease including multiple sclerosis [ ] . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at - months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains, while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age [ , , ] . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is also effective. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice - days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling, rolling and weakness. animals may develop typical flaccid paralysis of hindlimbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralyzed. experimental infections with low-virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr and dba/ strains are most susceptible to this chronic demyelinating disease. cba and c h/he are less susceptible strains, and strains a, c bl/ , c bl/ and dba/ are relatively resistant [ ] . differences in humoral immune responses play a role in resistance to tmev infection [ ] , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h- d region of the major histocompatibility complex [ ] . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low-virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of - weeks while adult mice often show no clinical signs of infection. the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralyzed limbs, or wasting or atrophy of the hindlimbs in long-term survivors. tmev infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis and poliomyelitis with neuronolysis, neuronophagia and microgliosis in the brainstem and ventral horns of the spinal cord [ ] . demyelination in immunocompetent mice is considered to be immunemediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by t cells that release cytokines leading to recruitment of monocytes and macrophages as a consequence of infection of macrophages and other cns-resident cells [ ] [ ] [ ] . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation [ , ] . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes [ ] . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. [ ] , kim et al. [ ] and lipton et al. [ ] . experimental infection of foxn nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs, including the typical hindlimb paresis, develop weeks after inoculation, and most animals die within weeks. in foxn nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes [ ] . demyelination and lethality are reduced after administration of neutralizing antibodies [ ] . histopathological changes in prkdc scid mice are very similar to those in foxn nu mice [ ] . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by days of age. intrauterine transmission to fetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection [ ] . all tmev isolates are closely related antigenically and form a single serogroup, as determined by complement fixation and hi [ ] . hemelt et al. 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diseases pathology and diagnosis of mousepox induction of natural killer cell responses by ectromelia virus controls infection agedependent susceptibility to a viral disease due to decreased natural killer cell numbers and trafficking h- -linked control of resistance to ectromelia virus infection in b congenic mice differential pathogenesis of lethal mousepox in congenic dba/ mice implicates natural killer cell receptor nkr-p in necrotizing hepatitis and the fifth component of complement in recruitment of circulating leukocytes to spleen identification of nitric oxide synthase as an innate resistance locus against ectromelia virus infection enhanced resistance in stat -deficient mice to infection with ectromelia virus surviving mousepox infection requires the complement system innate resistance to lethal mousepox is genetically linked to the nk gene complex on chromosome and correlates with early restriction of virus replication by cells with an nk phenotype different roles for cd þ 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detection of human orthopoxvirus infections and differentiation of smallpox virus with real-time pcr observations on the replication of ectromelia virus in mouse-derived cell lines: implications for epidemiology of mousepox reexamination of the efficacy of vaccination against mousepox effect of vaccination on the clinical response, pathogenesis and transmission of mousepox pathogenesis of vaccinia (ihd-t) virus infection in balb/cann mice mouse models for studying orthopoxvirus respiratory infections animal models of orthopoxvirus infection ectromelia virus: the causative agent of mousepox a protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost postexposure immunization with modified vaccinia virus ankara or conventional lister vaccine provides solid protection in a murine model of human smallpox mousepox in the c bl/ strain provides an improved model for evaluating antipoxvirus therapies a new mouse virus apparently related to the adenovirus group an adenovirus isolated from the feces of mice i. isolation and identification genotypic differences between the mouse adenovirus strains fl and k molecular cloning, physical mapping and cross-hybridization of the murine adenovirus type and type genomes genetic relationship between mouse adenovirus- (strain k ) and human adenovirus- mouse adenoviruses microbiological contamination of laboratory mice and rats in korea from to a serologic survey for viruses and mycoplasma pulmonis among wild house mice (mus domesticus) in southeastern australia comparative biological characterization of mouse adenovirus strains fl and k and seroprevalence in laboratory rodents pathogenesis of experimentally produced mouse adenovirus infection in mice age and susceptibility of swiss mice for mouse adenovirus, strain fl mouse adenovirus type causes a fatal hemorrhagic encephalomyelitis in adult c bl/ but not balb/c mice duodenal lesions associated with adenovirus infection in athymic 'nude' mice murine adenovirus infection of scid mice induces hepatic lesions that resemble human reye syndrome experimental adenovirus infection of the mouse adrenal gland. i. light microscopic observations electron microscope study of experimental enteric adenovirus infection in mice experimental infection with mouse adenovirus in adult mice intestinal resistance in the experimental enteric infection of mice with a mouse adenovirus. i. growth of the virus and appearance of a neutralizing substance in the intestinal tract fluctuation of antiviral resistance in the intestinal tracts of nude mice infected with a mouse adenovirus biological and biophysical characteristics of mouse adenovirus, strain fl serological relationship between mouse adenovirus strains fl and k a naturally occurring intestinal mouse adenovirus infection associated with negative serologic findings acceleration of scrapie disease in mice by an adenovirus a novel cardiotropic murine adenovirus representing a distinct species of mastadenoviruses serological diagnosis of murine adenovirus characterization of k virus and its comparison with polyoma virus murine pneumotropic virus vp virus-like particles (vlps) bind to several cell types independent of sialic acid residues and do not serologically cross react with murine polyomavirus vp vlps recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units polyoma viruses the major site of murine k papovavirus persistence and reactivation is the renal tubular epithelium distribution of k-papovavirus in infected newborn mice morphological and immunohistochemical studies of the central nervous system involvement in papovavirus k infection in mice chronic infection of nude mice by murine k papovavirus serial passage of murine k-papovavirus in primary cultures of mouse embryo cells comparison of an enzymelinked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to k-papovavirus in mice diagnostic polymerase chain reaction assays for identification of murine polyomaviruses in biological samples a model for mixed virus disease: co-infection with moloney murine leukemia virus potentiates runting induced by polyomavirus detection of dna and rna virus genomes in organ systems of whole mice: patterns of mouse organ infection by polyomavirus transplacental transmission of polyoma virus in mice persistence of polyomavirus in adult scid c.b- mice immunosuppression and murine polyomavirus infection reactivation of polyoma virus in kidneys of persistently infected mice during pregnancy ifn-g controls mouse polyomavirus infection in vivo heterogeneity among viral antigen-specific cd þ t cells and their de novo recruitment during persistent polyomavirus infection long-term infection of adult mice with murine polyomavirus following stereotaxic inoculation into the brain murine polyomavirus virus-like particles (vlps) as vectors for gene and immune therapy and vaccines against viral infections and cancer cd þ, and cd þ t cells can act separately in tumour rejection after immunization with murine pneumotropic virus chimeric her /neu virus-like particles molecular characterization of a newly recognized mouse parvovirus the rodent parvoviruses viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research. diseases lurking in the shadows: emerging rodent infectious diseases coping with parvovirus infections in mice: health surveillance and control identification and propagation of a putative immunosuppressive orphan parvovirus in cloned t cells molecular characterization of newly recognized rodent parvoviruses identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice temporal transmission studies of mouse parvovirus in balb/c and c.b- / icr-prkdc scid mice serologic prevalence of mpv in mouse strains in a commercial laboratory mouse colony determined by using vp antigen serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant vp proteins in vivo studies with an 'orphan' parvovirus of mice characterization of mouse parvovirus infection by in situ hybridization humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus experimental infection of mice with hamster parvovirus: evidence for interspecies transmission of mouse parvovirus effect of mouse strain and age on detection of mouse parvovirus by use of serologic testing and polymerase chain reaction analysis strainand age-associated variation in viral persistence and antibody response to mouse parvovirus in experimentally infected mice characterization of mouse parvovirus infection among balb/c mice from an enzootically infected colony expression of recombinant parvovirus ns protein by a baculovirus and application to serologic testing of rodents validation of an enzyme-linked immunosorbent assay for detection of mouse parvovirus infection in laboratory mice detection of newly recognized rodent parvoviruses by pcr detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays antemortem detection of mouse parvovirus and mice minute virus by polymerase chain reaction (pcr) of faecal samples polymerase chain reaction for detection of rodent parvoviral contamination in cell lines and transplantable tumors embryo transfer rederivation of c.b- /icr-prkdc scid mice experimentally infected with mouse parvovirus detection of mouse parvovirus in mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft rejection a minute virus of mice immunosuppressive activity of a subline of the mouse el- lymphoma. evidence for minute virus of mice causing the inhibition pathogenicity of fibroblastand lymphocyte-specific variants of minute virus of mice pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype minute virus of mice. ii. prevalence, epidemiology, and occurrence as a contaminant of transplanted tumors electron microscopic localization of virions in developing teeth of young hamsters infected with minute virus of mice experimentally induced infection with autonomous parvoviruses, minute virus of mice and h- , in the african multimammate mouse (mastomys coucha) pathogenicity of minute virus of mice (mvm) for rats, mice, and hamsters fetal infections of hamsters, rats, and mice induced with the minute virus of mice (mvm) in vitro myelosuppressive effects of the parvovirus minute virus of mice (mvmi) on hematopoietic stem and committed progenitor cells myeloid depression follows infection of susceptible newborn mice with the parvovirus minute virus of mice (strain i) severe leukopenia and dysregulated erythropoiesis in scid mice persistently infected with the parvovirus minute virus of mice reduced fecundity and death associated with parvovirus infection in b-lymphocyte deficient mice minute virus of mice: antibody response, viral shedding, and persistence of viral dna in multiple strains of mice presence of minute virus of mice in immunocompetent mice despite the onset of host immunity gender influences infectivity in c bl/ mice exposed to mouse minute virus a rapid and simple procedure to detect the presence of mvm in conditioned cell fluids or culture media risk assessment of minute virus of mice transmission during rederivation: detection in reproductive organs, gametes, and embryos of mice after in vivo infection transmission of mouse minute virus (mmv) but not mouse hepatitis virus (mhv) following embryo transfer with experimentally exposed in vivo-derived embryos antineoplastic activity of parvoviruses experience with viral contamination in cell culture the molecular biology of arteriviruses lactic dehydrogenase virus isolation of lactate dehydrogenase-elevating viruses from wild house mice and their biological and molecular characterization lactate dehydrogenaseelevating virus induces systemic lymphocyte activation via tlr -dependent ifnalpha responses by plasmacytoid dendritic cells distinct gamma interferon-production pathways in mice infected with lactate dehydrogenase-elevating virus lactate dehydrogenase-elevating virus replication persists in liver, spleen, lymph node, and testis tissues and results in accumulation of viral rna in germinal centers, concomitant with polyclonal activation of b cells transmissible agent associated with types of experimental mouse neoplasms from bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics lactic dehydrogenase virus (ldhv) contamination in human tumor xenografts and its elimination contamination of a monoclonal antibody with ldh-virus causes interferon induction infection of central nervous system cells by ecotropic murine leukemia virus in c and akr mice and in in utero-infected ce/j mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus lactate dehydrogenase-elevating virus regulation of transplacental virus infection by developmental and immunological factors: studies with lactate dehydrogenaseelevating virus transplacental lactate dehydrogenase-elevating virus (ldv) transmission: immune inhibition of umbilical cord infection, and correlation of fetal virus susceptibility with development of f / antigen expression regulation of maternal-fetal virus transmission in immunologically reconstituted scid mice infected with lactate dehydrogenase-elevating virus immunofluorescent antibody response to lactic dehydrogenase virus in different strains of mice characterization of lactate dehydrogenase-elevating virus orf protein expressed by recombinant baculoviruses enzymatic amplification of lactate dehydrogenase-elevating virus detection of lactate dehydrogenase-elevating virus in transplantable mouse tumors by biological assay and rt-pcr assays and its removal from the tumor cell false negative results using rt-pcr for detection of lactate dehydrogenase-elevating virus in a tumor cell line comparison of the sensitivity of in vivo antibody production tests with in vitro pcr-based methods to detect infectious contamination of biological materials detection and typing of lactate dehydrogenase-elevating virus rna from transplantable tumors, mouse liver tissues, and cell lines, using polymerase chain reaction comparison of the mouse antibody production (map) assay and polymerase chain reaction (pcr) assays for the detection of viral contaminants relationship between the lactic dehydrogenase-elevating virus and transplantable murine tumors removal of lactate dehydrogenase-elevating virus from human-in-mouse breast tumor xenografts by cell-sorting infection of mice with lactate dehydrogenase-elevating virus leads to stimulation of autoantibodies suppression of development of diabetes in nod mice by lactate dehydrogenase virus infection natural killer cell activation after infection with lactate dehydrogenase-elevating virus effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen lactate dehydrogenase-elevating virus infection at the sensitization and challenge phases reduces the development of delayed eosinophilic allergic rhinitis in balb/c mice suppression of acute anti-friend virus cd þ t-cell responses by coinfection with lactate dehydrogenase-elevating virus mammalian reservoirs of arenaviruses risk to humans through contact with golden hamsters carrying lymphocytic choriomeningitis virus (author's transl) lymphocytic choriomeningitis virus first outbreak of callitrichid hepatitis in germany: genetic characterization of the causative lymphocytic choriomeningitis virus strains arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin- expression and hepatocyte proliferation lymphocytic choriomeningitis virus spatial and temporal dynamics of lymphocytic choriomeningitis virus in wild rodents, northern italy antibodies to lymphocytic choriomeningitis virus in wild rodent sera in egypt seroepidemiological survey of lymphocytic choriomeningitis virus in wild house mice in china with particular reference to their subspecies lymphocytic choriomeningitis virus infection and house mouse (mus musculus) distribution in urban baltimore contamination of transplantable murine tumors with lymphocytic choriomeningitis virus human-rodent contact and infection with lymphocytic choriomeningitis and seoul viruses in an inner-city population seroprevalence of lymphocytic choriomeningitis virus in nova scotia lymphocytic choriomeningitis virus infection in a province of spain: analysis of sera from the general population and wild rodents mouse-tohuman transmission of variant lymphocytic choriomeningitis virus exposure to lymphocytic choriomeningitis virus lymphocytic choriomeningitis outbreak associated with nude mice in a research institute laboratory studies of a lymphocytic choriomeningitis virus outbreak in man and laboratory animals lymphocytic choriomeningitis virus in southern france: four case reports and a review of the literature lymphocytic choriomeningitis in laboratory personnel exposed to hamsters inadvertently infected with lcm virus pet rodents and fatal lymphocytic choriomeningitis in transplant patients outbreak of lymphocytic choriomeningitis virus infections in medical center personnel virus zoonoses and their potential for contamination of cell cultures transmission of lymphocytic choriomeningitis virus by organ transplantation lymphocytic choriomeningitis virus: an unrecognized teratogenic pathogen lymphocytic choriomeningitis virus: reemerging central nervous system pathogen lymphocytic choriomeningitis virus: emerging fetal teratogen persistence of lymphocytic choriomeningitis virus at very low levels in immune mice mechanisms of antibody-mediated protection against lymphocytic choriomeningitis virus infection: mother-to-baby transfer of humoral protection murine hepatitis caused by lymphocytic choriomeningitis virus. ii. cells involved in pathogenesis biology and pathogenesis of lymphocytic choriomeningitis virus infection lymphocytic choriomeningitis infection of the central nervous system murine infection with lymphocytic choriomeningitis virus following gastric inoculation timed appearance of lymphocytic choriomeningitis virus after gastric inoculation of mice lymphocytic choriomeningitis infection undetected by dirty-bedding sentinel monitoring and revealed after embryo transfer of an inbred strain derived from wild mice detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by elisa using purified recombinant nucleoprotein development of a reverse transcription-polymerase chain reaction assay for diagnosis of lymphocytic choriomeningitis virus infection and its use in a prospective surveillance study detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain reaction analysis quantitative pcr technique for detecting lymphocytic choriomeningitis virus in vivo centers for disease control and prevention and national institutes of health mechanisms of humoral immunity explored through studies of lcmv infection lymphocytic choriomeningitis virus and immunology viral persistence: parameters, mechanisms and future predictions stage of primary infection with lymphocytic choriomeningitis virus determines predisposition or resistance of mice to secondary bacterial infections reovirus type infection, liver, mouse the mouse in biomedical research. diseases reovirus infection in laboratory rodents direct spread of reovirus from the intestinal lumen to the central nervous system through vagal autonomic nerve fibers type reovirus neuroinvasion after intramuscular inoculation: direct invasion of nerve terminals and age-dependent pathogenesis reoviruses and the host cell orthoreoviruses and their replication passive immunity to fatal reovirus serotype -induced meningoencephalitis mediated by both secretory and transplacental factors in neonatal mice infectivity, disease patterns, and serologic profiles of reovirus serotypes , , and in infant and weanling mice reovirus-induced liver disease in severe combined immunodeficient (scid) mice. a model for the study of viral infection, pathogenesis, and clearance histopathological characterization of the naturally occurring hepatotropic virus infections of nude mice detection methods for the identification of rodent viral and mycoplasmal infections reverse transcriptionpolymerase chain reaction detection and nucleic acid sequence confirmation of reovirus infection in laboratory mice with discordant serologic indirect immunofluorescence assay and enzyme-linked immunosorbent assay results diagnosis of murine infections in relation to test methods employed reovirus not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease detection of reovirus type by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral l genome segment isolation of a non-pathogenic tumour-destroying virus from mouse ascites an oncolytic virus recovered from swiss mice during passage of an ascites tumour mouse hepatitis virus enterotropic mouse hepatitis virus effects of air temperature and relative humidity on coronavirus survival on surfaces asymptomatic infection of mouse hepatitis virus in the rat effects of experimental infection of the deer mouse (peromyscus maniculatus) with mouse hepatitis virus isolation of a latent murine hepatitis virus from cultured mouse liver cells induction of lytic plaques by murine leukemia virus in murine sarcoma virus-transformed nonproducer mouse cells persistently infected with mouse hepatitis virus mhv-s mouse hepatitis virus biology and epizootiology the cellular and molecular pathogenesis of coronaviruses enterotropic coronavirus (mouse hepatitis virus) in mice: influence of host age and strain on infection and disease response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice effective clearance of mouse hepatitis virus from the central nervous system requires both cd þ and cd þ t cells role of cd þ and cd þ t cells in mouse hepatitis virus infection in mice antibody prevents virus reactivation within the central nervous system mouse hepatitis virus enterotropic mouse hepatitis virus infection in nude mice persistent transmission of mouse hepatitis virus by transgenic mice duration of challenge immunity to coronavirus jhm in mice virus strain specificity of challenge immunity to coronavirus duration and strain-specificity of immunity to enterotropic mouse hepatitis virus passively acquired challenge immunity to enterotropic coronavirus in mice epizootic coronaviral typhlocolitis in suckling mice isolation of mouse hepatitis virus from infant mice with fatal diarrhea thymus involution induced by mouse hepatitis virus a in balb/c mice adverse effects of mouse hepatitis virus on ascites myeloma passage in the balb/ej mouse murine hepatitis virus strain produces a clinically relevant model of severe acute respiratory syndrome in a/j mice tolllike receptor deficiency increases disease and mortality after mouse hepatitis virus type infection of susceptible c h mice granulomatous peritonitis and pleuritis in interferon-gamma gene knockout mice naturally infected with mouse hepatitis virus pathogenesis of enterotropic mouse hepatitis virus in immunocompetent and immunodeficient mice vertical transmission of mouse hepatitis virus infection in mice tissue distribution and duration of mouse hepatitis virus in naturally infected immunocompetent icr (cd- ) and immunodeficient athymic nudenu mouse strains used for ovarian transplantation and in vitro fertilization rederivation of inbred strains of mice by means of embryo transfer risk assessment of mouse hepatitis virus infection via in vitro fertilization and embryo transfer by the use of zona-intact and laser-microdissected oocytes mouse hepatitis virus immunofluorescence in formalin-or bouin's-fixed tissues using trypsin digestion comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses monoclonal antibody solution hybridization assay for detection of mouse hepatitis virus infection detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection detection of rodent coronaviruses by use of fluorogenic reverse transcriptase-polymerase chain reaction analysis an immunofluorescence test for detection of serum antibody to rodent coronaviruses simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay differences in antibody production against mouse hepatitis virus (mhv) among mouse strains maternally-derived passive immunity to enterotropic mouse hepatitis virus mouse hepatitis virus: molecular biology and implications for pathogenesis maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus replication of murine coronaviruses in mouse embryonic stem cell lines. in vitro reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus efficacy of three microbiological monitoring methods in a ventilated cage rack rederivation of mhv and mev antibody positive mice by cross-fostering and use of the microisolator caging system elimination of mouse hepatitis virus from a breeding colony by temporary cessation of breeding evolution of mouse hepatitis virus (mhv) during chronic infection: quasispecies nature of the persisting mhv rna the elimination of mouse hepatitis virus by temporary transplantation of human tumors from infected athymic nude mice into athymic nude rats (rnun/rnun) development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase pcr assay to detect murine norovirus infection in mice stat -dependent innate immunity to a norwalk-like virus murine norovirus infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat -dependent interferon responses replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages persistent infection with and serologic cross-reactivity of three novel murine noroviruses murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence molecular detection of murine norovirus from experimentally and spontaneously infected mice naturally occurring murine norovirus infection in a large research institution soiled-bedding sentinel detection of murine norovirus the use of cross-foster rederivation to eliminate murine norovirus, helicobacter spp., and murine hepatitis virus from a mouse colony impact of murine norovirus on a mouse model of ibd virus-plus-susceptibility gene interaction determines crohn's disease gene atg l phenotypes in intestine murine norovirus: an intercurrent variable in a mouse model of bacteria-induced inflammatory bowel disease investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection effects of murine norovirus infection on a mouse model of diet-induced obesity and insulin resistance mouse adenovirus, k virus, and pneumonia virus of mice sendai virus and pneumonia virus of mice (pvm) pneumonia virus of mice: severe respiratory infection in a natural host pneumonia virus of mice infection, lung, mouse, and rat persistence of pneumonia virus of mice and sendai virus in germ-free (nu/nu) mice fatal pneumonia with terminal emaciation in nude mice caused by pneumonia virus of mice respiratory disease and wasting in athymic mice infected with pneumonia virus of mice pathogenesis of pneumovirus infections in mice: detection of pneumonia virus of mice and human respiratory syncytial virus mrna in lungs of infected mice by in situ hybridization differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains experimental pneumovirus infections: . hydrocephalus of mice due to infection with pneumonia virus of mice (pvm) detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis lethal exacerbation of pneumocystis murina. pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice handbook of animal models of infection mouse rotavirus murine rotavirus infection, mouse successful sanitation of an edim-infected mouse colony by breeding cessation role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhoea age-dependent diarrhoea induced by a rotaviral nonstructural glycoprotein the immunology of rotavirus infection in the mouse murine model of rotavirus infection evidence that resolution of rotavirus infection in mice is due to both cd and cd celldependent activities ifn-l determines the intestinal epithelial antiviral host defense persistent rotavirus infection in mice with severe combined immunodeficiency role of b cells and cytotoxic t lymphocytes in clearance of and immunity to rotavirus infection in mice comparison of methods for detection of serum antibody to murine rotavirus identification of nonspecific reactions in laboratory rodent specimens tested by rotazyme rotavirus elisa removal of inhibitory substances from human fecal specimens for detection of group a rotaviruses by reverse transcriptase and polymerase chain reactions synergistic rotavirus and escherichia coli diarrhoeal infection of mice infection of rabbits with sendai virus pathogenesis of sendai virus infection in the syrian hamster determinants of organ tropism of sendai virus sendai virus infection, lung, mouse, and rat multigenic control of resistance to sendai virus infection in mice naturally-occurring sendai virus infection of athymic nude mice signs and lesions of experimental sendai virus infection in two genetically distinct strains of scid/beige mice cooperation between cytotoxic and helper t lymphocytes in protection against lethal sendai virus infection. protection by t cells is mhc-restricted and mhc-regulated; a model for mhc-disease associations delayed clearance of sendai virus in mice lacking class i mhc-restricted cd þ t cells naturally occurring sendai virus disease of mice affinity of sendai virus for the inner ear of mice detection of nucleoprotein gene of sendai virus in the lungs of rats by touchdown nested reverse transcription polymerase chain reaction the effectiveness of a microisolator cage system and sentinel mice for controlling and detecting mhv and sendai virus infections the efficacy of a dirty bedding sentinel system for detecting sendai virus infection in mice: a comparison of clinical signs and seroconversion serological evidence that mus musculus is the natural host of theiler's murine encephalomyelitis virus comparison of mhg virus with mouse encephalomyelitis viruses genetic analysis of a theiler-like virus isolated from rats a serological indication of the existence of a guineapig poliovirus duration and patterns of transmission of theiler's mouse encephalomyelitis virus infection a spontaneous outbreak of theiler's encephalomyelitis in a colony of severe combined immunodeficient mice in the uk axonal loss results in spinal cord atrophy, electrophysiological abnormalities and neurological deficits following demyelination in a chronic inflammatory model of multiple sclerosis the theiler's murine encephalomyelitis viruses susceptibility of inbred mice to chronic central nervous system infection by theiler's murine encephalomyelitis virus role of the humoral immune response in resistance to theiler's virus infection the genetics of the persistent infection and demyelinating disease caused by theiler's virus infection with theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via nf-kappab activation: potential role for the initiation of demyelinating disease innate immune response induced by theiler's murine encephalomyelitis virus infection cardioviruses: encephalomyocarditis virus and theiler's murine encephalomyelitis virus effect of immunization with theiler's virus on the course of demyelinating disease protection of sjl/j mice from demyelinating disease mediated by theiler's murine encephalomyelitis virus immunosuppression promotes cns remyelination in chronic virus-induced demyelinating disease pathogenesis of theiler's murine encephalomyelitis virus mechanism of theiler's virus-induced demyelination in nude mice survival of athymic (nu/nu) mice after theiler's murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody vacuolar neuronal degeneration in the ventral horns of scid mice in naturally occurring theiler's encephalomyelitis evolution of the placental barrier to fetal infection by murine enteroviruses enhanced detection of theiler's virus rna copy equivalents in the mouse central nervous system by real-time rt-pcr spontaneous demyelinating myelopathy in aging laboratory mice key: cord- -ys n authors: whary, mark t.; baumgarth, nicole; fox, james g.; barthold, stephen w. title: biology and diseases of mice date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ys n today’s laboratory mouse, mus musculus, has its origins as the ‘house mouse’ of north america and europe. beginning with mice bred by mouse fanciers, laboratory stocks (outbred) derived from m. musculus musculus from eastern europe and m. m. domesticus from western europe were developed into inbred strains. since the mid- s, additional strains have been developed from asian mice (m. m. castaneus from thailand and m. m. molossinus from japan) and from m. spretus which originated from the western mediterranean region. laboratory animal medicine development of the 'modern' laboratory mouse. research use of mice has grown exponentially during the past and current century with the recognition of the power of the mouse for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized mammal on earth (silver, ; lyon et al., ; morse, a) . the current ability to create highly sophisticated, genetically engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal. historical reviews have documented the origins of the laboratory mouse, which extend thousands of years into antiquity (keeler, ; morse, ; silver, ) . the laboratory mouse belongs within the genus mus, subfamily murinae, family muridae, superfamily muroidea, order rodentia, and within the m. musculus clade collectively called the 'house mouse' (lundrigan et al., ) . anatomic features of molar teeth and cranial bones were traditionally used by zoologists to identify over different species within the genus, and to differentiate them from other murids. because of considerable phenotypic variation within a single mus species, this approach has proven to be inaccurate, and given way to contemporary genetic analysis. the native range of the genus mus is eurasia and north africa. members of this genus are generally classified as aboriginal, consisting of species that live independent of humans, or commensal, which includes taxa that have coevolved and geographically radiated with human civilization since the dawn of agriculture , years before present (bp). this close association with human agrarian society gave rise to the genus name, derived from sanskrit, mush: to steal. the commensal group is known as the 'house mouse' clade, consisting of several subspecies of mus musculus, including m. m. domesticus, m. m. musculus, m. m. castaneus, m. m. bactrianus , and a lesser known lineage, m. m. gentilulus (prager et al., ) . the japanese house mouse, m. m. molossinus, is a natural hybrid of m. m. musculus and m. m. castaneus. the progenitor of the m. musculus clade arose in the northern indian subcontinent and diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). there is debate whether these taxa are species or subspecies, and some have referred to them as 'incipient species,' but their genetic divergence is now blurring as they colonize the world and hybridize. the native ranges of these taxa are important for understanding the origins of various laboratory mice, whose genomes are mosaics derived from m.m. domesticus (~ %), m.m. musculus (~ %), and m.m. castaneus (~ %) (wade and daly, ; wade et al., ) . it is now apparent that the m.m. musculus and m.m. castaneus contributions to the laboratory mouse genome were primarily derived from m.m. molossinus japanese fancy mice (takada et al., ) . mus m. domesticus is indigenous to western europe and southwest asia, m.m. musculus to eastern europe and northern asia, m.m. castaneus to southeast asia, and m.m. molossinus to japan and the korean peninsula. the cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in china over years bp. by the s, mouse 'fanciers' in asia had created many varieties of fancy mice, as did european fanciers, who subsequently acquired asian stocks, particularly japanese fancy mice (m.m. molossinus) , to mix with european (m.m. domesticus) fancy mouse varieties. this genetic mixing for fancy variants was also occurring in the united states, and these mouse lines contributed to many of the major laboratory mice used today. meanwhile, the european colonial expansion era contributed to the worldwide dissemination of m.m. domesticus, which now occupies every continent of the world. it is well documented that wild-caught m.m. domesticus also contributed to the genetic composition of fancy and laboratory mice on multiple occasions. despite their diverse genetic origins and phenotypic differences, most laboratory mouse strains are closely related, since many were derived from a genetically mixed but small number of fancy mice from a single mouse breeder (abbie lathrop's granby mouse farm, massachusetts) at the beginning of the th century. most inbred laboratory mice share a common maternal mitochondrial genome derived from m.m. domesticus (ferris et al., ; yu et al., ) , and a common y chromosome contributed by m.m. musculus (bishop et al., ) through its contribution to the genome of m.m. molossinus (nagamine et al., ) . thus, the most inclusive name that can be assigned to the genetically mosaic laboratory mouse is m. musculus, the over-arching name for the entire commensal clade. there are exceptions, however. c bl/ mice contain minor genetic elements derived from m. spretus (hardies et al., ) , and a number of wild aboriginal species that are not members of the m. musculus clade, including m. spretus, m. caroli, and others, have been established as inbred lines of mice. genetic mapping in mice began in the early s with a focus on inheritance of coat color. the first autosomal genes, albino and pink-eyed dilution, were linked in (haldane et al., ) . extensive linkage maps and an impressive array of inbred strains are now available to expedite genetic research (table . ) (lyon et al., ) . mice have pairs of telocentric chromosomes that are differentiated by their size and patterns of transverse bands. the chromosomes are designated by arabic numbers in order of decreasing size. during the s, chromosome rearrangements were used to assign known genetic linkage groups -identified by roman numerals -to specific chromosomes and for determining locus order with respect to the centromere. genes can be located physically on chromosomes by fluorescent in situ hybridization (fish). development of quantitative trait loci (qtl) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (moore and nagle, ) . for more information on comparative genomics, see chapter animal models in biomedical research, subsection c. one of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. histocompatibility (h) loci control expression of cell surface molecules that modulate critical immune responses, such as the recognition of foreign tissue. for example, the time, onset, and speed of skin graft rejection are controlled by two groups of h loci. the major group is located in the major histocompatibility complex (mhc, h ) on chromosome . the h complex contains several loci, including k, d, l, i-a, and i-e. inbred strains of mice, being homozygous, each have unique sets of h alleles, termed h haplotypes. for example, the balb h haplotype is h d and the c bl h haplotype is h b . the international immunogenetics (imgt) information system provides details on h haplotypes for various inbred mice (www.imgt.org/imgtrepertoiremhc/polymorphism/ haplotypes/mouse/mhc/mu_haplotypes.html). minor h loci groups are scattered throughout the genome and are responsible for delayed graft rejection. genes associated with the h complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. immune-mediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the h complex (stuart, ) . non-mhc or minor histocompatibility systems also are under active study (roopenian et al., ) . mouse genomics have accelerated tremendously in the last two decades, heralded by the development of a robust physical map and high-quality genome sequence of the c bl/ j mouse in by the international mouse genome sequencing consortium (waterston et al., ) . the mouse genomes project/wellcome trust sanger institute is extending this effort to include the genomic sequences of key mouse strains. completed and evolving sequence data are available through the european nucleotide archive (www.ebi.ac.uk/ena/home). the burgeoning numbers of inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are cataloged in the mouse genome informatics (mgi) database: http://www.informatics.jax.org/mgihome). the growing number of mutant mice has fostered the development of a number of mouse repositories, from which specific mice can be located and acquired. in the united states, there are four regional national institutes of health (nih)-supported mutant mouse regional resource centers (http://www.mmrrc.org), which link to international repositories in europe, japan, china, australia, and canada, as well as additional resource programs in the united states through the international mouse strain resource (imsr; http://www.informatics.jax.org/ imsr/index.jsp) for depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community. in addition to numerous mutant mice produced independently by scientists in various academic institutions, three major targeted gene knockout programs, all utilizing c bl/ n embryonic stem cells, are under way internationally, and funded by the nih, the european community, and genome canada (collins et al., ; skarnes et al., ) . these include the knock out mouse project (komp; http://www.knockoutmouse.org), the european conditional mouse mutagenesis program (eucomm; http://www.eucomm.org), and the north american conditional mouse mutagenesis project (norcomm; http://norcomm.phenogenomics.ca/index. htm). these mouse lines will be available through laboratory animal medicine three distribution centers: the german resource center for genome research (rzpd; http://www.rzpd.de), the komp repository (https://komp.org), and the canadian mouse consortium (cmc; http://www.mousecanada. ca/index.htm). the repositories are all linked to the imsr, and provide access to mice, germplasm, genomic detail, and phenotypic data. genetic, genomic, and biological data are also available through the international mouse phenotyping consortium (impc; www.mousephenotype. org) and the mouse genome database (mgd; http://www. informatics.jax.org) (eppig et al., ) . inbreeding is a fundamental genetic tool applied to the laboratory mouse and detailed information is available on the web (table . ). the first inbred strain (dba) was developed by c.c. little in , with the subsequent creation of over inbred strains and stocks of mice (festing, ) . genetic origins, basic characteristics, references, and breeding performance of inbred strains of mice are available through michael festing's online version of inbred strain characteristics (http:// www.informatics.jax.org/external/festing/mouse/ strains.shtml). overviews of genetic manipulation for the creation of different types of mice are available (lyon et al., ; silver, ) . inbred mouse lines are termed strains, and are achieved by or more brother × sister (filial; f) generations (table . ). mice within an inbred strain, for practical purposes, are genetically identical (syngeneic or isogenic) to other mice of the same strain and sex. because of residual heterozygosity, a strain is not fully inbred until after f generations. most commonly used inbred mouse strains represent or more f generations, providing a high degree of experimental reproducibility. the mouse genome is not static, so when branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations result in genetic differences. therefore, if branches of an inbred strain are separated before f , if branches have separated for generations, or if genetic differences arise, the different branches become substrains. the same holds true if branches of a substrain diverge, resulting in substrains of the inbred substrain. when two inbred mouse strains are crossed, the f hybrids are genetically identical to one another (isogenic), but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parental strain), whereas f hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). with each subsequent f generation, mice once again approach inbred status. this technique is used for creating recombinant inbred (ri) strains. ri strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by singlepair random matings of sibling mice from the f generation, thereby creating separate breeding lines. each line created is maintained separately, and then propagated by brother-sister matings for generations, with each line becoming a separate inbred strain, but belonging to a set of ri strains. ri mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains (bailey, ) . ri sets are generally limited to two parental strains. an ongoing international effort has been undertaken to increase allelic diversity among ri strains by creating the collaborative cross (cc) in which a panel of ri strains are being generated mixing the genomes from eight disparately related inbred (octo-parental) mouse strains, including a/j, c bl/ j, s /svimj, nonobese diabetic (nod)/shiltj, nzo/ hlltj, cast/eij, pwk/phj, and wsb/eij. these eight strains capture nearly % of the known genetic variation present among laboratory mice. future applications of the cc will utilize ri intercrosses of pairs of ri cc lines (threadgill and churchill, ; welsh et al., ) . recombinant congenic strains are sets of inbred strains derived in a manner similar to that for ri sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the f generation, before inbreeding is begun. the other parental strain is designated as the donor strain. the proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (demant and hart, ) . advanced intercross lines (ails) are another type of ri lines. they are made by producing an f generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. the purpose is to increase the possibility of recombination between tightly linked genes. when a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele. frequently, a mutation that arose in one inbred strain may be desired within the genetic background of another inbred strain. this can be accomplished by backcrossing, in which an f hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. after backcross generations (n generations), the mutant mouse line is now congenic to the background inbred strain. backcrossing to create congenic strains of mice has been used extensively when targeted mutations have been induced in embryonic stem cells, with backcrossing onto c bl/ inbred mice. congenic mice are never co-isogenic, as the preserved locus in a congenic mouse is invariably surrounded by flanking dna, which may significantly influence phenotype (linder, ) . in contrast to inbred mice, outbred mice are genetically heterogeneous and are maintained by breeding systems that intentionally minimize inbreeding. outbred mice are called stocks, which are defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximal heterozygosity. outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. outbreeding can be achieved only in a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population. a small breeding population or passage through the genetic 'bottleneck' of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. in a population of breeding pairs, e.g., heterozygosity will decrease at % per generation with standard randomization techniques. random breeding involves the statistically random selection of breeders by using a random numbers table or computer program. an outbreeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. another 'circular' set of cages serves as the breeding nucleus for the n + generation. each mated pair in the nth generation contributes one female and one male to the n + generation. outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + generation. most outbred mouse stocks are of 'swiss' origin, derived from nine mice imported to the united states in , and are therefore quite homogeneous genetically (chia et al., ) . various lines of these mice have been maintained at different institutions, giving rise to numerous closely related stocks. although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many swiss mouse stocks are blind due to the homozygous recessive rd allele (serfilippi et al., b) . it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. in that regard, the diversity outbred mouse has been developed, which is a heterogeneous stock derived from the same eight founder inbred strains of the cc . additional types of inbred mice are utilized in research, including consomic and conplastic strains. consomic strains, also known as chromosome substitution strains, are inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits (singer et al., ) . conplastic mice are inbred mice that are congenic for different mitochondrial genomes (mtdna) contributed by other inbred strains, other subspecies, or other species of mus (yu et al., ). in addition to spontaneously occurring mutations that are maintained as co-isogenic strains (such as the c bl/ beige mouse), mutant lines of mice have been created by radiation mutagenesis, chemical mutagenesis, or transgenesis. radiation was one of the earlier methods for in vivo mutagenesis (silver, ) , but in vitro radiation of embryonic stem (es) cells is also performed (thomas et al., ) . chemical mutagenesis involves in vivo treatment of male mice or in vitro treatment of es cells with mutagenic chemicals such as ethylmethanesulphonate (ems) or n-ethyl-n-nitrosourea (enu) , which induce point mutations in dna (o'brien and frankel, ; justice et al., justice et al., , . technically, a transgenic mouse is any mouse in which foreign dna has been integrated into its genome, regardless of method. however, the term transgenic commonly refers to mice that are genetically altered by additive transgenesis through microinjection of foreign dna into the pronucleus of a fertilized egg. each ensuing embryo results in a genetically different founder mouse, since the transgene is integrated in random sites of the genome of each founder mouse. since the injected dna is not homologous to the mouse genome and is not an allele, transgenic founders are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene. transgenes typically integrate as tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line (tinkle and jay, ) . transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissuespecific expression of the cdna, so that the transgene expression pattern reflects the expression pattern of the promoter. transcriptional regulation of the transgene can be inducible by drug-dependent regulatory control, such as the widely used tetracycline (tet) regulatory system, in which treatment of mice with tetracycline or doxycycline induces up-or down-regulation of the transgene (jaisser, ) . es cells are used for the less efficient integration of genetic material by homologous dna recombination, but allow large-scale screening of es cell clones for transformation. integration can be achieved in a random fashion by gene trapping, or by targeted mutation. both methods involve homologous dna recombination. gene trapping is a high-throughput approach that randomly introduces insertional mutations within the genome. vectors contain a gene trapping cassette with a promoter-less reporter gene and/or selectable genetic marker flanked by an upstream ′ splice site and a downstream termination sequence. when inserted into an laboratory animal medicine intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a dna tag for the rapid identification of the disrupted gene (skarnes et al., ) . targeted gene mutations are achieved by homologous recombination of specific sites within the genome of es cells. homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration. a variation on this approach is site-specific recombinase (ssr) technology. two of the most common recombinases are cre from the coliphage p and flp from saccharomyces cerevisiae. cre and flp mediate recombination between target sites, termed loxp and frt, respectively. for example, cre loxp target sites are engineered to flank the gene target, which can be used in different ways to achieve different outcomes (conditional mutations), depending upon the orientation and location of the flanking loxp sites. if the loxp sites are oriented in opposite directions, cre recombinase mediates inversion of the floxed segment. if the loxp sites are on different chromosomes (trans), cre recombinase mediates a chromosomal translocation. if the loxp sites are oriented in the same direction on the same chromosome (cis), cre recombinase mediates deletion of the floxed segment. once the floxed mutation is created in es cells, the transformed es cells are developed into a mouse with the conditional mutation. the conditional mutant mouse is then genetically crossed with a cre transgenic mouse, in which cre recombinase is under the control of a ubiquitous or tissue-specific promoter. wherever and whenever cre is expressed, cre recombinase will recognize and recombine the loxp sites. this approach can include insertion of reporter genes and selectable markers, and can be under the control of inducible gene expression systems (http:// www.eucomm.org/docs/protocols/mouse_protocol_ _ sanger) (nagy, ) . es cells are pluripotent with the full genetic capacity to develop into mice when implanted into the blastocyst of a developing embryo. interest in 'embryonal carcinomas' (teratomas) that arose in relatively high frequency in the testes of mice and early gene transfer experiments in the late s and early s led to the development of es cell lines derived from several different strains. this early emphasis on teratomas prompted creation of 'better' mouse lines that were more prone to development of testicular teratomas, resulting in genetic corruption of the mouse (simpson et al., ; threadgill et al., ) . this realization gave rise to the need to revise mouse nomenclature (festing et al., ) . this was necessary because genetic variation significantly impacts homologous recombination in order to match genome sequence of the es cell line with the mouse from which it was derived. es cells can be created from any mouse strain or hybrid, but es cell lines have been commonly used. recent international knockout mouse program efforts use c bl/ n es cells. transformed es cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. the pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed es cells (chimeras). the goal is for male chimeric progeny to produce spermatozoa of es cell origin (containing the mutation), in order to create f progeny by mating the chimera with the desired background strain (http://www.eucomm.org/docs/protocols/mouse_protocol_ _sanger). for this reason, most es cell lines are xy, which favors male chimerism. if the es cells are of (or other) strain origin, the chimeras are often bred to a desired background mouse strain (commonly c bl/ ) and backcrossed for n generations, thereby creating congenic inbred mouse lines. recent international knockout mouse efforts utilize c bl/ n es cells, so that chimeric males are bred directly with c bl/ mice, thereby creating co-isogenic lines. the latter approach saves time and money, and creates a more genetically refined mutant mouse. an alternate approach is to allow es cells to aggregate with a developing embryo to form blastocysts in culture (aggregation chimera), then implant the chimeric blastocysts (tanaka et al., ) . rna interference (rnai), which functions through short double-stranded rna (dsrna), has also been utilized to produce transgenic mice, known as gene knockdown mice (gao and zhang, ; peng et al., ) . the dsrna is enzymatically processed into small molecules, termed small interfering rna (sirna), which find homologous target mrnas, resulting in interference. this phenomenon is believed to be a self-defense mechanism against viral infection. in order to adapt this approach to generation of transgenic mice, small hairpin rna (shrna) can be expressed in the same way as other transgenes in mice, resulting in processing of the shrna into sirna with gene-silencing effects. constructs are introduced into mouse es cells by electroporation or lentiviral infection. this method can be embellished conditionally, as with other transgenes. although rnai knockdown mice are genetically stable, rnai-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations (peng et al., ) . recent advances in engineered endonuclease (ee) technology, including zinc finger nucleases (zfns), laboratory animal medicine transcription activator-like effector nucleases (talens), and rna-guided endonucleases (rgens), have revolutionized the field of transgenics (sung et al., ; wijshake et al., ; gaj et al., ) . zfns and talens consist of engineered proteins that target dna fused to the nonspecific endonuclease, fok (cathomen and joung, ; joung and sander, ) . zfns are comprised of three to six tandem zinc finger proteins, each of which targets a specific bp nucleotide sequence. paired zfns are generated, with each half of the pair targeting opposite dna strands, allowing dimerization of fok which is required for introduction of double-stranded breaks (dsbs) in the dna of interest (cathomen and joung, ) . talens function similarly, but are composed of tandem repeats of - amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (rvd)', at positions and (joung and sander, ) . in contrast to zfns and talens, clustered regularly interspaced short palindromic repeats (crisprs) paired with crispr-associated (crispr/cas) systems are rgen systems that target specific dna sequences. cas proteins, rather than fok , produce dsb (hsu et al., ) . dsb generated by ee are repaired by host cells by either nonhomologous end joining (nhej) or, less commonly, by homologous recombination (hr). nhej is an error-prone repair system and results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption. hr is a less common repair pathway, but certain manipulations of the engineered nucleases can increase hr efficiency. for example, nucleases can be engineered to generate a break in a single strand of dna rather than inducing dsb, and the resulting nickases increase the incidence of hr with high fidelity (gaj et al., ; wijshake et al., ) . hr allows for the introduction of donor dna to generate knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxp sites (brown et al., ; wijshake et al., ) . vectors encoding the ee can be injected into mouse embryos by pronuclear injection of dna, intracytoplasmic injection of rna, or transfection of mouse es cells (sung et al., ; wijshake et al., ) . one advantage of ee technologies over more traditional transgenic methods is the ability to target dna and induce mutations in any background strain of mouse negating the need to backcross onto the desired strain. multiple genes can be targeted with crisprs simultaneously, thus avoiding the need to cross single knockout animals (zhou et al., ) . in addition, it is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation (zhou et al., ; wijshake et al., ) . vectors for generating ee are available through plasmid repositories; websites are available to assist in identifying appropriate dna sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (xie et al., ; sander et al., ; bae et al., ; reyon et al., ; herscovitch et al., ; wolfson, ) . crisprs tend to be particularly cost effective and easy to design, with minimal restrictions for targeting specific dna sequences. there are currently more than separate outbred stocks and traditional inbred strains, often with multiple substrains (table . ). in addition, there are thousands of induced mutant strains. therefore, it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion, and to ensure reproducibility of research results. as an example of substrain variation that makes precise nomenclature important, cba/j mice are homozygous for the retinal degeneration allele (rd ), whereas cba/caj mice do not carry this allele. the international committee on standardized genetic nomenclature for mice and rats, established in the early s, is responsible for genetic nomenclature rules. the rules are available online at the mgi website (http:// www.informatics.jax.org/mgihome/nomen). inbred mouse strains are designated by a series of capital letters and/or numbers, which often provide a shorthand description of the origin and history of the strain. the c bl/ j mouse serves as an example. the inbred strain c bl originated from abbie lathrop's female (and male ) at the cold spring harbor laboratory (c), and was the black (bl) line from this female. early in their history, inbred c bl mice split into major substrains, e.g., c bl/ and c bl/ . substrains are identified by appending a forward slash (/) after the inbred strain name. since , uniform international nomenclature has been built upon these historical names, so that substrains of an inbred strain are now designated using lab codes that are registered in the international laboratory code registry maintained at the institute for laboratory animal research (ilar) of the national academies (dels. nas.edu/global/ilar/lab-codes). laboratory codes are composed of one to five letters that identify an institute, laboratory, or investigator. each lab code starts with an uppercase letter, followed by lowercase letters if more than one letter is used (such as n, j, jci, crl, and tac). the j in c bl/ j means it is a substrain maintained at the jackson laboratory (j). another common substrain of c bl/ mice is c bl/ n, which is maintained at nih (n). substrains can be cumulative, reflecting the genetic history of the mouse strain. for example, there are a number of c bl/ j substrains (such as c bl/ jjci and c bl/ jjmsslc), and a number of c bl/ n substrains (such as c bl/ njci, c bl/ ncrlcrlj, dba/ j inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before generations of brother × sister breeding and is the subline maintained at the jackson laboratory (j) c h/hesn-ash/+ co-isogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on c h/hesn c bl/ j-tyrc- j /+ co-isogenic segregating inbred mutant strain carrying the albino j mutant allele of the cloned tyrosinase gene (tyr) aej/gnj-a e /a w-j inbred strain segregating for two alleles at the agouti gene congenic inbred strain in which the b haplotype at the h complex was transferred from c bl/ j (b ) to the akr background b .cba-d mit -d mit congenic strain in which the chromosomal segment between d mit and d mit was transferred from cba to b b .cg m lepr db /++ congenic inbred strain in which the linked mutant genes misty (m) and diabetes (lepr db ) were transferred from multiple, mixed, or unknown genetic backgrounds to b and are carried in coupling, i.e., on the same chromosome b .cg-m +/+ lepr db congenic inbred strain in which the m and lepr db mutations are carried in repulsion bxd- /ty recombinant inbred (ri) strain number in a set of ri strains derived from a c bl/ j (b) female mated to a dba/ j (d) male and made by taylor (ty) recombinant congenic (rc) strain number in a set made by crossing the balb/c (c) and sts (s) strains, backcrossing one or two times to balb/c and then inbreeding as with ri strains and c bl/ ntac). significant differences may exist among these substrains (mekada et al., ). thus, a string of substrain designations indicate the genetic progression of the substrain, which can be identified when reading the entire strain name. this nomenclature is highly nuanced, as c bl/ ncrlcrlj mice, whose last letter is a lowercase j, are not a substrain maintained at the jackson laboratory (j), but rather at charles river japan (crlj), underscoring the importance of upper-and lowercase lettering in rodent nomenclature. balb/c mice are another popular inbred strain with numerous substrains. like the ' ' in c bl/ , the 'c' that follows laboratory animal medicine of the mutational event as a superscript. for example, cftr tm unc is a targeted mutation (tm), first line ( ) congenic mice are often derived from es cells, backcrossed onto a background strain, such as c bl/ . under such circumstances, when the backcross generation is at n , the '.' symbol is used between the background inbred strain and the donor strain (e.g., c bl/ n. p /olahsd-abc tm zzz , abbreviated as b . -abc tm zzz . when backcrossing is incomplete but at the n generation, the mouse is an incipient congenic, designated with a ';' in lieu of a '.': b ; -abc tm zzz . if the background strain is mixed genetic origin, it is designated stock. -abc tm zzz . if the donor strain is mixed origin, it is designated 'cg'. for example, b .cg-abc tm zzz outbred stock that meets specific criteria is designated by placing the lab code before the stock symbol, separated by a full colon (':'). for example, hsd:icr designates an icr (swiss) outbred stock maintained by harlan sprague dawley (hsd). the above overview covers the nomenclature of commonly encountered types of mice. there are numerous additional specifications for nomenclature of mice. details are available at the mgi website (http://www. informatics.jax.org/mgihome/nomen). optimum housing conditions and husbandry practices for research mice should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, labor and financial resources, behavioral needs of mice, and investigator needs for consistent colony maintenance, including standardized husbandry practices and nutrition. the emerging interest in the mouse microbiome in combination with the immune competency of diverse genetically engineered mouse strains demands high standards of mouse care. mouse colonies are optimally maintained as specificpathogen-free (spf) which obligates veterinary and facility management to exclude specific organisms. housing options for spf immunocompetent mice typically include static or individually ventilated microisolator cages, which differ significantly in cost and labor required to maintain. severely immunodeficient strains such as nod.cg-prkdcscid il rgtm wjl/szj (nsg) mice require staff training, caging systems and husbandry practices that minimize risk for opportunistic infections the '/' in balb/c is a lowercase letter because of historical precedent. subsequent substrains follow accepted nomenclature, e.g., balb/cbyj and balb/cann. hybrids of two inbred strains are often used in research, and are particularly common with engineered mutations that are created in -derived es cells, followed by intercrossing the chimeric mice with c bl/ or other background strains of mouse. when an f hybrid is created, the female partner is listed first, e.g., a c bl/ j × s /svpas hybrid would be designated: c bl/ j s /svpasf . ri strain sets that are derived from two parental inbred strains are identified by an x between the two parental strains followed by a hyphen designating the specific ri line, e.g., c bl/ jxdba/ j- , c bl/ jxdba/ j- , etc. cc ri strains do not use the x between the parental strains because they are derived from eight parental strains, so they are designated cc- , cc- , etc. in order to simplify the complexity of this nomenclature, abbreviations are used for common inbred strains and substrains of mice (table . ), but it is important to include the full genetic nomenclature in publications. using the abbreviated nomenclature, c bl/ j s /svpasf mice would be b f and c bl/ jxdba/ j- ri mice would be bxd- . parental order is an important consideration in nomenclature, as a b mouse is genetically different from a b mouse due to mitochondrial dna (from the female) and y chromosome (from the male) differences. mutant genes are designated by a brief abbreviation for the mutation (e.g., bg for beige which arose at the jackson laboratory, j). the symbol for the parent gene is noted in italics, starting with an uppercase letter (e.g., lyst) and the mutant allele is designated in superscript (e.g., lyst bgj ). thus, the beige mutation arose in c bl/ j mice, so that c bl/ j beige mice, which are co-isogenic with c bl/ j mice, are designated c bl/ j-lyst bgj . a transgenic strain is designated by the strain and substrain name, followed by a symbol for the transgene. transgene symbols take the form tg(yyy)#zzz, where 'tg' indicates transgenic, yyy defines the transgene as a brief description of the inserted dna (such as a gene symbol), '#' is the assigned number in the series of events generated using a given construct, and 'zzz' is the lab code. for example, fvb/n-tg(mmtv-erb ) led mice are inbred fvb/n mice in which the rat erb gene was introduced under control of the mouse mammary tumor virus (mmtv) ltr promoter (mmtv-erb ), the first line ( ) created in the laboratory of phil leder (lab code led). when a transgene causes an insertional mutation in an identified endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (-abc tg zzz ). a targeted mutation, or knockout, is designated by the mutated gene with the identification laboratory animal medicine (foreman et al., ) . barrier practices and microisolator techniques may include autoclaved or irradiated feed and bedding, autoclaved or acidified water, cage-tocage transfer of mice using disinfected forceps, positive displacement change hoods, and verified sanitation of caging and equipment through tunnel or rack washers to prevent fomite transmission of infectious agents (compton et al., ) . in addition to husbandry staff, it is critical to maintenance of colony health status that investigators who handle cages are also trained in these techniques. the microenvironment for mice is the cage which will vary in design, size, and composition. vendors often successfully house production colonies in open-top cages to expedite detection of pathogen transmission should a break occur. end-users usually prefer filter-top microisolator cages which prevent (at least) gross contamination between cages by fecal contamination and aerosolized debris. the objective is to keep mice in an uncrowded, socially compatible, low-odor, dry and clean environment. ambient temperature should minimize any confounding impact on the animal model and energy expenditure for the mice, while also being suitable for staff and investigators. shoebox static cages made of polycarbonate, polypropylene, or polystyrene plastic (in order of decreasing cost and durability) with filtered microisolator tops continue to be used for housing and breeding mice. older cage designs are being rapidly supplanted by individually ventilated caging systems that promote the advantages of increasing housing capacity, decreasing labor costs, and mitigating exposure of mice to noxious gases such as ammonia and exposure of humans to allergens. as more advanced caging systems are developed, the level of biosecurity may be increased but at the cost of increased health surveillance efforts to detect the source of an infectious outbreak (shek, ) . disposable, recyclable polyethylene caging is a recent innovation, particularly for facilities not equipped with a cage wash facility. animal care programs should carefully consider the necessity for housing mice on wire-mesh flooring because of injury risk to limbs and thermoregulation issues in neonates and hairless mice which are more difficult to maintain without nesting material. solidbottom cages should contain sanitary bedding, such as hardwood chips, paper products, or ground corn cob. criteria for selecting bedding vary with experimental and husbandry needs. it may be preferable to irradiate or autoclave bedding, but if this is not done, the bedding should be used only after its origin and microbial content have been evaluated (table . ). germfree and gnotobiotic mice require positive pressure isolators, most usually flexible film, with additional protection provided by sterile air through high-efficiency particulate air (hepa) filters. this equipment can be negatively pressurized when the objective is to contain known or unknown pathogens. animal care programs should establish enrichment policies which for mice should include social housing when mice are compatible and experiments do not require single housing. species-specific behaviors are encouraged by nesting material and hiding places such as tubes or shacks. nutrient requirements for the mouse are influenced by genetic background, disease status, growth rate, pregnancy, lactation, and environmental factors such as ambient temperature. the best current estimate of nutritional requirements is shown in table . . nutritional requirements for laboratory mice are also published periodically by the national research council and have been reviewed by knapka and coworkers (knapka et al., ; knapka, ) . feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. mice consume about - g of feed per day after weaning, and maintain this intake throughout life. outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (figs. . and . ). diet is often neglected as a variable in animal-related research. diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. therefore, diet must provide a balance of essential kraft ( ) . knapka ( ) . b linoleic acid: . % is adequate. c john and bell ( ) . d theuer ( ) . e knapka et al. ( ). f nutrition ( . g hurley and bell ( ) . h pleasants et al. ( ) . nutrients, and contaminants must be kept to a minimum (see also chapter ). natural-product commercial diets for mice are usually satisfactory for breeding and maintenance. animal care programs should avoid using fresh produce, grains, fish meal, or other supplements to minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens (guerrero-bosagna et al., ) . mouse diets can be purchased as open-formula, fixedformula, constant nutrition, and closed-formula which laboratory animal medicine are designed to reduce variation in experimental data attributable to diet (reviewed in barnard et al. ( ) ). diets are supplied in standard, irradiated, or autoclavable formulations. irradiated diets will be virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria. autoclavable diets are higher in heat-labile nutrient content. many programs use sterilized mouse chow exclusively to minimize risk of opportunistic infections. because commercial diets vary in nutrient content, diets should be selected for optimal maintenance of adult mice or for growth and reproduction in breeding colonies. mice should have continuous access to potable water even if a high-moisture diet is fed. water is needed for lubrication of dry food and for hydration. adult mice drink - ml of water per day. decreased water intake will decrease food consumption. water imbalance may occur immediately post weaning and weanlings on automatic watering systems need extra attention. water intake will decrease in sick mice. therefore, dosing mice with medicated water requires careful assessment of hydration and clinical or experimental efficacy of the compound administered. the main reference used to update this section of the rd edition is volume iii; normative biology, husbandry and models in the mouse in biomedical research, nd edition, aclam series published by academic press. normative data on the mouse are presented in table . , and clinical chemistry reference ranges are summarized in table . . mice have a relatively large surface area per gram of body weight. this results in dramatic physiologic changes in response to fluctuations in the ambient temperature (t a ). the mouse responds to cold exposure, e.g., by nonshivering thermogenesis. a resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal. a mouse must generate about kcal/m per h to maintain body temperature for each °c drop in t a below the thermoneutral zone. mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. therefore, it is not advisable to conserve energy in animal quarters at night by lowering t a . because of the ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. its biological half-time for turnover of water ( . days) is more rapid than for larger mammals. water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. the conservation of water can preempt thermal stability. if the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. the mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. mice can partially compensate for changes in t a increases from °c to °c. it adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. its primary means of cooling in the wild is behavioral -retreat into a burrow. in the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of °c or higher. thus, the mouse is not a true endotherm. in fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before days of age. the thermoneutral zone for mice varies with strain and with conditioning but is about . - . °c, narrower than that of any other mammal measured thus far. thermoneutrality should not be equated with comfort or physiological economy. recent data have suggested that mice housed under routine vivarium conditions are chronically cold-stressed. mice maintained at °c were shown to expend more energy compared with mice housed at intermediate ( °c) and a higher temperature ( °c) with an increase in glucose utilization and activation of brown adipose tissue (david et al., ) . in contrast, other studies report that mice in a t a range of - °c grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone. the respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharnyx; the intermediate section consists of larynx, trachea, and bronchi, all of which have cartilaginous support; and the posterior portion of the respiratory tract consists of the lungs. the left lung is a single lobe. the right lung is divided into four lobes: superior, middle, inferior, and postcaval (cook, ) (fig. . loeb and quimby ( ) . a mouse at rest uses about . ml o /g/h, which is about times more o /g/h than is used by an elephant. to accommodate for this high metabolic rate, the mouse has a high alveolar p o ; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (rbc) concentration; high rbc hemoglobin and carbonic anhydrase concentrations; a high blood o capacity; a slight shift in the o -dissociation curve, enabling o to be unloaded in the tissue capillaries at a high p o ; a more pronounced bohr effect, i.e., the hemoglobin affinity for o with changes in ph is more pronounced; a high capillary density; and a high blood sugar concentration. the kidneys, ureters, urinary bladder, and urethra form the urinary system. the paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. the right kidney is normally located anterior to the left kidney. kidneys from males of many inbred strains are consistently heavier than kidneys from females. the glomeruli of mice are small, about μm in diameter, or about half the size of glomeruli in rats. there are, however, . times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. mice excrete only a drop or two of urine at a time, and it is highly concentrated (table . ). the high concentration is made possible by long loops of henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of henle in the medulla. the mouse can concentrate urine to mosm/l, whereas humans can concentrate to a maximum of mosm/l. mice normally excrete large amounts of protein in the urine. taurine is always present in mouse urine, whereas tryptophan is always absent. creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. the creatinine/creatine ratio for fasting mice is about : . . mice excrete much more allantoin than uric acid. the submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. the tubular portion of the gastrointestinal (gi) tract consists of esophagus, stomach, small intestine, cecum, and colon. the esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. the proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. gastric secretion continues whether or not food is present. the gastrointestinal flora consists of (at least) species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. the ceca of normal mice contain up to bacteria/g of feces. the bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions. gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (see chapter ). for certain studies, it is desirable to colonize germfree mice with a defined microbiota. in the mid- s, schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (schaedler and orcutt, ) . he subsequently supplied animal breeders with this group of microorganisms. these defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. the so-called extremely oxygen-sensitive (eos) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. of the defined microbiotas later used for gnotobiotic studies, the one known as the 'schaedler flora' was the most popular. in , the national cancer institute (nci) decided to revise the schaedler flora, or 'cocktail' consisting of eight bacteria, in order to standardize the microbiota used to colonize germfree rodents. the new defined microbiota, now known as the 'altered schaedler flora' (asf), consisted of four members of the original schaedler flora (two lactobacilli, bacteroides distasonis, and the eos fusiform bacterium), a spiral-shaped bacterium, and three new fusiform eos bacteria. studies have quantified the regional colonization of the asf strains along the gastrointestinal tract (sarma-rupavtarm et al., ) (fig. . ) . individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant lactobacillus murinus asf present at - cells/g of tissue in the upper gastrointestinal tract and obligate anaerobic asf strains being predominant in the cecal and colonic flora at - cells/g of tissue. it is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. it is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. in the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. with the advent of polymerase chain reaction (pcr) technology, the eight asf strains were identified taxonomically by s rrna sequence analysis . three strains were previously identified as lactobacillus acidophilus (strain asf ), l. salivarius (strain asf ), and bacteroides distasonis (strain asf ), based on phenotypic criteria. s rrna analysis and genome sequencing indicated that each of the strains differed from its presumptive identity (wannemuehler et al., ) . the s rrna sequence of strain asf is essentially identical to the s rrna sequences of the type strains of l. murinus and l. animalis (both isolated laboratory animal medicine from mice), and all of these strains probably belong to a single species. strain asf is a novel lactobacillus that clusters with l. acidophilus and l. lactis. strain asf is a parabacteroides sp. the spiral-shaped strain, strain asf , is in the flexistipes phylum, exhibits sequence identity with rodent isolates of robertson, and has been formally named, mucispirillum schaedleri (robertson et al., ) . the remaining four asf strains, which are eos fusiform bacteria, group phylogenetically with the low-g + c content gram-positive bacteria (firmicutes, bacillus-clostridium group) (asf -eubacterium plexicaudatium; asf -firmicutes bacterium; asf and asf -clostridium sp.) (fig. . ) . the s rrna sequence information was determined by dewhirst et al. ( ) and draft genome sequences for each member of asf were recently published (wannemuehler et al., ) . this genetic data will permit detailed analysis of the interactions of asf organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms. the lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes ( fig. . ) , and intestinal peyer's patches. mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. the typical lymph node is beanshaped and consists of a cortex and a medulla. the cortex is divided into b lymphocyte domains, called primary follicles, and t lymphocyte domains, known s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l section of the gi tract bacteroides sp. asf the sections are taken from the esophagus (esop.) (section e ), stomach (sections s and s ), small intestine (sections i to i ), ileocecal junction and apical cecum (sections c and c , respectively), and colon (sections l to l ). from sarma-rupavtarm et al. ( ). as the diffuse cortex. the mouse does not have palatine or pharyngeal tonsils. the spleen lies adjacent to the greater curvature of the stomach. different strains of mice have varying degrees of accessory splenic tissue. age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. male spleens, e.g., may be % larger than those of females. most lymphocytes enter and leave the spleen in the bloodstream. the so-called white pulp of the spleen is organized along the central arteriole and is subdivided into t-and b-cell zones. the periarteriolar sheath is composed mainly of cd + and cd + t cells, and lymph follicles, which often contain germinal centers, are located at the periphery. the red pulp consists of sinusoids and hemoreticular tissue. cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. the thymus is a bilobed lymphoid organ lying in the anterior mediastinum. it reaches maximum size around the time of sexual maturity and involutes between and days of age. the thymus plays a major role in maturation and differentiation of t lymphocytes. this function is not complete in newborn mice. thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. the mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (fogh, ) . the mucosa-associated lymph tissue (malt) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. the term malt designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. they include the peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the respiratory and genitourinary system. lymphatics drain these lymphoid-rich areas, thus providing a direct link with lymph nodes and the bloodstream. bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (pastoret et al., ) . normal hematologic values are listed in table . . bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin. the cardiovascular system of mice is reviewed extensively by hoyt et al. in the nd edition of volume iii; normative biology, husbandry and models in the aclam series the mouse in biomedical research (hoyt, ) . the heart consists of four chambers, the thin-walled atria and the thick-walled ventricles ( fig. . ) . mice conditioned to a recording apparatus have mean systolic blood pressures ranging from to mmhg. an increase in body temperature does not lead to an increase in blood pressure. heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. heart rates from to /min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. the skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. the normal vertebral formula for the mouse is c t l s c , with some variations among strains, especially in the thoracic and lumbar regions. normal mouse dentition consists of an incisor and three molars in each quadrant. these develop and erupt in sequence from front to rear. the third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. the incisors grow continuously and are worn down during mastication. the mouse brain has a typical mammalian structure as documented by a detailed study of the neuroanatomy of the c bl/ j mouse (sidman et al., ) . more recently, gene expression patterns have been used to study the functional anatomy of the mouse brain (bohland et al., ) . use of wild-type and genetically modified mice in behavior, learning, and memory paradigms has exponentially increased over the last decade. the male reproductive organs consist of paired testes, urethra, penis, prostate and associated ducts and glands ( fig. . ) . the female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands ( fig. . ). the clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. the female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region ( fig. . ). the mammary glands are often not appreciated for how far they extend over the cervical, axillary, and inguinoabdominal flank regions which become the following section summarizes normal reproduction in the mouse. the reader is referred to a more comprehensive text in the aclam series (pritchett and taft, ) and online resources such as the jackson laboratories publication of the biology of the laboratory mouse (http://jaxmice.jax.org/jaxnotes/ / j.html). external influences, such as noise, vibration, diet, light cycle, and cage density, and intrinsic factors, such as health status, genetics, and parity impact reproductive success by directly or indirectly influencing the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. genotype also dramatically affects the reproductive performance of the mouse. coincident with the explosion in the number of mouse strains, each with unique induced or spontaneous mutations, a sound breeding program must include training of care staff to recognize anticipated and unanticipated breeding performance and strain or stock characteristics. in the new age of genomics, older methods of confirming genetic purity of mouse lines are being replaced with formal genetic monitoring by comparing strain-specific panels of single-nucleotide polymorphisms (snps). follicle-stimulating hormone promotes gametogenesis in both sexes. luteinizing hormone promotes the secretion of estrogen and progesterone in the female and androgen in the male. prolactin promotes lactation and development of the ovary during pregnancy. these gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. the hypophysis is usually responsive to hormonal influence by day in the male and day in the female. ovarian follicle development begins at weeks of age and matures by days. rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as - days. puberty is slightly later in the male (up to weeks). sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary. the mouse is polyestrous and cycles every - days. in the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. the cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (table . ). patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus ( fig. . ) . irregularities of the estrous cycle occur during aging. seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. estrus is routinely observed in mice at about - h after parturition (postpartum estrus). however, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. mice are spontaneous ovulators. ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. the cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. reversing the timing cook ( ) . laboratory animal medicine of the light-dark cycle reverses the time of estrus, ovulation, and mating. pheromones (table . ) and social environment also affect the estrous cycle. for example, estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine ('whitten effect'). once exposed to male urine, most female mice will be in estrus within days with a second estrus in about days. hence, estrus can be synchronized by group-housing females prior to pairing with males. in contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females and is known as the 'bruce effect'. see section ii.c on behavior for more detail on the effect of pheromones on mouse reproductive behavior. mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. the plug usually fills the vagina from cervix to vulva (fig. . ). plug detection is often coupled with vaginal cytology to evaluate fertility and conception. when the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. secretion continues for about days. if fertilization has occurred, the placenta takes over progesterone production. if fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. fertilization usually takes place ( ) testis, ( ) head of epididymitis, ( ) caudal epididymitis, ( ) vas deferens, ( ) testicular vein, ( ) ampullary gland, ( ) seminal vesicle, ( ) anterior prostate, ( ) ureter, ( ) bladder, ( ) ventral prostate, ( ') dorsal prostate, ( ) urethra, ( ) bulbourethral muscle, ( ) ischiocavernosus, ( ) bulbourethral gland, ( ) diverticulum of bulbourethral gland, ( ) penis, ( ) preputial gland, ( ) glans penis, ( ) prepuce, ( ) testicular artery, and ( ) vas deferens artery. adapted from (komarek, ) . fallopian tube, ( ) uterine horn, ( ) endometrium, ( ) cervix, ( ) vagina, ( ) vaginal vestibulum, ( ) clitoris, ( ) clitoral gland, ( ) urethra, ( ) bladder, ( ) medial ligament of bladder, ( ) lateral ligament of bladder, ( ) left ureter, ( ') right ureter, ( ) mesovarium, ( ) mesometrium, ( ) ovarian artery, ( ) uterine horn artery, and ( ) ovarian artery and vein. adapted from (komarek, ) . adapted from komarek ( ) . in the ampulla or the upper portion of the oviduct. ova can be fertilized to produce normal embryos for - h after ovulation. gestation is usually - days. because of postpartum estrus, lactation and gestation can occur simultaneously. lactation can delay gestation because of delayed implantation. this may cause prolongation of gestation for up to - days in certain inbred strains. the effective reproductive life of some inbred strains approaches years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. therefore, females are usually retired by months of age. average litter size is strain dependent and commonly ranges from to pups. maternal care can account for about % of the variation in body weight of neonatal mice. nursing females usually lactate for weeks. milk production increases up to days postpartum and then declines until weaning at days. interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (nishimori et al., ) . some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. antibodies continue to be secreted in the milk throughout lactation. decay of maternally acquired immunity occurs within several months after weaning. loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions. mice are socially gregarious animals with strong family bonds who communicate through complex olfactory, auditory, tactile, and visual signals. wild mice aggregate into groups called demes with low exchange of individuals between different groups. each deme consists of kinrelated members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience, which all contribute to their remarkably successful environmental adaptability. the deme is composed of a dominant breeding male, a hierarchy of females, subordinate males, and juveniles. wild mice occupy territories measuring just a few square meters when food is abundant to several square kilometers. mice are crepuscular (active during the twilight hours of dawn and dusk), strongly territorial, and omnivorous. coprophagy contributes to approximately one-third of their ingesta as an essential nutritional activity. aside from territoriality, social interactions, breeding, burrowing (when conducive substrates are available), and nest building are major activities. in managing laboratory mice, it is important to understand the complex behavioral biology of their free-living counterparts (latham and mason, ) . chemo-olfactory communication is mediated through extremely diverse chemical factors that trigger innate (non-learned) social responses among conspecifics, known as pheromones (table . ). pheromones have been traditionally divided into two broad categories: releaser pheromones, which elicit an immediate behavioral response, and primer pheromones, which mediate a slowly developing and longer-lasting endocrine response. this original definition of pheromone categories has been expanded to another category, termed signaler pheromones, which convey individual or group identity, as well as mediating parent-offspring recognition and mate choice. the biology and genetics of pheromone signaling is being extensively studied in the mouse as a model of mammalian pheromone communication (brennan and zufall, ; rodriguez and boehm, ) . mouse pheromones are excreted in the urine, as well as plantar, salivary, lacrimal, preputial, and mammary glands. in the urine, major urinary proteins (mups), small peptides, mhc class i peptides, volatile chemicals, and sex hormones all contribute to chemosignals that communicate dominance, kinship, diversity, and gender. wild mice possess a great deal of individual variations of roberts et al. ( ) . c ferrero et al. ( ) . laboratory animal medicine these elements, providing a 'bar code' that distinguishes individuals. inbreeding of laboratory mice has reduced individual variation, but each inbred strain possesses a characteristic array of signals, and to a certain extent, unique signals exist among individuals within a strain (sharrow et al., ; sturm et al., ) . pheromones are detected by sensory neurons in the vomeronasal organ, the olfactory epithelium, and the lesser known septal organ of masera within the olfactory epithelium, and the gruenberg ganglion, which is located at the anterior end of the nasal cavity (breer et al., ; chamero et al., ; liberles and buck, ; restrepo et al., ) . neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and thence to the brain. mups are important components of chemosensory communication in mice, and also an important occupational hazard to human handlers. chromosome contains a cluster of mup genes, plus a number of pseudogenes. mups are small soluble proteins known as lipocalins, which bind small organic chemicals (pheromones) with high affinity, and function as pheromone transporters and stabilizers (thereby contributing to slow release), but also act as protein pheromones themselves. they are synthesized in the liver and excreted in the urine, as well as nasal mucosa, lacrimal glands, and salivary glands. their endogenous role on metabolic activity is not yet understood. male mice excrete significantly more mups in the urine than females. one wellcharacterized mup is 'darcin', named after fitzwilliam darcy, the romantic hero in pride and prejudice. as its name implies, it is a female attractant. mups also act as kairomones, which function as chemical signals between species. for example, cat and rat mups invoke fear in mice. mups are important in the laboratory animal management context, as they are excreted in copious amounts ( - mg/ml in urine) and are potent allergens for humans, particularly mus m (ag or ma ), which is encoded by the mup gene (sharrow et al., ) . chemosensory communication has numerous behavioral effects that influence mouse social interactions. one of the most studied behavioral effects is the bruce effect, or pregnancy block, which is a complex physiologic response in which recently conceived females resorb fetuses during early pregnancy in the presence of an unrelated male, particularly a dominant male. the continued presence of the original mate protects the female from this effect (bruce, ) . the vandenbergh effect results in acceleration of puberty of juvenile females in response to male urine (vandenbergh, ) . the lee-boot effect occurs among group-housed females that are isolated from males, in which there is suppression of estrus cyclicity (van der lee and boot, ) . the whitten effect results in synchronization of estrus among a group of females in response to a male (whitten et al., ) . the lee-boot and whitten effects are utilized in the laboratory to assist in induction of synchronized timed pregnancy, but the bruce effect can have deleterious consequences on breeding colonies when foreign males are introduced to a breeding colony, as pheromone communication can occur in the absence of direct contact. the above effects are well-defined pheromone-driven behavioral responses, but chemosensory communication has a myriad of other effects. estrus, pregnant, or lactating females also accelerate puberty among juvenile females. females use odor cues to avoid parasite-laden males, males prefer odors of estrus females, and estrus females prefer odors of dominant males. mice have strong mating and social preferences based upon mhc proteins, which indicate genetic relatedness. maternal recognition of young is also mhc-related, and pups prefer nest odors of maternal and sibling pups based upon mhc relatedness. male aggression against unrelated males is also a strong mhc-related phenomenon. mhc haplotypes determine not only mhc proteins in the urine, and mhc-specific olfactory receptors, but also the composition of volatile chemicals in the urine (kelliher and wersinger, ) . the complexity of social communication extends to auditory stimuli as well. male mice utilize ultrasonic 'birdsong' to vocally communicate and attract females. mouse vocalization patterns are largely genetically innate and unique to each strain of mouse, but they can also be modified, or learned, to a limited extent (arriaga et al., ) . the behavioral biology of the mouse is highly complex, and depends upon genetic, physiologic, social, and environmental variables, which all impact on how laboratory mice can best be managed in captivity. it is clear that this rich complexity cannot be fully addressed under laboratory conditions, but that does not mean that basic needs, such as nest building, burrowing, foraging, and olfactory environments, cannot be provided. for example, intermale aggression, which is particularly apparent in some strains of mice such as balb/c and swiss-origin stocks and strains, can be minimized by maintaining males from infancy as sibling groups, since adult siblings tend not be aggressive to one another. this sibling bond, however, can be easily broken by short-term separation. environmental enrichment often features provision of plastic houses, which may make vivarium managers feel good, but maximal enrichment can be provided by provision of nesting material, which includes structural scaffolding, such as crinkled cardboard, which facilitates construction of three-dimensional nests. mouse nests are replete with 'appeasement' pheromones, thereby contributing to harmony within the cage, whereas introduction of dirty bedding has the opposite effect. frequent cage changing, including removal of established nests, is highly stressful and disruptive to social harmony within a cage. provision of appropriate and adequate amounts of bedding material that is conducive to burrowing is desirable. it is important to remember that mice are socially gregarious, and that mouse welfare is optimally enriched by other mice within a socially harmonious deme (latham and mason, ; van loo et al., ) . a laboratory mouse ethogram, defined as an operationalized list of mouse behaviors, arranged by their adaptive meaning to the animal, is available on the web: www. mousebehavior.org. behavioral phenotyping, particularly of transgenic mice, is used extensively in genomic research. a wide variety of standardized test batteries and approaches are used, depending upon the focus of research (reviewed in crawley ) . initial behavioral evaluations include general health, body weight, body temperature, appearance of the fur and whiskers, and neurological reflexes assessment. specific tests include observations of home cage behaviors, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. novel and complex environmental enrichment in animal housing conditions facilitates enhanced sensory and cognitive stimulation as well as physical activity. environmental enrichment and exercise have beneficial effects such as cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders (pang and hannan, ) . the immune system of the mouse is very similar to that of humans. the availability of inbred mouse strains, in which each individual animal expresses identical mhc alleles so that tissues and cells can be transplanted without tissue rejection, greatly simplifies and indeed enables functional analyses of immune system components not possible with any other outbred mammalian species. in addition, the ability to genetically manipulate the mouse genome, adding to, altering, and deleting existing genes, enables unprecedented in vivo analysis of immune cell functions. it is for these reasons that the mouse is the primary animal model for immunology research. the immune system is an unusual organ system in that it consists of both solid tissues and various migrating cell populations. the bone marrow and thymus are considered primary lymphoid organs, as sites of hematopoiesis and b-and t-lymphocyte development, respectively. lymph nodes, spleen, and intestinal peyer's patches are considered secondary lymphoid tissues, as sites of immune response initiation. lymph nodes and spleen are analyzed frequently for studies of immune responses and as organs for immune cell isolation. tertiary lymphoid tissue sites are those that form in other solid organs in response to an insult or microbial exposure. among them are the lymphoid cell aggregates of the gastrointestinal and respiratory tract, also called 'gut-associated lymphoid tissue' (galt) and bronchusassociated lymphoid tissues (balt). leukocytes are classified as belonging to the innate or adaptive immune system. the innate immune system responds rapidly to an antigen insult via recognition of pathogen-associated molecular patterns (pamps), such as lipopolysaccharide, bacterial flagellin, single (s)-and double-stranded (ds) rna, and non-methylated dna, via extracellular or intracellular pattern recognition receptors (prrs). receptors include the toll-like receptors (tlrs), such as tlr (recognizing lps), tlr / (ss and dsrna) and tlr (dna), nod-like receptors (nod / ), and rig-like receptors (rig-i, mda- ) among others (takeuchi and akira, ) . cells of the innate immune system are monocytes/macrophages, granulocytes and dendritic cells as well as innate-like lymphocyte populations (ilc) , and , which include natural killer (nk) cells (spits et al., ) . cells of the adaptive immune system (t and b lymphocytes) express a highly antigen-specific receptor that has arisen through gene rearrangement (t-cell and b-cell receptors, respectively). b cells of the b- lineage and γδ t cells are regarded as innate-like cells, as they express a rearranged antigen receptor but seem to respond in an innate-like manner. leukocytes are identified and classified by sets of monoclonal antibodies (mab) against uniquely expressed surface receptors, typically measured by flow cytometry. identification of a unique receptor by one or more mab of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation (cd)'. for example, t cells are differentiated into two subsets based on their expression of either cd or cd . cd + t cells (t helper cells) recognize peptides presented in mhc class ii and promote b-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines (see below). cd + t cells recognize antigenic peptides presented in mhc class i and serve as cytotoxic cells during the cell-mediated immune response where they can destroy infected cells (e.g., against cells containing infectious agents). the major function of b cells is to respond to an encounter with an antigen/pathogen with the production of highly antigen-specific immunoglobulins (ig; antibodies), which can bind to and inactivate pathogens and toxins. activation of b cells can lead to their differentiation to plasma cells, which produce large amounts of ig. five laboratory animal medicine classes or ig 'isotypes' can be distinguished, which differ in effector function: igm, igg, iga, ige, and igd. the latter is expressed only on the surface of b cells in mice. the igg class, the most abundant antibody class in the serum, is further divided into subtypes: igg , igg a/c , igg b , and igg . polymorphisms exist on the ig locus such that some strains of mice produce the igg a subtype (e.g., balb/c), whereas others produce igg c (e.g., c bl/ ) (zhang et al., ) . additional allelic polymorphisms of the locus also exist. for example, balb/c and sv mice express the igh-a allotype, whereas c bl/ mice express the igh-b allotype. recombinant inbred strains of mice exist for both balb/c and c bl/ , which harbor the reciprocal igh locus (i.e., igh-b for balb/c and igh-a for c bl/ mice). these mice are useful tools for tracking b cells following adaptive cell transfer via allotype-specific mab (see below). immunoglobulin isotype production varies according to the type of immunogen used to evoke the response. igm is secreted short term after initial exposure to an antigen, followed by the other ig isotypes. in viral and intracellular bacterial infections, igg a/c is dominant, whereas in extracellular bacterial infections igg dominates the response. igg b and igg are usually induced to carbohydrate or lipid antigens. ige is linked to parasitic infections and to allergy. serum antibodies specific for an immunogen can often be measured for the life of the animal. while serum iga levels are low, iga is the highest produced ig in mice. iga production, however, occurs in plasma cells lodged in the lamina propria of mucosal tissues, from where the iga is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' iga (brandtzaeg, ). cytokines are secreted signaling molecules involved in cell-cell communication in a complex biological system (table . ). these include the large family of interleukins (ils, currently il- to il- ), tumor necrosis factors (tnfs), interferons (type i, ii, and iii) and growth factors such as granulocyte-macrophage colonystimulating factor (gm-csf) and stem cell factor (scf). cytokine secretion often occurs in response to recognition of antigen via prr or tcr. because of their importance in modulating immunity to antigenic stimuli, mice with specific deletions or overexpression of individual cytokines have been made and have contributed to a detailed understanding of many of their often pleotropic functions (akdis et al., ) . chemokines are a similarly large group of small, secreted molecules that regulate cell trafficking to sites of antigen encounter but also facilitate cell-cell contact by acting as chemoattractants. chemokines are grouped according to the number of cysteines and disulfide bonds in the molecule into c-x-c-, c-c, c, and cx cl chemokine ligands (l) and receptors (r) and designated accordingly as cxcr - /cxcl - and ccr - /ccl - (allen et al., ) . immune responses must be coordinated to provide the most appropriate effector functions for the type of pathogen/antigen encountered. immune effector responses differ depending on the life cycle (facultative or obligate intracellular, extracellular, localized, systemic, etc.) and antigen types displayed by the encountered antigen/ pathogen, because this affects the type of prr engaged and activated. prr engagement leads to cytokine and chemokine responses by the first responders, i.e., epithelial cells, local macrophage populations and other innate cells. the type of cytokines and chemokines produced then dictates the types of cells recruited to the site of infection and their subsequent differentiation and functions. the prr engagement also leads to antigen uptake, activation and migration of dendritic cells (dcs) from the site of insult to the regional lymph nodes, where dcs present antigen peptides on mhc molecules to t cells. in addition, the dcs secrete cytokines induced by the initial prr activation, which cause the differentiation of cd t cells towards a particular effector response. for example, secretion of il- in response to activation of tlr or will result in the induction of interferongamma (ifn-γ) production by cd t cells, whereas il- and tgf-β production by dc will induce cd t cells to secrete il- (kara et al., ) . because the dc translates signals from prr at the site of infection into differentiation signals for t cells in the lymph tissues, these cells are regarded as a 'bridge' between the innate and adaptive immune systems. the specific ig isotype secreted in response to a pathogen depends to a large degree on the type of cytokine produced by cd t cells that provide 't-cell help' for b cells. t cells that interact with b cells are identified as a discrete subset termed 't follicular helper cells (t fh )' and it is their cytokine profile that directs b cells to secrete a particular ig isotype (kara et al., ) . the classic t h /t h dichotomy outlined above was in part shaped by the observation that ifn-γ production will lead to switching of b cells to secrete igg a/c, whereas production of il- leads to the secretion of igg . interestingly, it appears that the cytokine profile induced by the effector t-cell population is mirrored by the innate immune response. innate-like lymphocytes also have effector phenotypes that correspond to those of cd t cells and are induced by the same signals and transcriptional regulators (spits et al., ) and the same appears to be true also for macrophages and other innate immune cells (sica and mantovani, ) . while initial studies identified two particular antagonistic effector response types (termed t h and t h and classified by t-cell production of ifn-γ and il- , respectively), more recent studies now demonstrate a much wider array of effector responses in which innate and adaptive immunity acts together to reinforce an immune response phenotype as well as modulate its size by induction of t regulatory cells (t regs ) that generate inhibitory cytokines (kara et al., ; sica and mantovani, ; spits et al., ) . the use of cytokine-deficient and reporter mice that enabled the identification of cytokine-producing cells via expression of a fluorescent reporter was particularly valuable for the development of this more nuanced view of the quality of immune responses. spontaneous mouse models of immune deficiencies have been used extensively in research. their use, plus the expanding number of knockout, transgenic, and dominant negative mouse mutants, has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system (table . ). interbreeding of multiple immune-deficient mice has allowed the development of 'humanized' mice in which immune cells of the mouse are replaced with those of humans. while many challenges remain to fully replenish mice with components of the human immune system, the use of immune-deficient nod/severe combined immunodeficient (scid)/il- rγ -/recipients for transfer of human peripheral blood lymphocytes, cordblood or bone marrow-derived cd + stem cells with human liver and thymus (blt-mice) is yielding promising results (akkina, ) . investigators using genetically engineered mice are constantly reminded that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. this may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. as with other biological systems, compensation mechanisms also may mask the phenotype. experimental approaches are being increasingly used to refine the knockout technology by restricting a specific genetic deficiency to a particular tissue of interest using the cre-lox system, in which tissue-specific or temporal restricted expression of the cre recombinase induces the deletion of a 'floxed' gene (mak et al., ) . transgenic mice are available that restrict cre expression to various hematopoietic cells or tissue or drive cre recombination following injection of tamoxifen. other approaches are the generation of 'bone marrow irradiation' chimeras. here, inbred wild-type mice or mice deficient in certain immune cells (table . ) are lethally irradiated by exposure to a gamma-irradiation source to deplete the hematopoietic stem cells. these are then replaced by transfer of bone marrow cells to the irradiated mice. reconstitution of the hematopoietic system is usually achieved within about weeks, during which time mice are provided with antibiotic-containing drinking water to avoid infections of these temporarily immune-compromised animals. transfer of bone marrow from a congenic knockout restricts the genetic defect to the hematopoietic system. a mix of bone marrow from two sources is also often used to generate tissue-specific knockouts. for example, mixing a bone marrow from t-cell-deficient mice ( %) with that of a gene knockout ( %) generates 'mixed bone marrow chimeras' in which all t cells only develop from the knockout, thus lack the gene of interest, whereas most of the other cells t from the wild-type source, effectively constraining the genetic defect to the t-cell population. sets of congenic mice with defined allotypic differences are often used to confirm the source of individual cells. such markers include the gene locus cd . /cdc . or cd . /cd . (thy . /thy . ). alternatively, cells may express a fluorescent transgene, such as green-fluorescent protein (gfp). identification is usually performed by flow cytometry, or less commonly by immunofluorescence or immunohistochemistry. generation of bone marrow chimeras circumvents the time-consuming breeding of cre recombinase-expressing flx/flx mice. however, numerous controls are needed to exclude off-target effects due to irradiation damage. repeat injection of antibodies targeting specific cell populations is another rapid approach that avoids the potential for irradiation damage and allows short-term depletion of individual cell subsets. its main disadvantage is the need to identify mab that bind to surface receptors uniquely expressed by a cell subset of interest and the verification of the efficacy of the depletion. frequently used is antibody treatment for the short-term depletion of t-cell subsets using mab against cd or cd as well as individual cytokines. contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. the widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology. many microbial agents have the potential to cause disease in mice or interfere with mouse-based research. housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. surveillance should encompass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. because surveillance strategies will vary with research needs and operating conditions, it is prudent to consult a number of sources, such as the federation of european laboratory animal science associations (felasa) (nicklas et al., ) and commercial laboratories, for guidance. detailed discussion of microbial quality control is provided in chapter . there are also recommendations regarding specific agents in following sections. diagnostic methods involve gross and microscopic pathology, parasitology, microbial isolation and culture, serology, and pcr. serology is particularly important for viral surveillance, and now relies principally on enzyme-linked immunosorbent assay (elisa), multiplex fluorescent immunoassay (mfi) for simultaneous detection of antibodies to multiple agents (hsu et al., ) , indirect fluorescent antibody (ifa) assay, or hemagglutination inhibition (hai), with the latter two methods generally used for confirmation (livingston and riley, ; pritchett-corning et al., ) . mouse antibody production (map) testing has been historically used for testing biological materials for contamination by infectious agents. pcr panels for murine infectious agents are now commercially available and have cost and time-saving advantages as well as improved assay sensitivity and specificity. beyond the classic bacterial and viral murine infections, pcr assays are now available for endo-and ectoparasites (see chapter ). etiology mousepox is caused by ectromelia virus (ectv), an orthopoxvirus that is antigenically and genetically closely related to a number of other poxviruses, including vaccinia, variola, and cowpox viruses. the original isolate of ectv, known as the hampstead strain, was discovered by j. marchal in (marchal, as the cause of epizootic disease among laboratory mice in england. the disease featured amputation of extremities, which marchal termed ectromelia (from the greek, ectro, amputation and melia, limb). other strains of the virus include moscow, nih- , washington university, st. louis , beijing , ishibahsi i-iii, and naval (nav) strains, which vary in virulence, but are essentially indistinguishable genetically and serologically, suggesting a common origin. virus can be isolated from infected tissues by inoculation of cell cultures (bs-c- , hela, l cells) or embryonated eggs. the natural host (and original source of infection of laboratory mice) of ectv remains unknown. clinical signs the expression of clinical signs reflects an interplay among virus-related factors, including virus strain, dose and portal of entry, and host-related factors, including age, genotype, immunological competence, and gender (brownstein et al., a) . during natural epizootics, it was observed that a, bc, dba/ , dba/ , and cba strains developed acute fatal infections, whereas c bl/ mice were resistant to severe disease (briody, ) . experimental studies have shown that all strains of mice are susceptible to infection, but balb/c, a, dba/ , and c h/he mice were highly susceptible, akr and sjl mice were moderately susceptible, and c bl/ mice were highly resistant to lethal infection (bhatt and jacoby, c; wallace and buller, ) . the mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including nk cells (brownstein et al., a; jacoby et al., ) . the nuances of cytokine and cellular immune responses to ectv infection have received recent attention (reviewed in buller and fenner ( ) and esteban and buller ( ) ). outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality in susceptible strains of mice. clinical signs such as ruffled fur or prostration may occur for only a few hours before death. mice that survive acute infection may develop chronic disease characterized by a focal or generalized rash anywhere on the body ( fig. . ). conjunctivitis also may occur. skin lesions usually recede within several weeks, but hairless scars may remain. additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. epizootiology mousepox is not a common disease. outbreaks occur sporadically and recent outbreaks have been traced to the importation of contaminated mice or mouse products. for example, contaminated mouse serum was responsible for recent outbreaks in the united states (dick et al., ; . natural exposure is thought to occur through direct contact and skin abrasions. cage-to-cage transmission is low and can be virtually nil if filter-topped cages are used (bhatt and jacoby, b) . ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (bhatt and jacoby, d) . aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. virus-free progeny can be obtained laboratory animal medicine from immune dams (bhatt and jacoby, b) . however, intrauterine infection and fetal deaths, albeit rare, have been reported. natural transmission is facilitated by intermediately resistant mice, which survive long enough to develop skin lesions that can shed virus for relatively long periods of time. the risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. although virus excretion typically lasts for about weeks, virus has been found in scabs and/or feces for up to weeks. resistant mouse strains also are dangerous because they can shed virus during subclinical infections. however, infections in resistant mice tend to be short-lived. highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. thus, juxtaposition of resistant or intermediately resistant infected mice with highly susceptible mice can provoke explosive outbreaks. infant and aged mice are usually more susceptible to lethal infection than young adult mice. maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. such mice may subsequently transmit infection by contact exposure. pathology the classic descriptions of ectv pathogenesis by fenner remain timely, including the frequently cited and reproduced figure summarizing the pathogenesis of infection ( fig. . ) (fenner, b) . interest in smallpox has renewed the interest in ectv as a model of host response to infection (esteban and buller, ) . ectv multiplies in the cell cytoplasm and produces two types of inclusion bodies. the a type (marchal body) is well demarcated and acidophilic in histological sections. it is found primarily in epithelial cells of skin ( fig. . ) or mucous membranes and can also be found in intestinal mucosa. the b type of inclusion is basophilic and can be found in all ectromelia-infected cells. however, it is difficult to visualize unless cells are stained intensely with hematoxylin. ectv antigen can be readily visualized by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (esteban and buller, ; jacoby and bhatt, ) . following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues. splenic and hepatic involvement begin within - days, whereupon larger quantities of virus are disseminated in blood to the skin. this sequence takes approximately week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. the primary lesion is due to the development of antiviral cellular immunity. severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. white spots indicative of necrosis develop throughout the liver ( fig. . ). in nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe. necrosis and scarring of red and white pulp can produce a macroscopic 'mosaic' pattern of white and red-brown the primary skin lesion, which occurs - days after exposure, is a localized swelling that enlarges from inflammatory edema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viremia. they are often multiple and widespread and can be associated with conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. the skin lesions also can ulcerate and scab before scarring. diagnosis mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. observation of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. several serological tests are available to detect mousepox. historically, the standard test was hai, using vaccinia antigen as a source of hemagglutinin. elisa is more sensitive and specific and has replaced hai for serological monitoring among nonvaccinated mice (buller et al., ) . ectv infection also can be detected by ifa (buller et al., ) and pcr. serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. thus, serum from vaccinated mice may react by elisa but should not react by hai. differential diagnosis mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. these include sendai pneumonia, mouse hepatitis, and tyzzer's disease. the latter two can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. the skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, ascariasis, and bite wounds. prevention and control mousepox is a dangerous disease because of its virulence for susceptible mice. therefore, infected colonies should be quarantined immediately. depopulation has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. materials should be autoclaved or, preferably, incinerated. disinfected rooms should be challenged with susceptible sentinel animals that are observed for clinical signs and tested for seroconversion after several weeks. depopulation and disinfection must be carried out vigorously. because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice. such mice can be quarantined along with cessation of breeding to permit resolution of infection (bhatt and jacoby, b) . sequential testing with contact-exposed sentinels should be employed with this option. additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virus-free mice from previously infected dams, with the caveat that progeny will be transiently seropositive with maternally derived antibody. vaccination can control or prevent clinically apparent mousepox. the hemagglutinin-deficient strain of vaccinia virus (ihd-t) is used to scarify skin on the dorsum of the tail. 'takes' should occur in previously uninfected mice by - days, but not in infected mice (bhatt and jacoby, a) (fig. . ). infected mice should be quarantined separately or eliminated. vaccination may not prevent infection, although infection in vaccinated mice is often transient. furthermore, vaccinia virus can be shed from scarification sites for at least several days. therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. additionally, seroconversion evoked by vaccination must be taken into account in serological monitoring of vaccinated colonies. finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure. research complications the primary threat from mousepox is mortality in susceptible mice. the loss of time, animals, and financial resources can be substantial. (shellam, , ) mice are naturally susceptible to two herpesviruses from the subfamily betaherpesvirinae and in the genus muromegalovirus, the two species murid herpesvirus (of which one of the members is mouse cytomegalovirus (mcmv)) and murid herpesvirus (of which one of the members is mouse thymic virus (mtv)). they are species-specific viruses and distinct from each other and from other rodent herpesviruses. mctv has received considerable attention as a model of human cmv infection. etiology mouse cytomegalovirus (mcmv) is a mouse-specific betaherpesvirus. it can, however, replicate in cell cultures from several species, including mouse (fibroblasts and t cells), hamster, rabbit, sheep, and nonhuman primate. cocultivation may be required to rescue latent virus. clinical signs mcmv causes subclinical infection in adult immunocompetent mice, but experimental inoculation of neonates can cause lethal disease due to multisystemic necrosis and inflammation. epizootiology the prevalence of mcmv in laboratory mice is probably uncommon but undefined, since infection is clinically silent and serological surveillance is not widely practiced. wild mice are commonly infected and serve as a natural reservoir for infection, which implies that the entry of virus into a modern vivarium is most likely to occur from contaminated animal products. persistence is a central feature of nonlethal infection. persistently infected mice excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. vertical transmission does not appear to be a common factor in natural infection. further, maternal immunity protects sucklings from infection. pathology mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. infection can occur in young and adult mice. however, the pathogenicity of mcmv for mice decreases with age. neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. immunodeficient mice, however, remain susceptible to pathogenic infection as adults. persistent infection often affects the salivary glands and pancreas. the persistence of salivary gland infection appears to be dose dependent. there is experimental evidence that mcmv can produce latent infection of b cells, probably t cells as well as aforementioned tissues. persistent infection may lead to immune complex glomerulonephritis. latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression. diagnosis mcmv antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. neutralizing antibody titers are low during acute infection and difficult to find during chronic infection. serology and pcr-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. detection of enlarged cells with intranuclear inclusions, especially in salivary glands, is diagnostic, if they are present. in situ hybridization can be used as an adjunct to routine histopathology. differential diagnosis mcmv infection must be differentiated from infection with mtv. the latter virus can produce necrosis of thymic and peripheral lymphoid tissue when infant mice are experimentally inoculated. lytic lesions of lymphoid tissues are not a hallmark of mcmv. the viruses can also be distinguished from each other serologically. sialoadenitis with inclusions can occur during infection with mouse polyoma virus. like mcmv, mtv infects the salivary gland as its primary target organ. prevention and control control measures for mcmv have not been established, because it has not been considered an important infection of laboratory mice. cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. the exclusion of wild mice is essential. research complications mcmv can suppress immune responses. apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as pseudomonas aeruginosa. etiology mouse thymic virus (mtv) is a herpesvirus (murid herpesvirus ) that is antigenically distinct from mcmv. no suitable in vitro method for cultivation has been developed; therefore, viral propagation depends on mouse inoculation. clinical signs natural infections are subclinical. epizootiology the prevalence of mtv is thought to be low. mice can be infected at any age, although lesions develop only in mice infected perinatally. mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. excretion of virus in saliva is considered the primary factor in transmission. seroconversion occurs in adults but does not eliminate infection. infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. the mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. mtv also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. prenatal transmission has not been found. pathology mtv causes severe, diffuse necrosis of the thymus and lymphoid tissue with tropism for cd + t cells in mice inoculated within approximately week after birth. the severity of thymic and lymph node necrosis can be mouse strain-dependent. grossly, the thymus is smaller than normal. infected thymocytes display mtv-positive intranuclear inclusions. necrosis is followed by granulomatous inflammation and syncytium formation. reconstitution of lymphoid organs takes - weeks. diagnosis thymic necrosis associated with intranuclear viral inclusions is the hallmark lesion. viral antigen can be detected by immunohistochemistry. serologic detection is effective, but generally not utilized, and is potentially negative in neonatally exposed mice. suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. inoculated mice should be examined for thymic necrosis - days later. pcr or the mouse antibody production (map) test can also be used to detect infection. differential diagnosis reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. prevention and control because mtv induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. research complications mtv transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal t lymphocytes. parvoviruses are among the most common viral infections in contemporary laboratory mouse populations (livingston et al., ) (pritchett-corning et al., ) , and pose major challenges to both detection and control. the mouse parvoviruses are composed of two antigenically and genetically distinct but related groups, including minute virus of mice (mvm) and mouse parvovirus (mpv), with each group containing a number of strains. the international committee on taxonomy of viruses classifies mpv, mvm, and several other rodent parvoviruses into one genus, protoparvovirus, and species, rodent protoparvovirus , but these viruses will be treated separately herein. etiology minute virus of mice (mvm) is a small ( -kb) single-stranded dna virus. the prototypic strain is designated mvmp. an allotropic variant with immunosuppressive properties in vitro is named mvmi, and additional laboratory animal medicine named strains include mvmc and mvmm. the genome encodes two nonstructural proteins, ns- and ns- , which are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in serological assays that utilize whole virus antigen. the viral capsid proteins, vp- and vp- , are virus-specific and form the basis for serological differentiation of mvm from mpv. mvm has a broad in vitro host range. it replicates in monolayer cultures of mouse fibroblasts (a cells), c rat glial cells, sv (simian virus )-transformed human newborn kidney ( k cells), t-cell lymphomas (el ), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. clinical signs natural mvm infections are subclinical. neonatal mice of some inbred strains are experimentally susceptible to lethal renal and/or intestinal hemorrhage during mvmi infection, but this syndrome has not been reported in natural outbreaks. experimental inoculation of adult c.b- -prkdc scid (scid) mice with mvmi results in lethal infection (lamana et al., ; segovia et al., ) , and similar severe illness has been noted in naturally infected b-cell-deficient nod. cg-h h -igh null mice (naugler et al., ) . epizootiology mvm is a common virus that naturally infects laboratory mice, but appears to be less common than mpv (besselsen et al., ; livingston et al., ) . mvm is moderately contagious for mice, its only known natural host. virus can infect the gastrointestinal tract and is excreted in feces and urine. the resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. therefore, contamination of caging, bedding, food, and clothing must be considered a risk for the spread of infection. transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within weeks, but limited exposure may delay seroconversion. young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. mvm, in contrast to mpv, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than weeks (smith, ; smith and paturzo, ). infection appears to last less than month, even in oronasally inoculated neonatal mice, but immunodeficient mice may be persistently infected. there is no evidence that mvm is transmitted in utero. pathology natural infections or experimental inoculation of adult mice appears to be nonpathogenic. contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. experimental infection of neonatal balb/c, swr, sjl, cba, and c h mice with mvmi can cause renal hemorrhage and infarction (brownstein et al., b) . dba/ mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. c bl/ neonates are resistant to vascular disease. this lesion has been attributed to viral infection of endothelium. infection of immunodeficient mice, including scid and b-cell-deficient mice, results in lethal damage to granulomacrophagic, megakaryocytic, and erythrocytic hematopoietic tissue with severe leukopenia (lamana et al., ; naugler et al., ; segovia et al., ) . intranuclear viral inclusions and viral antigen have been observed in splenic mononuclear cells of b-cell deficient mice (naugler et al., ) . diagnosis serology is the primary method of detecting infection, which utilizes recombinant mvm and mpv major capsid viral proteins (vp ) as antigens, which discriminate between the two groups of mouse parvoviruses. in contrast, the conserved nonstructural protein, ns can be used to detect antibody to both groups, but is less sensitive than vp assays (livingston et al., ) . mvm infection also can be detected by pcr, in situ hybridization, and immunohistochemistry. pcr assays can be used to detect mvm-or mpv-specific vp or all rodent parvovirus group specific ns exons (besselsen, ; besselsen et al., ) . mvm can be isolated from the spleen, kidney, intestine, and other tissues by inoculation of the c rat glial cell line. it also can be detected by the mouse antibody production test. prevention and control because mvm does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. mpv has been shown to be successfully eliminated by a cage-bycage test (serology and fecal pcr) and cull approach, although there are no published reports confirming the success of this strategy for eliminating mvm (macy et al., ) . cesarean rederivation or embryo transfer may also be used to rederive virus-free progeny. prevention of mvm infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. research complications mvm contamination of transplantable neoplasms can occur; therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. failure to establish long-term cell cultures from infected mice or a low incidence of tumor 'takes' should alert researchers to the possibility of mvm contamination. mvmi has the potential to inhibit the generation of cytotoxic t cells in mixed lymphocyte cultures. etiology mouse parvovirus (mpv) is among the more common viruses detected within contemporary laboratory animal medicine mouse colonies, and is more common than mvm (livingston et al., ; livingston and riley, ; pritchett-corning et al., ) . mpv was initially isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. the virus grew lytically in a cd + t-cell clone designated l and inhibited the proliferation of cloned t cells stimulated with antigen or interleukin (il- ) (mckisic et al., ) . molecular analysis of mpv indicates that regions encoding the ns proteins are similar to those of mvm (and other rodent parvoviruses). however, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity. the prototype isolate was first called an 'orphan' parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (mpv). immortalized t cells (l ) are the only cells found thus far to support replication of mpv. there are three genetically distinct variants of mpv, including mpv- , mpv- , and mpv- . mpv- includes a number of closely related variants, including mpv- a, mpv- b, and mpv- c. in addition, a hamster parvovirus isolate is closely related to mpv- , which is infectious to mice and likely to be of mouse origin (besselsen et al., ; christie et al., ) . clinical signs mpv infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice (besselsen et al., ) . immunologic perturbations are the most likely signs of infection (mckisic et al., ) . epizootiology mpv causes persistent infection in infant and adult mice, a property that differentiates it from mvm. in situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. experimental studies following inoculation of neonatal balb/c and c.b- -prkdc scid (scid) mice revealed that balb/c mice shed high levels of virus for weeks, with transmission to sentinels exposed during the first weeks of infection. thereafter, balb/c mice shed extremely low virus intermittently. in contrast, scid mice shed high levels of virus until weaning, but lower levels at weeks of age, yet they effectively transmitted infection to sentinels at all stages of infection (besselsen et al., ) . others have shown that transmission of mpv by sencar mice inoculated as infants was intermittent up to weeks, whereas transmission by mice inoculated as weanlings occurred during the first weeks of infection . transmission to balb/c progeny from infected dams was shown to occur, but embryo transfer rederivation was found to be successful in experimentally infected scid mice (besselsen et al., ) . humoral (e.g., passively or maternally acquired) immunity can protect against mpv infection. however, immunity to mvm may not confer cross-immunity to mpv (hansen et al., ) . pathology mpv appears to enter through the intestinal mucosa, which is a site of early virus replication ( fig. . ). acute infection is widespread but mild, involving the lung, kidney, liver, and lymphoid organs. histological lesions are not discernible. lymphocytotropism is a characteristic of acute and persistent mpv infection in infant and adult mice. during acute infection, virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (fig. . ) . diagnosis because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. serology that utilizes mpv vp as antigen is a sensitive and specific assay that differentiates mpv from mvm (livingston et al., ) . the map test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. as noted for mvm, pcr for murine parvoviruses, using nucleoprotein gene sequences that are conserved among murine parvoviruses, can be used as a screening test. pcr also can be used to detect mpv-specific sequences in the vp gene. although diagnostic pcr is sensitive and specific, it is effective only in actively infected animals. it can be used on feces to detect virus shedding, or applied to tissues, such as mesenteric lymph nodes, obtained at necropsy. differential diagnosis mpv infection must be differentiated from mvm infection. because both viruses are enterotropic and lymphocytotropic, serology and pcr must be used to distinguish between them. prevention and control the persistence of mpv in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of mpv infection. quarantine of infected rooms is appropriate. elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced, but a cage-by-cage test and cull approach has been shown to be successful under natural conditions (macy et al., ) . for mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. control of infection also should include environmental decontamination. chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. with the increasing use of mouse germplasm, it is important to note that mouse sperm, oocytes, ovarian tissue, and preimplantation embryos from enzootically mpvinfected mouse colonies may have a high prevalence of mpv contamination, based upon pcr (agca et al., ) . research complications murine parvoviruses can distort biological responses that depend on cell proliferation. for mpv, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses. etiology adenoviruses are nonenveloped dna viruses that produce intranuclear inclusions in vitro and in vivo. two adenovirus species in the genus mastadenovirus have been associated with mice: murine mastadenovirus a (with the representative strain being mav- or fl) and murine mastadenovirus b (with the representative strain being mav- or k ). both strains replicate in mouse kidney tissue culture but are antigenically distinct. clinical signs mav- can cause severe clinical disease after experimental inoculation of infant mice. signs include scruffiness, lethargy, stunted growth, and often death within days. mav- virus is enterotropic and is responsible for virtually all naturally occurring infections in contemporary mouse populations. infection is usually subclinical in immunocompetent mice, with the possible exception of transient runting among infant mice. wasting disease can occur in athymic mice infected with mav- . epizootiology the prevalence of adenovirus infection in mouse colonies is low, particularly mav- riley, , pritchett-corning et al., ) . transmission occurs by ingestion. adult mice experimentally infected with mav- may remain persistently infected and excrete virus in the urine for prolonged periods. adult mice experimentally infected with mav- excrete virus in feces for at least weeks but eventually recover. athymic mice can shed mav- for at least weeks and episodically for at least months. pathology infection with mav- causes multisystemic disease characterized by necrosis. infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of brown fat, myocardium, adrenal cortex, salivary gland, and kidney, with the development of intranuclear inclusions. more mature mice usually develop subclinical infection leading to seroconversion; however, athymic and scid mice can develop intestinal hemorrhage and wasting, with fatal disseminated infection (lenaerts et al., ) . infection with mav- produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine (fig. . ) . inclusions are easier to detect in infant mice than in adults. infection of c.b- -prkdc scid mice with mav- results in enteric infection, but also hepatic lesions resembling reye's syndrome (pirofski et al., ) . diagnosis although mav strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. cross-neutralization tests have revealed that antiserum to mav- neutralizes both strains, but antiserum to mav- neutralizes mav- weakly at best. therefore, mav- antigen should be used for the serological detection of adenovirus infection irrespective of the assay employed. mav also can be detected by pcr. differential diagnosis intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic laboratory animal medicine and differentiate mav- infection from other known viral infections of mice. infection may resemble rotavirus infection, with runting and abdominal bloating in infant mice. prevention and control prevention requires serological monitoring of mice and examination for contamination of animal products such as transplantable tumors. because mav- infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. however, rederivation coupled with subsequent barrier housing is a more conservative approach. research complications mav infection is unlikely to affect research using immunocompetent mice. however, it has the potential for pathogenicity in immunodeficient mice. mice can incur natural infection with two polyomaviruses: polyoma virus (pyv) and k virus. these viruses belong to the family polyomaviridae. k virus belongs to the genus polyomavirus and the species murine pneumotropic virus, while the classical polyoma virus belongs to the species murine polyomavirus. etiology polyoma virus (pyv) is a small dna virus that derives its name 'polyoma' (many tumors) from its ability to experimentally induce multiple types of tumors in mice experimentally infected as neonates. its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. the transformative activity is mediated by 't' (tumor) antigens, encoded by large t, middle t, and small t genes, with middle t (mt) being considered the major viral oncogene, and as a result has been used extensively in transgenic constructs. clinical signs natural infections in immunocompetent mice are usually subclinical. however, tumor induction, neurological disease, and wasting can occur in naturally exposed immunodeficient mice (mccance et al., ; sebesteny et al., ) . epizootiology modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. pyv is used for experimental studies and thus can inadvertently be introduced to mouse colonies. inoculation of mice with contaminated biologicals or cell cultures is a potential source of entry and spread. natural transmission occurs via the respiratory route. exposure of neonatal mice results in persistent infection and shedding of the virus in urine, feces, and saliva, thereby contaminating the environment for spread to other mice. infection of adult mice is transient, with minimal virus shedding, although pcr has revealed infection lasting up to months in cba mice inoculated with virus as adults (berke and dalianis, ) . maternal antibody is highly effective at preventing infection of newborn mice, but as maternal antibody wanes, mice are partially susceptible, with transient virus shedding. thus, the natural cycle of transmission in enzootically infected populations requires contamination of bedding and nesting material in order to infect and be inefficiently transmitted, which is readily precluded by modern husbandry. intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. as in immunologically immature neonatal mice, pyv infection can persist in adult immunodeficient mice. pathology pyv-induced tumors are essentially a laboratory phenomenon, optimized by virus strain and mouse strain, with akr, c h, c , cba, swr, and others being most susceptible, and c bl/ being among the most resistant to pyv oncogenesis. intranasal inoculation of neonatal mice results in initial replication in pulmonary respiratory epithelium (gottlieb and villarreal, ) followed by viremic dissemination and acute, lethal disease. tumors appear - months after inoculation of surviving mice. tumors of both epithelial and mesenchymal origin arise in multiple organs, particularly mammary carcinomas, basal cell tumors of the skin, carcinomas of salivary glands, thymomas, and various types of sarcomas. athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. intranuclear inclusions may be present in cytolytic lesions. demyelinating disease and skeletal tumors have been reported in experimentally . laboratory animal medicine inoculated and naturally exposed athymic mice, and myeloproliferative disease has been reported in experimentally inoculated c bl/ -scid mice (szomolanyi-tsuda et al., ) . diagnosis pyv can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically. additionally, pcr and immunohistochemistry can be used. differential diagnosis wasting in athymic mice can be caused by other infectious agents, including coronaviruses, sendai virus (sv), and pneumocystis. intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and k virus. prevention and control control depends on elimination of infected mice and material, together with prevention of airborne spread. biological material destined for mouse inoculation should be tested for pyv by the map test or molecular diagnostics. research complications pyv infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread. k virus infection k virus has historical importance, and is apparently absent from contemporary mouse populations (livingston and riley, ; pritchett-corning et al., ) , but it continues to be tested for, adding to the expense of infectious disease surveillance. oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by viremic spread. vascular endothelium is the primary target in affected tissues, which often include the lung, liver, spleen, and adrenal glands. dyspnea occurs from pulmonary infection because of edema and hemorrhage. infection of immunocompetent adult mice is subclinical and results in a vigorous immune response. however, both adults and infant mice develop persistent infection. the primary organ for persistence is the kidney, with shedding of virus from tubular epithelium, and shedding can be reactivated by immunosuppression (greenlee et al., ) . additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. gross lesions are limited to pulmonary hemorrhage and edema. histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. mild hepatitis with hepatocyte degeneration also may develop. infection can be detected by serology or pcr. prevention and control measures, if ever to be found within a mouse population, are similar to those described for pyv. etiology lactate dehydrogenase-elevating virus (ldv) is a mouse-specific small enveloped rna virus belonging to the family arteriviridae. infected mice are persistently viremic, resulting in increased concentration of several serum enzymes, most notably lactate dehydrogenase (ldh). infection is common among wild mice, but is now rare in contemporary laboratory mouse populations. however, surveys of biologic material indicate that ldv may be a common contaminant of biologic materials (nicklas et al., ) . clinical signs infection is subclinical. however, poliomyelitis has occurred in immunosuppressed c and akr mice inoculated with ldv, and has recently been observed in icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . epizootiology the primary mode of mouse-tomouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. it is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva. pathology viremia peaks within day after inoculation, then persists at a diminished level. the elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. ldv selectively targets mature f /f -positive macrophages, which are continually produced by uninfected progenitor cell populations, thereby maintaining persistent infection. virus also escapes immune clearance by evolution of neutralizing antibody-resistant quasi-species. no lesions are seen in naturally infected mice. the only significant lesion that can arise from experimental infection is poliomyelitis. this syndrome requires a combination of immunosuppression (due to age, genetics or induced means), mouse strain (c , akr, c h/fg, and pl), neurotropic ldv strains, and endogenous ecotropic murine leukemia virus. the mouse strain-dependent element is homozygosity for the fv- n allele, which permits replication of endogenous n-tropic ectotropic murine leukemia virus. mice develop spongiosis, neuronal necrosis, and astrocytosis of the ventral spinal cord and brain stem, with axonal degeneration of ventral roots. lesions contain both ldv and retrovirus. although this syndrome is largely experimentally induced, a natural outbreak of poliomyelitis has been reported in fv- homozygous icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . diagnosis plasma ldh levels are elevated, a response that is used to detect and titrate ldv infectivity. of the five isoenzymes of ldh in mouse plasma, only laboratory animal medicine ldh-v is elevated. sjl/j mice in particular show spectacular increases in ldh levels ( - times normal), a response controlled by a recessive somatic gene. ldv is detected by measuring ldh levels in mouse plasma before and days after inoculation of specific pathogenfree (spf) mice with suspect material. it is important to use nonhemolyzed samples because hemolysis will produce falsely elevated readings. plasma enzyme levels are measured in conventional units/ml, conventional unit being equivalent to . international units (iu). normal plasma levels are - iu, whereas in ldv infection, levels as high as iu can occur. ldv also interferes with the clearance of other serum enzymes and results in their elevation in serum. in a recent survey, serum samples were tested by serum ldh enzyme assays, among which % were deemed potentially positive. however, pcr revealed that all were false-positives (pritchett-corning et al., ) , emphasizing the inaccuracy of traditional enzyme assays. infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and biologic materials. however, inhibitory factors in cells and serum may cause falsenegative results in pcr testing, so appropriate quality control measures are essential if this method is used (lipman and henderson, ) . prevention and control transplantable tumors have been a common source of ldv historically. therefore, tumors or cell lines destined for mouse inoculation should be monitored for ldv contamination. although ldv can contaminate tumor cell lines, it does not replicate in the tumor cells. therefore, one can attempt to free tumors of virus by passaging them through athymic nude rats, which are not nonpermissive to ldv but are permissive to xenografts. research complications ldv has numerous potential effects on immunological function. it may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth. etiology the house mouse is the natural host for lymphocytic choriomeningitis virus (lcmv), an old world member of the arenaviridae family that has spread worldwide along with m. musculus. lcmv virions are pleomorphic, containing single-stranded rna, and bud from the cell membrane. disease associated with infection is due to host immune response to the otherwise non-cytolytic virus. its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. lcmv is a zoonotic virus that may cause a variety of clinical manifestations in humans, including meningitis. it has been extensively studied as an experimental model of virus-induced immune injury, using a number of closely related strains, including ones that have been selected for their relative neurotropism or viscerotropism. lcmv can be propagated in a variety of mammalian, avian, and even tick cell lines, with minimal cytopathic effect. these characteristics favor its propensity to persistently and silently contaminate biologic products, such as tumor cell lines. clinical signs natural infection in immunocompetent adult mice is usually self-limiting and subclinical. during enzootic infection of a mouse population, lcmv is transmitted in utero from persistently infected dams to their fetuses or to neonates, which are persistently infected and immunologically tolerant to lcmv. since lcmv is non-cytolytic in and of itself is minimally pathogenic, congenitally infected mice grow into adulthood, reproduce, and therefore transmit infection to the next generation. however, with age, immune tolerance breaks down, and mice develop a syndrome known as 'late disease' in which mice will progressively lose weight and die. in utero infection results in a low level of fetal mortality and maternal cannibalism of infected pups. the immune tolerance to lcmv is virus-specific, with the mice capable of eliciting effective immune responses against other agents. clinical signs following experimental inoculation of lcmv vary with age and strain of mouse, route of inoculation, and strain of virus. when virus is inoculated intracerebrally into immunocompetent adult mice, mice develop immune-mediated lymphocytic choriomeningitis, characterized by illness beginning - days after inoculation. sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the rear legs. spontaneous convulsions also can occur. animals usually die or recover in several days. a visceral form of infection can occur in adult mice inoculated by peripheral routes with 'viscerotropic' strains. it can be subclinical or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. if mice survive, recovery may take several weeks. surviving mice may have immune exhaustion due to consumption of lymphoid tissue, in contrast to immune tolerance that occurs when mice are infected in utero or as neonates. runting and death from lcmv infection may occur in neonatally infected mice and can lead to transient illness or to death. clinical signs are nonspecific, recovery is slow, and survivors may remain runted. this early form of disease is attributed to endocrine dysfunction caused laboratory animal medicine by lcmv infection. late-onset disease can occur in previously subclinical carrier mice that develop immune complex glomerulonephritis. it is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are - months old. clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. epizootiology lcmv is distributed widely in wild m. musculus throughout the world. among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. lcmv infection is rare in laboratory mice produced and maintained in modern quarters (livingston and riley, ; pritchett-corning et al., ) . infection is usually introduced through inoculation of virusinfected biologicals, such as transplantable tumors, or by feral mice. wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. naturally infected carrier mice can have persistently high concentrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. thus introduction of a single lcmv carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. infection in adult mice, in contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. mice can transmit lcmv to hamsters, which can remain viremic and viruric for many months, even if they contract infection as adults. infected hamsters can transmit virus to other hamsters and mice and are the primary source of human lcmv infection. persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission. pathology lcmv disease is a prototype for virusinduced, t-lymphocyte-mediated immune injury, noncytolytic endocrine dysfunction, and immune complex disease. however, lesions comparable to experimentally induced disease are rare during natural infection. intracerebral inoculation of virus into immunocompetent adult mice induces nonsuppurative leptomeningitis, choroiditis, and focal perivascular lymphocytic infiltrates. host tissues are damaged during the course of the cellular immune response to the virus. the character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. in severe infection, nonsuppurative inflammation can occur in many tissues. the severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. liver lesions can include hepatocyte necrosis accompanied by nodular infiltrates of lymphoid cells and kupffer cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. necrosis of cortical thymocytes can lead to thymic involution. lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. lymphoid nodules can form in various organs. lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. the lesions of acute and persistent lcmv infection reflect separate immunopathologic processes. in adult mice with acute lcmv infection, virus multiplies in dcs, b cells, and macrophages, whereas t cells are resistant. internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. thus, elimination of virus and virus-associated immunological injury are both t-cell-mediated. this apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. antibody can be detected by week after infection but does not play a significant role in eliciting acute disease. lesions of lcmv infection appear to develop from direct t-cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector t cells. lcmv also can suppress humoral and cellular immunity in acutely infected mice. persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. prenatal or neonatal infection induces immunological tolerance to lcmv, which can then replicate to high titer in many tissues. nevertheless, persistently infected mice develop humoral antibody to lcmv. antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. immune complex glomerulonephritis exemplifies this process, as noted above. diagnosis lcmv infection can be diagnosed serologically. whereas immunocompetent adult mice will normally seroconvert after exposure, carrier mice may develop poor humoral immune responses. therefore, testing must avoid false-negative results. employment of adult contact sentinel mice is a useful strategy for detecting lcmv infection by seroconversion. tissues, including biologic products and cell lines, can be tested laboratory animal medicine by pcr. a traditional method for detection involved collection of small blood samples from persistently infected live suspects, which are often viremic, and using them to inoculate cultured cells or adult and neonatal mice. intracerebral inoculation of lcmv-positive tissues should elicit neurological signs in adult mice within days, whereas infant mice should remain subclinical. histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the map test. virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (n- ) cells, bhk- cells, and l cells. application of immunofluorescence staining to detect lcmv antigen in inoculated cultured cells yields results more quickly than animal inoculation. of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel (see chapter ). the use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. differential diagnosis neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from bacterial infection. trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. late-onset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus infection, tyzzer's disease, or husbandry-related insults. prevention and control adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. if mice are easily replaced, however, depopulation is a safer and more reliable option. valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. infection of immunodeficient mice poses similar risk. lcmv can be transmitted to human beings, who can contract flu-like illness or severe cns disease. more frequently, human infection is subclinical. the zoonotic potential of lcmv infection makes it especially important to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. serum banking and periodic serological testing of highrisk human populations, such as those working with lcmv experimentally, are recommended. research complications lcmv may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. introduction of immune cells to a carrier animal may elicit an immunopathological response. immune complex disease can complicate longterm experiments and morphological interpretations. illness and death in mice and zoonotic risk to humans are obvious research-related hazards. etiology sv is a paramyxovirus that is antigenically related to human parainfluenza virus . viral particles are pleomorphic, contain single-stranded rna, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. sv grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney , and mouse fibroblast [l]). virus replicates in the cytoplasm and by budding through cell outer membranes. once common in laboratory rodent populations, sv is now rare or absent (livingston and riley, ; pritchett-corning et al., ) . clinical signs clinically affected adult mice often assume a hunched position and have an erect hair coat. rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. although highly susceptible adults may die, lethal infection is more common in suckling mice. sex differences in susceptibility have not been found. genetically resistant mice usually have subclinical infection. athymic mice and immunodeficient mice are at high risk for development of a wasting syndrome. they develop illness later than their immunocompetent counterparts, since clinical signs in immunocompetent mice are related to immune-mediated destruction of respiratory epithelium. opportunistic infections can complicate the clinical presentation. for example, secondary bacterial infections of the ear can cause vestibular signs. epizootiology sv is transmitted by aerosol and is highly contagious. morbidity in infected colonies is commonly %, and mortality can vary from % to %, partly because strains of mice vary greatly in their susceptibility to lethal sv infection. for example, c bl/ mice are highly resistant to clinically apparent infection, whereas dba/ mice are highly susceptible. aerogenic infection is promoted by high relative humidity and by low air turnover. prenatal infection does not occur. enzootic infection is commonly detected in postweaned mice ( - weeks old) and is associated with seroconversion within - days and the termination of infection. therefore, entrenched infection is perpetuated by the introduction of susceptible animals. there is no evidence for persistent infection in immunocompetent mice, but prolonged infection is common in immunodeficient mice. maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. rats, hamsters, and guinea pigs also are susceptible to sv infection. therefore, bidirectional cross-infection is a risk during outbreaks. pathology viral replication is nominally restricted to the respiratory tract and peaks by the first week after infection. gross lesions feature partial to complete consolidation of the lungs (fig. . ) . individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. sv targets airway epithelium and type ii pneumocytes. type i pneumocytes are less severely affected. histologically, the pattern of pneumonia is influenced by mouse genotype. susceptible mice usually have significant bronchopneumonia and interstitial pneumonia, whereas the interstitial component may be less prominent in resistant mice. typical changes begin with inflammatory edema of bronchiolar lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. necrosis and exfoliation of bronchiolar epithelium ensue, frequently in a segmental pattern (fig. . ). alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia (fig. . ) . lymphoid cells also invade peribronchiolar and perivascular spaces. the lymphocytic response to sv infection reflects the fact that cellular immunity contributes both to lesions and to recovery. local immunoglobulin synthesis by infiltrating cells also occurs. the extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with clinically susceptible hosts mounting a more florid immune response than resistant hosts. additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. multinucleated syncytia are occasionally seen in affected sucklings and scid mice, and inclusion bodies have been reported in infected athymic mice. regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. proliferation of cuboidal laboratory animal medicine epithelium may give terminal bronchioles an adenomatoid appearance. repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (jacoby et al., ) . diagnosis sv is notable for its ability to cause epizootics of acute respiratory distress in adult genetically susceptible strains. serology is an effective means to detect infection in all strains of immunocompetent mice. antibody can be detected by days postinfection and coincides with development of clinical signs related to the immune-mediated necrotizing bronchiolitis and alveolitis. repeated serologic sampling over several weeks can help stage infection within a population. alternatively, sentinel animals can be added to seropositive colonies to detect active infection. irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections), and, where possible, virus isolation should be used to confirm infection. virus can be isolated from the respiratory tract for up to weeks, with peak titers occurring at about days postinfection. nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or bhk- cell monolayer cultures. sv infection of cultured cells is non-cytolytic, so erythrocyte agglutination or antigen detection methods must be used. rt-pcr also can be used to detect virus in infected lungs. differential diagnosis respiratory infection caused by pneumonia virus of mice (pvm) is generally milder or subclinical. histologically, necrosis of airway epithelium is less severe. bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. because sv pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult. control and prevention sv infection is self-limiting in surviving immunocompetent mice. suckling mice from immune dams are protected from infection by maternal antibody until after weaning. control and eradication measures must eliminate exposure of susceptible animals, so that infection can 'burn out.' this is most easily accomplished by a quarantine period of - weeks wherein no new animals are introduced either as adults or through breeding. control also is aided by the fact that sv is highly labile. barrier housing is preferred for prevention and for control of transmission. vaccination with formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. research complications sv can cause immunosuppression and can inhibit growth of transplantable tumors. this effect has been attributed to virus-induced modification of tumor cell surface membranes. pulmonary changes during sv pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. they also have been associated with breeding difficulties in mice. this sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection. clinical signs natural pvm infection in mice is subclinical. therefore, its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. however, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with pvm (weir et al., ) . pvm is used experimentally as a model to study acute respiratory infection, using highly pathogenic strains of the virus (dyer et al., ) . epizootiology pvm causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. serological data indicate that pvm was once common, but is now relatively uncommon (livingston and riley, ; pritchett-corning et al., ). pvm appears to spread less rapidly than sv. intimate contact between mice is probably required for effective transmission. this characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. infection is acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice. pathology pvm replicates exclusively in the respiratory tract and reaches peak titers in the lung - days after infection. although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. histological lesions can occur in the upper and lower respiratory tract. they consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occur within weeks after exposure to virus and are largely resolved by weeks. the predominant inflammatory infiltrate is comprised of mononuclear cells, but some neutrophils laboratory animal medicine are usually present. immunohistochemistry on paraffinembedded tissues can be used to detect viral antigen in bronchiolar epithelium, alveolar macrophages, and alveolar epithelium during acute infection. residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. severe progressive pneumonia, with wasting, can occur in immunodeficient mice. it is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with desquamated alveolar pneumocytes and leukocytes filling alveolar spaces (fig. . ) . diagnosis diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. virus replication in bhk- cells is detected by immunofluorescence or other antigen detection methods. virus also can be detected in tissues by rt-pcr. differential diagnosis because pvm is antigenically distinct from other murine viruses, serology is the most useful method to separate pvm infection from other respiratory infections of mice. however, in immunodeficient mice, where clinical signs and lesions are typical, it must be differentiated from other pneumonias, especially those due to sv and pneumocystis. additionally, pvm can coexist with and exacerbate pneumocystis infection in immunodeficient mice (bray et al., ) . prevention and control pvm infection is acute and self-limiting in immunocompetent mice, but persistent in immunodeficient mice. seropositive mice should be viewed as either immune or in the final stages of acute infection. therefore, control and prevention follows guidelines applicable to sv infection. research complications pvm can exacerbate pneumocystosis, as noted above. (ward et al., ) two members of the family reoviridae infect laboratory mice: reovirus per se (species: mammalian orthoreovirus) and murine rotavirus (species: rotavirus a), also known as epizootic diarrhea of infant mice (edim) virus. etiology reoviruses of mammals, although taxonomically considered one type species, have been divided into three cross-reacting prototypic serotypes: reovirus , and , which can be differentiated by cross-serum neutralization. mice can be infected with any serotype, but reovirus is emphasized because it has been associated with naturally occurring disease. natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. a number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from virtually every mammal tested, as well as birds, reptiles, and insects. the virion contains segmented, double-stranded rna and is relatively heat stable. reoviruses replicate well in bhk- cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. clinical signs clinical disease is rare and age dependent. acute disease affects sucklings at about weeks of age, whereas adults have subclinical infection. signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. incoordination, tremors, and paralysis occur just before death. convalescent mice are often partially alopecic and are typically runted. alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. infants born to immune dams are protected from disease by maternal immunity. epizootiology the prevalence of reovirus infection in contemporary mouse colonies is rare (livingston and riley, ; pritchett-corning et al., ). reoviruses are highly contagious among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. transmission is inefficient among adult mice. there is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. the course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined. pathology reovirus can cause severe pantropic infection in infant mice. after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (m cells) that cover peyer's patches. virus can be carried to the liver in leukocytes, where it is taken up by kupffer cells prior to infecting hepatocytes. in acute disease, livers may be large and dark, with yellow foci of necrosis. the intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. myocardial necrosis and pulmonary hemorrhages have been reported. myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. the brain may be swollen and congested. central nervous system lesions have a vascular distribution, and are most prevalent in the brain stem and cerebral hemispheres. neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. severe encephalitis may evoke focal hemorrhage. in the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. orally infected suckling mice can develop multifocal hepatocyte necrosis, which may include the accumulation of dense eosinophilic structures resembling councilman bodies. hepatocytomegaly, kupffer cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. in experimentally inoculated mice, necrotic foci can persist in the liver for at least weeks. chronic active hepatitis may develop after acute infection and result in biliary obstruction. acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in the pancreas may be caused by obstruction rather than by viral invasion of parenchyma. pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. oronasal inoculation of infant mice with reovirus results in a similar distribution but significantly milder lesions compared to reovirus . in contrast, reovirus is highly enterotropic, inducing mild enteritis without lesions in other tissues, similar to epizootic diarrhea of infant mice (edim) . diagnosis serology uses reovirus as antigen, which detects seroconversion to all serotypes, and viral rna can be detected by rt-pcr. a presumptive diagnosis of reovirus infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. the presence histologically of multisystemic necrosis is consistent with severe reovirus infection but should be confirmed by immunohistochemistry or virus isolation. differential diagnosis reovirus infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, edim virus, salmonella spp., or clostridium piliforme. prevention and control although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, map testing, or other appropriate means, to rederive or replace infected stock. prevention depends on adequate barrier husbandry coupled with adequate serological monitoring. research complications reovirus infection can interfere with research in several ways. infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. virus has been commonly recovered from transplantable neoplasms and is suspected of being oncolytic. the potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research. etiology rotaviruses are double-stranded, segmented rna viruses that have a wheel-like ultrastructural appearance. edim virus is a group a rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. edim virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. these agents tend to be species-specific under natural conditions and can be differentiated by serum neutralization tests. cultivation of edim virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide. clinical signs clinical signs occur in infant mice less than weeks old. this age-related susceptibility also applies to infection in immunodeficient mice. furthermore, clinical signs occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease. the cardinal signs are bloated abdomens with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. despite high morbidity, mortality is low because affected mice continue to nurse. transient weight loss does occur, and there may be a delay in reaching adult weight. recovery from infection usually occurs in about weeks and, once weight is regained, is clinically complete. epizootiology edim virus appears to be infectious only for mice and occurs episodically in mouse colonies, and infection is probably widespread geographically (livingston and riley, ; pritchett-corning laboratory animal medicine et al., ) . all ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. the virus is highly contagious and is transmitted by the oral-fecal route. subclinically infected adult mice can shed virus in feces for at least days, an interval that may be extended in immunodeficient mice. after oral inoculation, virus is essentially restricted to the gastrointestinal tract, although small amounts of virus may be present in the liver, spleen, kidney, and blood. nursing dams can contract infection from their litters. transplacental transmission has not been demonstrated. pathology gross lesions occur primarily in the gastrointestinal tract, but thymic involution can result from infection-related stress. the intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. the stomach contains curdled milk, except in terminal cases with anal impaction due to caking of dried feces. virus preferentially infects terminally differentiated enterocytes in the small and large intestines, which accounts for the agerelated susceptibility to disease; the number of such cells decreases as the intestinal tract matures. characteristic histological lesions are often very subtle, but are most easily discerned in the small intestine in mice less than weeks old. they consist of vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (fig. . ) . the vacuoles must be differentiated from normal absorption vacuoles in nursing mice. the lamina propria may be edematous, but necrosis and inflammation are not prevalent. diagnosis edim virus infection is readily detected serologically. clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in the intestine or in intestinal filtrates or smears. rotavirus antigen can be detected in feces by elisa, but certain dietary ingredients can cause false-positive reactions. infection can also be diagnosed by rt-pcr. differential diagnosis edim virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis virus) infection, reovirus infection, tyzzer's disease, and salmonellosis. the presence of milk in the stomach can be helpful in differentiating edim virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. the possibility of dual infections must also be considered. thymic necrosis in edim virus-infected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (mtv) infection or other stressors. prevention and control the spread of edim can be controlled effectively by the use of microbarrier cages and good sanitation. because infection appears to be acute and self-limiting, cessation of breeding for - weeks to allow immunity to build in adults while preventing access to susceptible neonates also is recommended. alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. the duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. therefore, such animals should be eliminated. litters from immune dams are more resistant to infection. if edim virus is allowed to become enzootic within a colony, clinical signs will disappear within the population, which may be an appropriate management approach in conventional colonies. prevention of edim virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance. research complications the research complications of edim infection pertain to clinical illness with diarrhea and retarded growth. transient thymic necrosis may perturb immunological responses. infection (barthold, a,b) etiology coronaviruses are large, pleomorphic, enveloped rna viruses with radially arranged peplomers (spikes). in mice, early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (mhv). during that time, enteritis in infant mice was recognized as a separate entity caused by an uncharacterized virus, known as lethal intestinal virus of infant mice (livim). subsequent studies revealed that hepatitis-causing mhv and enteritis-causing livim were closely related coronaviruses, now collectively termed mhv. mhv isolates differ in biologic behavior according to their organ tropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but may progress to multisystemic dissemination, including the liver and brain. these differences are often reflected in their cell tropism in vitro. however, natural isolates may contain features of both biotypes. several prototype polytropic strains have been extensively studied as experimental models of hepatitis and encephalitis. they include jhm (mhv ), mhv- , mhv- , mhv-s, and mhv-a . numerous additional strains have been identified that differ in virulence, tissue tropism, and antigenicity. differentiation by strain, particularly under natural conditions, is irrelevant, since mutation is common among coronaviruses, and even named prototype strains differ significantly depending upon passage history. although mhv isolates and strains share internal antigens (m and n), they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens. mhv shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. mhv also is related to human coronavirus oc . a number of established cell lines may be used for propagating polytropic mhv strains in vitro. however, field isolates are difficult to maintain in vitro. nctc mouse liver cells are useful for growing many polytropic strains. mhv can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and susceptibility to infection. enterotropic strains, because of their tendency to be strictly enterotropic, have been grown in cmt- cells derived from a rectal carcinoma in a c bl mouse, but are generally difficult to propagate in cell culture. irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of mhv infection (fig. . ) . clinical signs clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus. as with many murine viruses, infection is often clinically silent among immunologically competent mature mice. clinical morbidity is most often associated with suckling mice less than weeks old or with immunodeficient mice. suckling mice infected with enterotropic mhv develop inappetence, diarrhea, and dehydration, often terminating in death (fig. . ) . epizootics of enterotropic mhv have been known to result in % mortality among neonatal mice in a breeding colony. older mice ( - weeks of age) may have ruffled pelage and runting. neurotropic strains such as mhv-jhm may induce flaccid paralysis of the hindlimb, but this sign is rarely encountered alone during natural infection. conjunctivitis, convulsions, and circling may be seen occasionally. enterotropic strains may not cause acute disease in athymic mice when exposed as adults, whereas mildly pathogenic polytropic strains can cause a progressive wasting syndrome that may be accompanied by progressive paralysis. epizootiology mhv infection, despite constant surveillance and preventive programs, continues to be a common threat to laboratory mouse populations (livingston and riley, ; pritchett-corning et al., ). there are no reports of natural transmission from mice to other species, but suckling rats have been found to develop necrotizing rhinitis after intranasal inoculation with mhv-s. mhv is highly contagious, with natural transmission occurring by respiratory or oral routes. mouse appears normal and has a milk-filled stomach. lower mouse is runted and dehydrated and has an empty stomach. from barthold et al. ( ) . enterotropic biotypes predominate in natural infections in contemporary laboratory animal facilities, since they tend to be the most contagious due to copious excretion of virus in feces, whereas polytropic strains generally spread by direct respiratory contact. natural vertical transmission has not been demonstrated. introduction of mhv through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect b lymphocytes and, by implication, hybridomas nonlytically. infection in immunocompetent mice is self-limiting. immune-mediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within - weeks. humoral and cellular immunity participate in host defenses to infection, and t-cell-dependent immunity is an absolute requirement. thus, age-related resistance to mhv correlates with maturation of lymphoreticular tissues, but intestinal proliferative kinetics are critical determinants of disease susceptibility with enterotropic mhv. enzootic infection had been construed to include persistent infection in individual mice. current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with mhv variants that arise by natural mutation. mutation is favored by immune pressure in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. thus, mice that have developed immunity to one strain of mhv can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection. this caveat has practical importance for breeding colonies. maternal immunity protects suckling mice against homologous mhv strains but not against antigenically variant strains. however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. therefore, the susceptibility of young mice to infection increases significantly at weaning. strain differences in resistance and susceptibility to polytropic mhv can be inherited as an autosomal dominant trait. for example, dba/ mice are highly susceptible to mhv- and die acutely even as adults, whereas a/j mice develop resistance to lethal infection shortly after weaning. however, genetic resistance is also virus strain-dependent. therefore, mice resistant to one strain of mhv may be susceptible to another strain. it also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. this pertains to mhv as well as other agents. for example, mhv infection has presented as granulomatous peritonitis and pleuritis in interferongamma (ifn-γ) knockout mice (france et al., ) . pathology polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. at necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. liver involvement may be accompanied by icterus and peritonitis. histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells (fig. . ) . syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. syncytia formation is a hallmark of infection in many tissues, including the intestine (fig. . ) , lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice ( fig. . ) . neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. immunodeficient mice can develop progressive necrotic lesions in the liver and elsewhere. compensatory splenomegaly may occur because of expansion of hematopoietic tissue. enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also figure . necrosis, inflammation, and syncytium in the liver of a mouse infected with mhv. courtesy of s.w. barthold. cause systemic lesions, especially in the liver and brain. the most common sites are terminal ileum, cecum, and proximal colon. the severity of disease is age-related, and dependent upon intestinal proliferative kinetics, similar to edim, with young infants being at highest risk for lethal infection. pathogenic strains can cause lesions ranging from villus attenuation to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within a few days (fig. . ) . the stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. syncytia are a consistent feature in viable mucosa (fig. . ) and not only are formed in intestine but also may be present in mesenteric lymph nodes and endothelium of mesenteric vessels. enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. surviving mice develop compensatory mucosal hyperplasia, which eventually recedes, but may contribute to clinical signs due to osmotic, secretory, and malabsorptive diarrhea. older mice are equally susceptible to infection, but are resistant to severe disease due to their mature (more rapid) intestinal proliferative kinetics. pathology may be subtle, consisting of transient syncytia without necrotic lesions. in adult mice, syncytia can be found most often in the surface mucosal epithelium of the ascending colon. the exception occurs in immunodeficient mice, such as athymic and scid mice, which can develop chronic proliferative bowel disease of varying severity with mhv antigen in mucosal epithelium (figs. . and . ). this may not always be present, as athymic nude mice exposed as adults may only manifest a few enterocytic syncytia without hyperplasia. diagnosis because mhv infection is often subclinical, serological testing is the most reliable diagnostic tool. many animal resources rely on sentinel mouse protocols for continuous serological surveillance. serology is well established, sensitive, and reliable. neutralization tests are used to differentiate individual virus strains in the research laboratory but are inappropriate for routine use, because of cost, technical complexity, and serologic identification per se does not predict biological behavior, including virulence or tissue tropism. serology also can be used in the context of map testing in which adult mice are inoculated with suspect tissues to elicit seroconversion. rt-pcr protocols to detect virus in tissues or excreta are available. the detection of syncytia augmented, when possible, by immunohistochemistry to laboratory animal medicine detect mhv antigen is a useful and practical means to confirm infection. this strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. the option of using immunodeficient mice as sentinels can be considered, because they sustain prolonged infection. however, they should be securely confined because they also amplify virus loads. if properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture. differential diagnosis mhv infection must be differentiated from other infectious diseases that cause diarrheal illness, runting, or death in suckling mice and wasting disease in immunodeficient mice. these include edim, mousepox, reovirus infection, tyzzer's disease, and salmonellosis. neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious cns lesions, such as neoplasms, including polyoma virus-induced tumors in athymic mice. prevention and control control and prevention of mhv infection can be difficult because of the numerous variables that influence its expression. perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. there is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. such mice can be expected to develop immunity and eliminate virus within days. therefore, selective quarantine at the cage (not room) level with the temporary cessation of breeding can be used effectively to eliminate infection. quarantine at the room basis is likely to fail, since mutations arise and continually reinfect the mouse population. additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters. careful testing with sentinel mice should be used to assess the effectiveness of quarantine or 'natural rederivation,' as just described. immunodeficient mice, in contrast, are susceptible to chronic infection and viral excretion. mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction may impact on mhv infection and its control. such colonies, which may contain highly valuable or irreplaceable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of mhv infection is subsequently ruled out. although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. the prevention of mhv requires procurement of animals from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance program. control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection. research complications numerous research complications have been attributed to mhv, and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. for example, apart from its clinical impact, mhv may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of subclinically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. it also can alter tissue enzyme levels. additionally, the ubiquitous threat of mhv infection and uncertainty about its potential effects on a given research project provoke concerns that may exceed its true impact. for example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. this is not to say that subclinical or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak. etiology mice are susceptible to infection by two members of the cardiovirus genus within the theilovirus. emcv has a less selective host range and can infect wild mice, but is not known to infect laboratory mice. tmev is a small, nonenveloped, rna virus that was discovered by max theiler during experimental studies of yellow fever virus in mice. established prototype strains include to (theiler's original), fa, da, and gd vii, the last of which is named after george martine (george's disease), an assistant in theiler's laboratory. tmev is rapidly destroyed by temperatures over °c and by alcohol but not by ether. it can be cultivated in vitro in several continuous cell lines, but bhk- cells are routinely used for isolation and propagation. tmev is antigenically related to emcv. as with other nonenveloped viruses, tmev is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection. clinical signs the development of clinical disease depends on virus strain, mouse strain, and route of exposure, but natural disease is exceedingly rare (estimated at . - . % of infected mice). when clinical signs occur, they are expressed as neurological disease. the characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice that are otherwise alert (fig. . ) . some mice may recover, but death frequently ensues, often because of failure to obtain food or water. furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. epizootiology infection occurs primarily in laboratory mice with the exception of the mgh strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. the prevalence of tmev in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse, but it continues to be among the more common viral contaminants of mouse colonies (livingston and riley, ; pritchett-corning et al., ) . tmev infection is acquired by ingestion and replicates primarily in the intestinal mucosa. enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months . mice often become infected shortly after weaning, but virus is seldom recovered in mice over months of age. however, neurologic infection can persist in the brain and spinal cord for at least year. immunity to one strain of tmev provides cross-protection to other strains. there are no reports of differences in mice with respect to susceptibility to infection under natural conditions. prenatal transmission has not been found. pathology intestinal tmev infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells and neurons, neuronophagia, and perivascular inflammation, which occur particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. during the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. the white-matter lesions are due to immune injury. spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with mev infection, has also been reported in aged mice. virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease. diagnosis infection is usually detected serologically or by pcr of feces, but virus shedding from infected mice may be intermittent. clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. histological lesions in the cns and especially the spinal cord are characteristic when present. differential diagnosis neurotropic variants of mhv may, on occasion, cause similar neurological signs. injury or neoplasia affecting the spinal cord can also produce posterior paralysis. polyoma virus infection in athymic mice can induce tumors or demyelination in the cns, which may result in clinical signs resembling those of tmev infection. prevention and control disease-free stocks were originally developed by foster-nursing infant mice. this technique, cesarean rederivation, or embryo transfer can be used successfully to eliminate infection. in either case, foster mothers should be surveyed in advance to ensure their mev-free status. selective culling can be considered as an option to eliminate infection, because laboratory animal medicine infection spreads slowly. however, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable. research complications the principal hazard from tmev for research relates to its potential effects on the cns. noroviruses are nonenveloped rna viruses that belong to the family caliciviridae. they are notoriously resistant to environmental inactivation, and cause significant gastrointestinal morbidity in humans. noroviruses are species-specific, including mnv, which exclusively infects mice. until the discovery of mnv, replication of noroviruses in vitro has not been possible. for this reason, mnv has emerged as an important small animal model of norovirus pathogenesis. mnv was relatively recently discovered in , and subsequent surveillance has revealed that it is the most common adventitious virus infection in laboratory mice (hsu et al., ; pritchett-corning et al., ) . over mnv isolates have been found in mouse research colonies around the world, which display nearly % genetic identity, comprising a single genetic cluster. although genetically homogeneous, significant biological differences exist among mnv strains (thackray et al., ) . mnv effectively replicates in macrophages and dendritic cells, including the mouse macrophage-like raw . cell line, as well as a microglial cell line (wobus et al., ) . clinical signs clinical signs of infection in immunocompetent mice are usually absent, but infection leads to systemic disease with high mortality in interferon αβγ receptor and stat null mice. affected mice have loss of body weight, ruffled fur, and hunched posture (ward et al., ) . experimental infection of and c h mice with mnv- caused mild diarrhea (kahan et al., ) . epizootiology mnv is transmitted by the fecaloral route, and contaminates the environment as an environmentally resistant virus. for this reason, it can efficiently infect sentinel mice with soiled bedding (manuel et al., ) . duration of infection varies with mnv strain, mouse immunocompetence and mouse genotype. experimental studies have revealed that several mnv strains persist in various tissues of c bl/ j, hsd:icr, and jcl:icr and c.b- -prkdc scid mice, with fecal shedding for at least - days (goto et al., ; hsu et al., ; thackray et al., ) . although not clinically ill, rag null mice are unable to clear infection . comparative studies with mnv- and mnv- have shown differences in virus replication and shedding (kahan et al., ) . mnv has a tropism for macrophages and dendritic cells, and virus can be detected in the intestine, intestinal lymphoid tissue, liver, and spleen (hsu et al., ; kahan et al., ; wobus et al., ) . pathology naturally and experimentally infected stat or ifnγr null mice may develop splenomegaly and multifocal pale spots on the liver. microscopic findings include varying degrees of hepatitis, focal interstitial pneumonia, vasculitis, peritonitis, and pleuritis (karst et al., ; ward et al., ) . encephalitis, cerebral vasculitis, pneumonia, and hepatitis have also been described in intracerebrally infected stat null mice (karst et al., ) . infection of immunocompetent mice may be associated with mild inflammation of the intestine, splenic hypertrophy, and lymphoid hyperplasia of spleen and lymph nodes (mumphrey et al., ) . diagnosis mnv infection can be detected by serology or rt-pcr. sentinel mouse surveillance, using soiled bedding, is an effective strategy for detecting mnv (manuel et al., ) differential diagnosis the mild change in fecal consistency associated with mnv in adult mice may mimic rotavirus, coronavirus, helicobacter spp., citrobacter rodentium, or other enteric diseases. disseminated lesions in stat or ifnγr null mice must be differentiated from other polytropic viral diseases in immunodeficient mice, including mhv. prevention and control depopulation and decontamination has been shown to be effective at eliminating mnv from an enzootically infected colony, whereas testand-removal of positive mice was found to be ineffective (kastenmayer et al., ) . embryo transfer and cesarean rederivation are also effective (goto et al., ; perdue et al., ) . neonatal mice are resistant to infection, so that cross-fostering neonates onto uninfected dams is another effective means of rederivation mnv-free mice (artwohl et al., ; compton, ) . research complications the tropism of mnv for macrophages and dendritic cells is likely to modify immune responses, and mnv infection may interfere with studies involving enteric disease. hantaviruses are rna viruses belonging to the very large bunyaviridae family. they differ from other members of this family by not being arthropod-borne. each hantavirus is antigenically distinct and maintained within single or at most a few rodent or insectivore hosts, but are infectious for other hosts. infection is lifelong, and virus is transmitted by shedding of virus in urine, feces, and saliva. several hantaviruses are zoonotic and may cause severe disease in humans. although there is overlap, hantaviruses in asia and europe cause hemorrhagic fever with renal syndrome (hfrs) in humans, a multisystem disease with significant renal involvement, and hantaviruses that are endemic in the americas cause laboratory animal medicine hantavirus pulmonary syndrome (hps) in humans, which is a multisystem disease with pulmonary involvement. among the better-known old world hfrs hantaviruses are hantaan, seoul, puumala, and dobrava-belgrade viruses. sin nombre virus is the best known new world phs hantavirus, among many others. most notably from the perspective of laboratory animal medicine, the norway rat, rattus norvegicus, serves as a reservoir host for hantavirus in the wild, but infection has also been associated with laboratory rats. in addition to being endemic in wild rats in asia, it has been found to be endemic in wild rats in the eastern united states and associated with human cases of hfrs (childs et al., ; leduc et al., ; tsai et al., ) . over cases of hantavirus infection have been transmitted to humans from laboratory rats in japan, belgium, and the united kingdom (desmyter et al., ; kawamata et al., ; lloyd et al., ; umenai et al., ) . m. musculus is not considered to be a primary reservoir host, but hantavirus infection has been documented serologically in conventional and barrier-maintained laboratory mice and rats in korea (won et al., ) , infection of wild m. musculus has been documented in the united states (baek et al., ) , and infection of wild mice in europe has been associated with human exposure (diglisic et al., ) . hantaviruses are difficult to culture in vitro. infection in rodents is subclinical and is detected by serology or rt-pcr. the main research complication from natural infection is the zoonotic risk and potentially subclinical effects on the immune response associated with viral defenses such as cd + t cell (taruishi et al., ) and nk function as demonstrated in human studies (braun et al., ) . the mouse is host to a number of enveloped rna viruses of the family retroviridae, subfamily orthovirinae, including the two type species (and their variants) mouse mammary tumor virus (mmtv) and murine leukemia virus (mlv). these viruses belong to a diverse assemblage of related mobile dna elements that are integrated into the host genome, and collectively termed 'retroelements', which include retrovirus-related elements and nonviral elements. during cell division, retroelements are transcribed into rna, and subsequently reverse-transcribed into dna copies that become integrated into a new location within the genome. this process utilizes reverse transcriptase, which is encoded by the retroelement. over millennia, retroelements have been repeatedly integrated within the genome in large numbers, comprising approximately % of the mammalian genome. various families of mouse retroelements share sequence similarity, despite their random distribution throughout the mouse genome, and the majority of them are truncated, mutated, and methylated to become incapable of infectivity. nevertheless, many of them continue to be mobile within the genome. noninfectious retrovirus-related retroelements include iap, vl , musd, and etn elements. replication-competent retroviruses represent the pinnacle of the retroelement constellation and are best considered as the most evolutionarily recent members. these include mmtv and mlv. mtv and mlv share similar genetic structure, except that the long terminal repeat (ltr) region of the mmtv genome encodes an additional superantigen (sag). both mmtv and mlv include exogenous viruses, which are horizontally transmitted, replication-competent viruses, and endogenous viruses, which are closely related to exogenous viruses, encoded within the mouse genome, and transmitted by mendelian inheritance. exogenous mmtv and mlv exist in wild mouse populations, but have been eliminated from contemporary laboratory mice. however, they may continue to be used experimentally, including bittner mmtv, and gross, friend, moloney, and rauscher mlvs. in particular, mouse colonies may be purposely infected with mmtv for mammary cancer research and are termed 'mmtv-positive', reflecting their exogenous virus status, even though the mice may also carry endogenous mmtv. the genomes of all inbred strains of mice encode one or more (over in some mouse strains) endogenous mmtv loci, the distribution of which is unique to each inbred strain of mouse. most mmtv genomic loci do not encode infectious virus or are transcriptionally inactive, except for mouse strains (dba, c h, grs) that carry mtv or mtv loci. these loci encode infectious virus, which can be visualized as b-type particles by electron microscopy. likewise, all mouse strains carry endogenous mlv loci within their genome, but not all mice carry replication-competent mlv sequences. some endogenous mlvs encode infectious virus, which can be visualized as c-type particles by electron microscopy. mice have often evolved mechanisms to counter the deleterious effects of retroviruses by preventing reentry or replication of virus into other cells. if an endogenous retrovirus is still infectious to other mouse cell targets, it is termed ecotropic, whereas if it is no longer infectious for mouse cells, but can infect cells of other species, it is termed xenotropic. viruses capable of infecting cells of mice as well as other species are termed polytropic. the combinations of endogenous replication-competent mlvs and cell tropism factors are a reflection of selective breeding of mouse strains for susceptibility to various types of cancer. clinical signs mice were originally inbred for specific phenotypes, including mammary tumors and lymphomas. thus, some strains of mice were genetically selected for unique combinations of endogenous mmtv and mlv in concert with susceptibility factors laboratory animal medicine that favored their expression and disease manifestations. in addition, noninfectious retroelements continue to reintegrate randomly within the genome during cell division as retro transposons. these ongoing integrations contribute to genetic drift, spontaneous mutations, and well-recognized mouse strain phenotypes, including the athymic nude allele, the hairless allele, and the rodless retina allele, among others. epizootiology exogenous mmtv and mlv are horizontally transmissible, primarily through the milk of lactating females. endogenous retroviruses and retroelements are inherited through the genome. replicationcompetent endogenous mmtvs and mlvs are also transmissible like their exogenous counterparts, but differ by being integrated within the genome of the mouse. pathology replication-competent mmtv and mlv, regardless of their exogenous or endogenous origin, are usually clinically silent. their ability to cause neoplasia is a reflection of genetic selection for susceptibility factors that are genetically encoded within individual mouse strain genomes. mmtv derives its name from its association with induction of mammary carcinomas in mammary cancer-susceptible strains of mice. mlv is associated with lymphomas, the pattern of which is mouse strain specific. for example, akr mice develop % prevalence of thymic lymphoma between and months of age, whereas aging balb/c mice commonly develop multicentric lymphoma. in these strains of mice, multiple endogenous mlvs are coexpressed in tissues and undergo recombination events that allow them to target and transform cells into neoplasia. despite its name, mmtv can induce lymphomas in some strains of mice, such as sjl mice which develop lymphomas arising from enteric lymphoid tissue and mesenteric lymph nodes. diagnosis exogenous retroviruses have been eliminated from contemporary mouse populations, unless purposely introduced for experimental purposes. because endogenous retroviruses and retroelements are encoded within the genome, and reflect the unique genetic composition of each strain of mouse, they are not targets of diagnostic pursuit. differential diagnosis patterns of some types of neoplasia within individual inbred strains of mice are a reflection of their endogenous retroviral integration. prevention and control exogenous retroviruses have been eliminated from laboratory mice by cesarean rederivation and foster nursing. mmtv-s, the 'bittner agent', continues to be purposely maintained in some mouse breeding populations, but can be eliminated by foster-nursing or other means. caution is advised when re-deriving such mouse colonies for other purposes, as elimination of exogenous mmtv will be an unintended consequence. research complications endogenous retroviruses and retroelements influence the life span of individual strains of mice, and random integrations during cell division can give rise to spontaneous mutations and genetic drift. it is estimated that significant mutations may arise due to mobile retroelement integrations every generations. astroviruses are small, nonenveloped, singlestranded rna viruses that have been associated with human gastroenteritis and detected in association with other enteric pathogens. the viral family astroviridae is split into two genuses: avastrovirus for those astroviruses infecting avians and mamastrovirus for those infecting mammals. astrovirus infection has been detected in research mice (muastv) using metagenomic analyses and appears to have a wide geographical, institutional, and host strain distribution. clinical signs none reported. epizootiology pcr screening has found muastv infection in up to % of a variety of mouse strains housed in vendor and academic facilities in the united states and japan. the virus has been detected most commonly in immunocompromised mice (nsg, nod-scid, nsg- gs, c bl -timp- −/− , and upa-nog), but also in immuncompetent strains (b j, icr, bash , and balb/c). both immunodeficient and immunocompetent mice are susceptible to muastv, but adaptive immunity is required to clear the virus. based on human epidemiology indicating children are at highest risk for infection, the virus may preferentially infect young mice. pathology immunodeficient mice showed no sign of pathology based on histopathology. diagnosis pcr data has indicated that muastv causes a systemic, chronic infection in immunocompromised mice, indicating samples from most tissues will be pcr positive. yokoyama et al. ( ) detected high viral load (up to genome copies) per fecal pellet from immunocompetent mice. differential diagnosis none, in the absence of lesions and clinical disease. prevention and control because immunocompetent mice clear the infection, quarantine may be successful but lack of routine screening for muastv in laboratory mice will allow for uncontrolled spread of the infection. research complications based on limited surveys, muastv may have a high prevalence in laboratory mice. the impact of infection on both innate and adaptive immune responses warrants further investigation to assess the potential for confounding research data. this section briefly describes the etiology, clinical signs, epizootiology, pathology, diagnosis, differential diagnoses, prevention and control, and research complications of the most common bacterial diseases encountered in research colonies of mice. as sequencing technology becomes more available, the number and genus/species classification of bacteria potentially responsible for infections, in particular, opportunistic infections, will grow (benga et al., ) . potential candidates include members of pasteurellacaeae, bordetella hinzii, streptococcus danielae, acinetobacter spp., and others, for which little is currently known about their pathogenic potential. etiology lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, is not a pathogen encountered in research colonies of mice but has been reported to infect wild mice and rats in close contact with infected livestock (collins et al., ) . clinical signs none reported but should consider lawsonia as a differential in necropsy cases with gross or histologic evidence of proliferative lower bowel lesions. epizootiology although mice are experimentally susceptible to infection and develop classic lesions of hyperplastic ileitis and typhlocolitis (murakata et al., ) , susceptibility varied with mouse strain and source of inoculum from rabbits or swine, suggesting important differences in l. intracellularis strains. pathology lawsonia infection may result in hyperplastic ileitis, typhlitis and/or colitis, and hemorrhagic intestines may be noted (percy and barthold, ) . diagnosis lawsonia spp. has been diagnosed using a variety of techniques, including pcr, immunohistochemistry, in situ hybridization, and warthin-starry silver stains. differential diagnosis bacterial infections associated with hyperplastic intestinal epithelium, including c. rodentium and enterohepatic helicobacter species in susceptible (typically immunodeficient) mouse strains. prevention and control species separation from hosts more commonly associated with natural infection (hamsters, ferrets, pigs). research complications none reported. the following section describes infection due to mycoplasma pulmonis and summarizes infections associated with other murine mycoplasmas including m. arthriditis, m. neurolyticum, m. collis, and m. muris. antigenic cross-reactivity among these species, and especially between m. pulmonis and m. arthriditis, mandates that reliable diagnostic strategies incremental to serology (elisa, ifa, mfia) such as culture (often false negative) and pcr be employed to distinguish potentially pathogenic infections. when screening cell lines for opportunistic pathogens, pcr is the most efficient method to discriminate between m. pulmonis and mycplasma contaminants associated with cell culture. etiology m. pulmonis is a pleomorphic, gram-negative bacterium that lacks a cell wall and has a single outer limiting membrane. it causes murine respiratory mycoplasmosis (mrm). clinical signs mice are relatively resistant to florid mrm; thus, subclinical infection is more common. when clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are 'chattering' and dyspnea, due to rhinitis and purulent exudate in nasal passages. otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. experimental inoculation of scid mice has caused systemic infection accompanied by severe arthritis (evengard et al., ) . epizootiology mrm historically was a common infectious disease of mice, but improved housing, husbandry, and health surveillance have reduced its prevalence dramatically. serologic data from a large diagnostic laboratory indicated m. pulmonis infection affects about . % of conventionally housed mouse colonies in the united states and . % in europe (pritchett-corning et al., ) . m. pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. therefore, infection should be considered highly contagious. mice injected with cells harvested from m. pulmonis contaminated cell cultures may develop disease. m. pulmonis can also be transmitted venerally; in utero infection has been demonstrated in rats but not in mice. because transplacental infection occurs in rats, the same route may be possible in mice, particularly immunocompromised strains. concomitant viral pneumonia (sv, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to mrm. m. pulmonis also infects rats, hamsters, guinea pigs, and rabbits. among these species, only rats are significant reservoirs of infection for mice. pathology m. pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. the organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to laboratory animal medicine cell death have also been described. detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. experimental infection of mrm is dose dependent. doses of colony-forming units (cfus) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. additionally, m. pulmonis strains can differ in virulence. survivors of severe infection may develop chronic bronchopneumonia with bronchiectasis and spread infection to other mice. intravenous inoculation of m. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. strains derived from a c bl background appear to be resistant to pathogenic infection, whereas balb/c, c h, dba/ , swr, akr, cba, sjl, and other strains have varying degrees of increased susceptibility (cartner et al., ; lai et al., ) . the initial lesion of mrm is suppurative rhinitis, which may involve the trachea and major airways. early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. syncytia can sometimes be found in nasal passages, in association with purulent exudate (fig. . ) . affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumen. chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (fig. . ) . chronicity also increases the prevalence of bronchiectasis and abcessation. diagnosis accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. clinical signs are variable but can be characteristic when they occur. serology is sensitive but although antibodies do not clear the infection, seroconversion may be weak or take months and may not accurately differentiate between m. pulmonis infection and m. arthriditis infection (cassell et al., ) . therefore samples for culture and pcr of the upper respiratory tract should be obtained to confirm diagnosis. buffered saline or mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. specimens for culture from the genital tract are warranted if this site is suspected. mycoplasma spp. may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. immunohistochemistry should be considered to supplement basic histopathologic examination. immunofluorescence and immunoperoxidase techniques are available to identify mycoplasma antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (brunnert et al., ) . pcr assays for m. pulmonis at veterinary diagnostic laboratories and pcr kits to screen cell culures for mycoplasma are readily available. differential diagnosis mrm must be differentiated from bronchopneumonia associated with ciliaassociated respiratory (car) bacillus. silver stains may reveal car bacilli adherent to the respiratory epithelium. sv also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. other causes of respiratory infection include pvm, corynebacteriosis and, in immunodeficient mice, pneumocystis murina infection. combined infections with known pathogens or secondary opportunists also must be considered. prevention and control mice mount an effective immune response to m. pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (cartner et al., ) . antibodies of various classes are produced locally and systemically, but clearance of the infection has been attributed to innate immune responses (love et al., ; sun et al., ) . there is some evidence that antibody may facilitate phagocytosis of m. pulmonis. t-cell responses, however, appear to exacerbate m. pulmonis in mice, because immunity cannot be transferred with immune cells. in addition, athymic and neonatally thymectomized mice are not more susceptible than immunocompetent mice to m. pulmonis pneumonia. nude and scid mice develop less severe respiratory disease than immunocompetent mice but infection becomes systemic and they may develop suppurative disease in multiple organs and joints (arthritis). host immunity aside, effective control and prevention of mrm depend primarily on maintenance of mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, pcr, and histopathology. cesarean or embryo rederivation may eliminate infection, although vertical transmission may occur in immunocompromised mice. treatment with tetracycline suppresses clinical disease but does not eliminate infection. earlier interest in developing dna-based vaccines against m. pulmonis has not achieved clinical application (lai et al., ) . research complications m. pulmonis can interfere with research by causing clinical disease or death. experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. additionally, affected mice are at greater risk during general anesthesia. m. pulmonis may alter immunological responsiveness. for example, it is mitogenic for t and b lymphocytes and can increase nk cell activity. perhaps one of the most important complications of mycoplasma infection is contamination of cell lines and transplantable tumors. other murine mycoplasmas cell lines are often contaminated with mycoplasma species such as m. arginini, m. hyorhinis, m. orale, or m. fermentans that can distort the results of in vitro assays (garner et al., ) . initial evidence of a contamination is often by pcr evidence of mycoplasma at the genus level when cell lines are pcr screened for opportunistic murine pathogens prior to use in mice. other than m. pulmonis, these mycoplasmas are not normally considered mouse pathogens in immunocompetent mice. in contrast, injection of mycoplasma contaminated cells into immunodeficient mice (e.g., xenografts) may result in clinical disease or confounding effects on immune responses (peterson, ) . mycoplasma contamination of murine embryonic stem cells has adversely affected germline transmission and postnatal health of chimeric progeny (markoullis et al., ) . mycoplasma arthritidis is antigenically related to m. pulmonis. therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. differentiation is important because m. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. mycoplasma collis has been isolated from the genital tract of mice but does not appear to cause natural disease. mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of m. neurolyticum exotoxin. characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. the rolling becomes more constant, but mice occasionally leap or move rapidly. after - h of rolling, animals become comatose and usually die within h. all published reports of rolling disease are associated with experimental inoculation of m. neurolyticum or exotoxin. large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. because animals are frequently inoculated with biological materials by parenteral routes, contamination with m. neurolyticum may induce rolling disease inadvertently. diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. clinical signs must be differentiated from rolling associated with pseudomonas-and p. pneumotropica-caused otitis. m. pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease. hemotropic mycoplasmas ribosomal rna sequencing has reclassified hemobartonella muris and eperythrozoon coccoides as mycoplasma hemomuris and mycoplasma coccoides, respectively (neimark et al., ; percy and barthold, ) . distinct from the mycoplasmas just discussed, these agents are trophic for red blood cells and cause anemia and hemolytic disease. these laboratory animal medicine infections could be encountered in wild mice but are rarely found in research mice. diagnosis is by morphologic assessment of blood smears and pcr. clinical signs mice infected with m. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. hemotropic mycoplasma infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or subclinical infection that can be reactivated by splenectomy. the carrier state may be lifelong. epizootiology the primary natural vector of m. coccoides, historically, is the mouse louse, polyplax serrata. infection was associated with primitive housing and husbandry conditions that no longer occur in modern vivaria. although the risks for infection have been reduced substantially by modern animal care procedures, m. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. diagnosis splenectomy or inoculation of test material into splenectomized mice is the most sensitive means of detecting m. coccoides infection. these procedures provoke mycoplasmemia, usually within - days. because mycoplasmemia may be transient, blood smears stained by the romanowsky or indirect immunofluorescence procedures of the blood should be prepared every h, beginning at h after splenectomy of index animals or inoculation of test specimens into splenectomized animals to ensure that mycoplasmemia is not missed. prevention and control treatment of m. coccoides infection is not practical. control is based on culling or rederivation of infected stock. if replacement animals are readily available, euthanasia is a more prudent course. suspect biological materials destined for animal inoculation should be screened for mycoplasma contamination by inoculation of splenectomized mice. research complications subclinical infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. conversely, m. coccoides may potentiate coincident viral infections in mice. this effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and ldv. active infection also may suppress interferon production. etiology car bacillus is a slender, gram-negative, non-spore-forming bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of mrm (see chapter ). clinical signs chronic respiratory disease has been produced in mice by experimental inoculation, but natural clinical disease is rare (griffith et al., ; pritchett-corning et al., ) . furthermore, putative natural cases were reported in mice that were seropositive for sv and pneumonia virus of mice. therefore, car bacillus may exacerbate respiratory disease as an opportunist rather than as a primary pathogen. on balance, it is assumed that mice contract natural infection, but attributing severe chronic respiratory disease in mice solely to car bacillus should be supported by screening for other respiratory pathogens. epizootiology car bacillus is transmitted by direct contact; dirty bedding transfer to sentinel mice may not reflect colony infection status. pathology lung lesions are typically mild in mice and are similar to respiratory mycoplasmosis. uncomplicated car bacillus infection results in peribronchiole cuffing with lymphocytes and plasma cells. severe bronchiolitis and pneumonia are possible (fig. . ) . fatal bronchopneumonia was reported in ob/ob mice (griffith et al., ) . diagnosis an elisa for serological screening is routinely used; pcr and histology are used for definitive diagnosis. in active infection, histologic assessment using warthin-starry or similar stains will reveal argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium along with the presence of peribronchial lymphocytes (fig. . ) . alternatively, immunohistochemistry assays have also been used successfully to detect infection. recovery of car bacillus requires cell culture or culture in embryonated eggs. differential diagnosis respiratory mycoplasmosis, bordetella (avium, hinzii). prevention and control given car bacillus does not form spores, disinfection of the environment should be effective. treatment using sulfamerazine ( mg/l) in drinking water may eradicate infection (matsushita and suzuki, ) but culling or embryo rederivation is recommended. research complications infection is most often subclinical, but like other infectious agents for mice, may confound studies particularly when mice are immunocompromised (griffith et al., ) . etiology the causative agent of transmissible murine colonic hyperplasia, c. rodentium (formerly citrobacter freundii strain ), is a nonmotile, gramnegative rod that ferments lactose but does not utilize citrate or does so marginally (barthold, ; schauer et al., ) . clinical signs c. rodentium infection can be a selflimiting colitis with sterilizing immunity or lead to severe colitis with life-threatening dehydration. clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (barthold et al., ) . epizootiology c. rodentium is not detected in the gastrointestinal flora of normal mice, and therefore, there is not a carrier state. it is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. c. rodentium shares several pathogenic mechanisms, such as attaching and effacing lesions mediated by the intimin receptor, with select escherichia coli (reviewed in collins et al. ( ) ). c. rodentium is used experimentally to model colitis caused by enteropathogenic (epec) and enterohemorrhagic e. coli (ehec) in humans (mallick et al., ; collins et al., ) . host genotype can influence the course and severity of disease (barthold et al., ) . for example, dba, nih swiss, and c bl mice are relatively resistant to mortality, whereas c h/hej mice are relatively susceptible both as sucklings and as adults. interestingly, c bl mice obtained from different commercial sources have varying susceptibility to c. rodentium (ostensibly due to the presence or absence of segmented filamentous bacteria). diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. pathology c. rodentium attaches to the mucosa of the descending colon and displaces the normal flora. attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (schauer and falkow, ; newman et al., ) . colonization results in prominent mucosal hyperplasia, by unknown mechanisms. the characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for - weeks in surviving animals ( fig. . ) . affected colon segments are rigid and either are empty or contain semiformed feces. histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (fig. . ). lesions subside after several weeks. intestinal repair is rapid and complete in adults but slower in sucklings. diagnosis diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of c. rodentium from the gastrointestinal tract or feces. the organism can be cultured on macconkey's agar during early phases of infection, whereas the intestine may be free of c. rodentium during later stages of the disease. c. rodentium also can be detected by molecular hybridization (schauer et al., ) . barthold et al. ( ) . diagnosis transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, salmonella, c. piliforme, and helicobacter spp. prevention and control some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (barthold, ; silverman et al., ) . because c. rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. additionally, the employment of microbarrier caging can reduce transmission. surveillance for c. rodentium should be incorporated into quality-assurance programs, and the organism screened for during quarantine of incoming mice from atypical sources. research complications the potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (barthold and beck, ) . c. rodentium infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in t-cell receptor transgenic mice (maggio-price et al., ) . immunocompromised mice infected with c. rodentium will die from sepsis. etiology pseudomonas aeruginosa is a motile, gramnegative rod. clinical signs p. aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (brownstein, ) . p. aeruginosa can, e.g., cause severe or lethal infections in athymic and scid mice. sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. immunosuppressed mice may also develop gastrointestinal ulcers. generalized infection is associated with severe leukopenia, especially neutropenia. neurologic signs are rare, but there are reports of central nervous system infection. chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis. epizootiology p. aeruginosa is not considered a component of the normal flora. however, it is an opportunist that inhabits moist, warm environments such as water and skin. once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. pathology pathogenic infection is most common in immunodeficient mice. organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. bacteremia is followed by necrosis or abscess formation in the liver, spleen, or other tissues. if otitis media occurs, the tympanic bullae may contain green suppurative exudate. the bowel may be distended with fluid, and gastrointestinal ulceration has been reported. diagnosis infection is diagnosed on the basis of history (e.g., immune dysfunction or recent immunosuppression), clinical signs, lesions, and isolation of p. aeruginosa from affected mice. carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for - h and then culturing the sipper tubes. p. aeruginosa can also be cultured from feces. differential diagnosis pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. these include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and tyzzer's disease. prevention and control infection can be prevented by acidification or hyperchlorination of the drinking water (homberger et al., ) . these procedures will not, however, eliminate established infections. entry of infected animals can be prevented by surveillance of commercially procured colonies. maintenance of pseudomonas-free animals usually requires barrierquality housing and husbandry. p. aeruginosa has a long history in the literature of antibiotic resistance and resistance to quaternary amine disinfectants. research complications p. aeruginosa infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. viral infections that alter host defense mechanisms, such as mcmv may enhance susceptibility to pseudomoniasis. (lindsey et al., a; percy and barthold, ) etiology pasteurella pneumotropica is a short, gramnegative rod. clinical signs many early observations concerning the pathogenicity of p. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. infection is usually subclinical. therefore, p. pneumotropica is most properly viewed as an opportunistic pathogen. studies of experimental p. pneumotropica suggest that it may complicate pneumonias due to mycoplasma pulmonis or sv. it has also been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. epizootiology p. pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. litters from infected dams can become infected during the first week after birth. pathology infections can cause suppurative inflammation, which may include abcessation. dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to p. pneumotropica. preputial and orbital abscesses also occur, especially in athymic mice (fig. . ). its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. cutaneous lesions can occur without systemic disease. they include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk. diagnosis diagnosis requires isolation of the organism on standard bacteriological media. although infection can be detected serologically by elisa (wullenweber-schmidt et al., ; boot et al., a, b) , subclinical carriers often do not seroconvert. pcr assays also are available (dole et al., ) and have shown that p. pneumotropica did not transmit from infected mice to contact or dirty bedding sentinels (ouellet et al., ; dole et al., ) . differential diagnosis suppurative lesions in mice may be caused by other bacteria, including staphylococcus, streptococcus, corynebacterium, klebsiella, and mycoplasma. treatment antibiotic sensitivity testing in vitro indicated p. pneumotropica was significantly more sensitive than p. aeruginosa to enrofloxacin (sasaki et al., ) . enrofloxacin in the drinking water at mg/kg daily for days eliminated clinical signs and infection in a closed breeding colonic of transgenic mice and after days of treatment there were no detectable carriers when the colony was screened weeks later (matsumiya and lavoie, ) . prevention and control because p. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. achieving this goal will normally require barrier housing supported by sound microbiological monitoring. rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. research complications clinically severe infection in immunodeficient mice is the major complication. although clinically silent, experimental evidence has shown that p. pneumotropica infection in immunocompetent mice (c bl/ ) stimulated transcription of multiple proinflammatory cytokines for at least days with residual elevation detectable days later (patten et al., ) . pioneering studies conducted in the s first linked a novel microaerobic bacterium, helicobacter hepaticus, with chronic active hepatitis and hepatic tumors in a/jcr mice (fox et al., , ward et al., ) . the organism could be visualized by electron microscopy in the bile canaliculi of the liver in susceptible mouse strains (fig. . ). subsequently, it was associated with inflammatory bowel disease in several murine models (table . ) which were further developed to examine the role of immune cell subsets, such as t regulatory cells, in the pathogenesis of inflammatory bowel disease (ibd) and colon cancer (fig. . ). helicobacteriosis is laboratory animal medicine now appreciated to be a common infection of laboratory mice. it is caused by a growing list of helicobacter spp. that vary in clinical, pathologic, and epidemiologic significance (whary and fox, ; fox et al., ) . because recognition and investigation of helicobacteriosis continues to evolve, many important questions about the impact of this infection on mice remain unresolved. h. hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic helicobacter spp. (ehs) (fox et al., , ward et al., ; suerbaum et al., ) . however, current information about other murine helicobacters is summarized in the concluding section. etiology helicobacter spp. are gram-negative, microaerophilic, curved to spiral-shaped organisms that have been isolated from the gastrointestinal mucosa of many mammals, including humans and mice whary and fox, ) . to date, the genus includes formally named helicobacter spp. assigned on the basis of s rrna analysis, complemented by biochemical, molecular, and morphological characteristics. the organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere ( % co , % n , % h ). there are currently formally named helicobacter species have been isolated from laboratory mice, as well as several other novel helicobacter spp. awaiting formal naming. species isolated from mice include h. hepaticus, h. bilis (which also infects rats), h. muridarum, h. rappini, and h. rodentium, h. ganmani, h. mastomyrinus, h. magdeburgensis, and h. typhlonius , each of which cahill et al. ( ) tcrα, β mutants defective t-receptors typhlocolitis chin et al. ( ) scid icr-defined flora b t-and b-cell deficient typhlocolitis shomer et al. ( ), shomer et al. ( c bl/il- −/−c lacks il- typhlocolitis burich et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) c blrag mice infected with h. bilis also developed ibd (shomer et al., ) . c ibd also developed in c bl/il- −/− mice experimentally infected with a novel urease-negative helicobacter spp. now named h. typhlonius (franklin et al., ) ; also ibd produced with h. trogontum (whary et al., ) and h. cinaedi (shen et al., ) . d h. bilis produces ibd (maggio-price et al., and colon cancer (maggio-price et al., ) . have been formally named (except for h. rappini) (fox and lee, ; franklin et al., ; whary and fox, ) . most recently, helicobacter pullorum, a human pathogen, has been isolated from commercial, barriermaintained mice (boutin et al., ) . these ehs are most commonly urease-, catalase-, and oxidase-positive. however, h. rodentium, h. typhlonicus, and another novel helicobacter sp. are urease-negative. clinical signs helicobacteriosis in adult immunocompetent mice is usually asymptomatic. liver enzymes are elevated in h. hepaticus-infected a/jcr mice (fox et al., a) . infection of immune-dysregulated mice with h. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea (miller et al., ) . epizootiology recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (shames et al., ; fox et al., b; taylor et al., ; lofgren et al., ) . furthermore, h. hepaticus (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. these results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. pathology helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. h. hepaticus and h. bilis, e.g., can cause inflammation in the gastrointestinal tract, which is expressed as ibd and colon cancer in immunodeficient mice or typhlitis in a/jcr mice infected with h. hepaticus (ward et al., ; knutson et al., ; shomer et al., ; erdman et al., b; nguyen et al., ) . thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, proctitis, and lower bowel carcinoma. these lesions can occur without coincident hepatitis. indeed, helicobacter spp. induced ibd and colon carcinoma are increasingly popular models to study pathogenesis of the disease in humans (table . ). helicobacter spp. also can cause liver disease. bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis. it is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis (fig. . ) . inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. additionally, phlebitis may affect central veins. this lesion has been linked to the presence of organisms in bile canaliculi by silver stains and electron microscopy. age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (fox et al., a) . this lesion may laboratory animal medicine increase susceptibility to hepatomas and hepatocellular carcinomas among aged male a/jcr and b c f mice from infected colonies. an increased incidence of hepatic hemangiosarcoma also has been noted in h. hepaticusinfected male b c f mice. in this context, a/jcr, c h/ hencr, and sjl/ncr mice are susceptible to hepatitis, whereas c bl/ mice are resistant (ward et al., ) . the finding of severe liver disease and tumor induction in b c f mice infected with h. hepaticus infers that genetic susceptibility to h. hepaticus-induced neoplasia has a dominant pattern of inheritance. studies with h. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (hailey et al., ; fox and lee, ; ihrig et al., ; franklin, ; hillhouse et al., ) . diagnosis rapid generic diagnosis can be accomplished by pcr detection of the highly conserved s rrna region of the helicobacter genome in feces or tissues, using suitable oligonucleotide primers (fox et al., a; shames et al., ; beckwith et al., ) . however, genus-specific pcr does not differentiate among different helicobacter spp. molecular speciation can be accomplished by s rrna sequencing, restriction fragment length polymorphism analysis of the pcr product or use of species-specific pcr assays. this procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. an igg elisa using the outer membrane protein as the antigen has been proposed for serological diagnosis, but shared antigens among ehs create lack of specificity for the assay. as noted above, helicobacters can be isolated on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically, and by helicobacter species-specific pcr. isolation of h. hepaticus and from other helicobacter spp. with spiral to curved morphology from feces should be preceded by passing slurried samples through a . -μm filter before plating. if infection with larger fusiform helicobacters (h. bilis, h. rappini) is suspected, filtration at . μm is preferred. helicobacters grow slowly and require prolonged incubation of cultures (up to weeks) before they can be deemed negative. signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (whary and fox, ) . differential diagnosis clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. coronavirus infection, clostridium piliforme, and salmonella spp. can cause enterocolitis and/or hepatitis. c. rodentium also causes colonic hyperplasia, which can present as rectal prolapse. infections caused by other helicobacters of mice h. bilis has been isolated from the livers and intestines of aged mice and experimentally induces ibd in scid mice as does h. hepaticus. h. bilis also experimentally produces lower bowel cancer in immunocompromised mice (nguyen et al., ) . helicobacter muridarum colonizes the ileum, cecum, and colon. it appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. h. 'rappini' has been isolated from the feces of mice without clinical signs. h. rodentium also colonizes the intestine and may be a component of normal flora. a dual infection of h. bilis and h. rodentium was noted in a natural outbreak of ibd in immunocompromised mice (shomer et al., ) . a novel urease negative helicobacter, which has been named h. typhlonius, causes ibd in il- −/− and scid mice (franklin et al., (franklin et al., , fox et al., ) . decreased reproductive efficiency has been reported in il knockout mice infected with h. rodentium and/or h. typhlonius (sharp et al., ) . prevention and control eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. the best results have been obtained by triple therapy with amoxicillin, metronidazole, and bismuth given for weeks (del carmen martino-cardona et al., ) . this strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated h. hepaticus from naturally infected mice. antibiotic impregnated wafers have been used to eradicate helicobacter spp. in mouse colonies (kerton and warden, ) . wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. careful husbandry procedures can limit infection within a colony (whary et al., ) . therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. in contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. this condition could occur during simultaneous infection with two or more helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and helicobacter spp. if highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. research complications chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. additionally, it may impede the development and assessment of noninfectious disease models, such as ibd models in mice with targeted deletions in t-lymphocyte receptors (fox et al., ) . h. hepaticus infections provoke a strong th proinflammatory response, which may perturb other immunological responses. h. hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in a/ jcr, b c f , ab f , b af , and carko mice (hailey et al., ; fox et al., a; garcia et al., garcia et al., , h. salmonellosis (ganaway, ; lindsey et al., etiology the genus salmonella contains two species, s. bongori which infects mainly poikilotherms and rarely, humans, and s. enterica which includes approximately serovars and are a major cause of food-borne illness in humans (fookes et al., ) . the salmonella of historical importance in mice that are now rare include s. enterica subsp. enterica serovar typhimurium (aka s. typhimurium) and serovar enteritidis (s. enteritidis). s. enteritidis is a motile, gram-negative rod that rarely ferments lactose. the genomes of many strains have been sequenced. virulence factors carried on pathogenicity islands and plasmids include antimicrobial resistance genes, type iii secretion systems, vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and factors essential for a intracellular life cycle in macrophages (de jong et al., ) . pathogenassociated molecular patterns (pamps) unique to salmonella interact with tlrs and nod-like receptors (nlrs) which recruit neutrophils and macrophages leading to inflammasome formation and release of pro-inflammatory il- , il- β, tnf-α, and ifn-γ. clinical signs acute infection is especially severe in young mice (casebolt and schoeb, ) . it is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. gastroenteritis is a common sign, but feces may remain formed. subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. chronic disease is expressed as anorexia and weight loss. enzootic salmonellosis in a breeding colony can produce episodic disease with alternating periods of quiescence and high mortality. the latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. epizootiology s. typhimurium is commonly used experimentally and cross-contamination in a mouse facility is a risk. modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. however, the organisms are widespread in nature. therefore, cross-infection from other species or from feral mice remains a potential hazard. salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. infection occurs primarily by ingestion. salmonella have a broad host range and vermin, birds, feral rodents, and human carriers are potential sources of infection. other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. conversely, murine salmonellosis presents a zoonotic hazard to humans. the induction and course of infection are influenced by the virulence and dose of the organism, route of infection, host sex and genetic factors, nutrition, and intercurrent disease. suckling and weanling mice are more susceptible to disease than mature mice. immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. nutritional iron deficiency has an attenuating effect on salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. resistance to natural infection is increased by the presence of normal gastrointestinal microflora. resistance to infection also can be an inherited trait among inbred strains. among the most important considerations is that mice that recover from acute infection can become subclinical carriers and a chronic source of contamination from fecal shedding. pathology the virulence of s. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. organisms reach peyer's patches within h after inoculation and spread quickly to the mesenteric lymph nodes. bacteremia results in spread to other lymph nodes, spleen, and liver within several days. in chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. bacteria reaching the intestine can reinvade the mucosa and can be shed intermittently in the feces for months. s. enteritidis infection also has been associated with chronic arthritis. acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. if mice survive for up to several laboratory animal medicine weeks, the intestine may be distended and reddened, whereas the liver and spleen are enlarged and contain yellow-gray foci of necrosis. affected lymph nodes are also enlarged, red, and focally necrotic. focal inflammation can develop in many organs, including the myocardium (percy and barthold, ) . histologic lesions reflect the course of disease and the number of bacteria in affected tissues. during acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. thrombosis from septic venous embolism may occur, especially in the liver. granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver. diagnosis diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. in mice with systemic disease, bacteria may persist in the liver and spleen for weeks. during acute stages, bacteria can also be isolated from the blood. subclinically infected animals can be detected by fecal culture using selective enrichment media (selenite f broth plus cystine followed by streaking on brilliant green agar). culture of the mesenteric lymph nodes may be more reliable, because fecal shedding can be intermittent. isolates can be speciated with commercial serotyping reagents. alternatively, isolates can be sent to a reference laboratory for confirmation. antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. however, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera. pcr-based assays are also available. differential diagnosis salmonellosis must be differentiated from other bacterial diseases, including tyzzer's disease, helicobacter spp., pseudomoniasis, corynebacteriosis, c. rodentium, and pasteurellosis. viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. among noninfectious conditions, mesenteric lymphadenopathy is an agingassociated lesion in mice and is not indicative of chronic salmonellosis. prevention and control salmonellosis can be prevented by proper husbandry and sanitation. contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. diets should be cultured periodically to check for inadvertent contamination. contaminated colonies should be replaced to eliminate infection and its zoonotic potential. research complications apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. this includes transmission among laboratory species, but especially between mice and the personnel working with them. i. streptobacillosis (lindsey et al., e; percy and barthold, ) etiology streptobacillus moniliformis is a nonmotile, gram-negative, pleomorphic rod that can exist as a nonpathogenic l-phase variant in vivo. however, it can revert to the virulent bacillus form. clinical signs streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. the arthritis can leave joints deformed and ankylosed. hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. breeding mice may have stillbirths or abortions. epizootiology streptobacillosis has historical importance as a disease of rats and mice, but modern husbandry, production, and health surveillance strategies have reduced its impact dramatically (wullenweber, ) . subclinical, persistently infected rats are the most likely source of dissemination to mice, but mouse-tomouse transmission then ensues. transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. s. moniliformis is also pathogenic for humans, causing rat bite fever (haverhill fever). pathology during acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in the liver, spleen, and lymph nodes. histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints. diagnosis diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism on blood agar. the organism has been recovered from joint fluid as long as months after infection. isolation from chronic lesions requires serumenriched medium. s. moniliformis as a cause of septic joints in humans has been diagnosed using pcr and electrospray-ionization followed by mass spectrometry (mackey et al., ) . differential diagnosis clinical signs must be differentiated from septicemic conditions, including mousepox, tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. prevention and control control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. bacterins and antibiotic therapy are not adequately effective. the potential laboratory animal medicine for cross-infection is a reason not to house rats and mice in the same room. research complications infection can be disabling or lethal in mice and has zoonotic potential for humans. j. corynebacteriosis (lindsey et al., ; weisbroth, ; percy and barthold, ) etiology corynebacteria are short gram-positive rods. corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (clifford et al., ; scanziani et al., ; dole et al., ) . clinical signs c. kutscheri infection is often subclinical in otherwise healthy mice. active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. clinical signs include inappetence, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. c. bovis infection causes hyperkeratotic dermatitis characterized by scaly skin, which is accompanied by alopecia in haired mice. severe infection may cause death. corynebacterial keratoconjunctivitis has been reported in aged c bl/ mice (mcwilliams et al., ) . epizootiology subclinically infected animals harbor c. kutscheri in the upper alimentary tract, colon, respiratory tract, regional lymph nodes, middle ear, and preputial gland. c. bovis colonizes skin and is shed in feces. therefore, transmission is by direct contact, fecaloral contact, and aerosol. resistance to infection appears to be under genetic control in some mouse strains. rats are susceptible to c. kutscheri, so cross-infection to mice may occur. pathology lesions caused by c. kutscheri develop from hematogenous spread to various internal organs and appear as gray-white nodules in the kidney, liver, lung, and other sites. cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. septic, necrotic lesions often contain caseous material or liquefied exudate. histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. colonies of gram-positive organisms with 'chinese letter' configurations can usually be demonstrated using tissue gram stains of caseous lesions. mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing pan arthritis. c. kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. subcutaneous abscesses have also been reported. hyperkeratotic dermatitis caused by c. bovis is characterized grossly by skin scaliness and alopecia. microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (fig. . ) . hyperkeratosis is typically more severe in glabrous athymic mice than in haired mice. organisms can be demonstrated in hyperkeratotic layers by gram stain. diagnosis c. kutscheri is usually diagnosed by culture and tissue gram stains on lesions from clinically apparent cases. agglutination serology is available, and immunofluorescence, immunodiffusion, and elisa tests have been reported (boot et al., a) . pcr of skin swabs or feces is a sensitive and specific method for the detection of c. bovis infection in mice (dole et al., ) . differential diagnosis the caseous nature of c. kutscheri-induced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. because mice can sustain natural infections with mycobacterium avium, histochemical techniques for acidfast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. diffuse scaling dermatitis in athymic nude mice is classic for c. bovis infection; however, in one case report staphylococcus xylosus was instead isolated in high numbers from the skin lesions (russo et al., ) . hyperkeratotic dermatitis caused by laboratory animal medicine c. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. prevention and control c. kutscheri infection occurs sporadically and infected colonies should be culled or rederived into an spf facility as treatment is not curative and control is difficult. c. bovis can be endemic in athymic nude mouse colonies. prevention and control are difficult because both immunocompetent and athymic mice as well as humans can carry c. bovis on the skin and in the upper respiratory system, respectively. c. bovis readily contaminates the environment as aerosolization within a class ii biosafety cabinet was shown to spread the bacterium during cage-change procedures (burr et al., ) . antibiotic treatment has been unrewarding (burr et al., ) research complications corynebacteriosis can cause morbidity and mortality, especially among immunodeficient mice. dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. etiology staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. the two most frequently encountered species are staphylococcus aureus, which can be highly pathogenic, and s. epidermidis, which is generally nonpathogenic. species subtypes are identified by phage typing and biochemistry profiles. pathogenic staphylococci are typically coagulase-positive, although s. xylosus has caused serious infections and is coagulasenegative (gozalo et al., ) . clinical signs staphylococcosis causes suppurative conjunctivitis, periorbital and retroorbital abscesses, preputial adenitis, and pyoderma in mice, particularly in immunocompromised strains such as nude mice. some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. because lesions are often pruritic, scratching causes additional trauma and autoinoculation. staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. these occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. male mice also may develop septic balanoposthitis secondary to penile self-mutilation. retrobulbar abscesses caused by s. aureus are frequently noted in athymic mice. sjl mice, which are nk cell deficient, are prone to necrotic dermatitis on the tail secondary to s. xylosus infection. epizootiology staphylococci are ubiquitous and can be carried on the skin and in the nasopharnyx and gastrointestinal tract. they also can be cultured from cages, room surfaces, and personnel. the prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with staphylococcus spp. c bl/ , c h, dba, and balb/c mice are among the most susceptible strains. age may also influence susceptibility, with young mice being more susceptible than adults. immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (fig. . ). as noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. phage typing can help to determine the source of infection. human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. pathology gross lesions are typified by suppurative, ulcerative and necrotic dermatitis involving the head and neck but may extend to the shoulders and forelegs (percy and barthold, ) . superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, e.g., in the external male genitalia. histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. chronic lesions contain lymphocytes, macrophages, and fibroblasts. deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies. infected athymic mice may develop laboratory animal medicine furunculosis of the muzzle and face accompanied by regional lymphadenitis. diagnosis diagnosis is made by documenting gross and histological lesions, including gram staining of suspect tissues, complemented by isolation of grampositive, coagulase-positive (s. aureus), or coagulasenegative staphylococcus species. differential diagnosis staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. ectoparasitism, fight wounds, and self-mutilation per se should also be considered. prevention, control, and treatment removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. in affected animals, nail trimming can reduce self-inflicted trauma. conditions that facilitate aggressive or self-mutilating behavior should be avoided. research complications staphylococcosis can cause illness and disfigurement in mice. immunodeficient mice are at increased risk. etiology streptococci are ubiquitous commensal gram-positive organisms and in some cases, primary pathogens. pathogenic streptococcal infections in laboratory mice are caused by β-hemolytic organisms in lancefield's group c, but epizootics caused by group a streptococci have occurred, and group g organisms have been isolated occasionally. group d has been reclassified as an enterococcus. alpha-hemolytic streptococci can cause systemic disease in scid mice, and group b streptococcus sp. infection has been reported to cause meningoencephalitis in athymic mice (schenkman et al., ) . additionally, streptococcus dysgalactiae subsp. equisimilis has lancefield group g or c antigens and was isolated from visceral abscesses of immunocompetent mice (greenstein et al., ) . clinical signs cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. epizootiology mice can carry streptococci subclinically in their upper respiratory tracts. lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. pathology systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (percy and barthold, ) . streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. infection with α-hemolytic streptococci can cause inflammatory lesions affecting kidney and heart. diagnosis diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. differential diagnosis streptococcosis must be differentiated from other suppurative infections of mice, including staphylococcosis, pasteurellosis, corynebacteriosis, and pseudomoniasis. prevention and control removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. research complications immunodeficient mice are at increased risk for streptococcosis. etiology e. coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. epizootiology infection is considered nonpathogenic in immunocompetent mice. however, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in scid mice infected with a non-lactose-fermenting e. coli (waggie et al., ; arthur et al., ) . clinical signs affected mice develop lethargy and fecal staining. pathology gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. diagnosis diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting e. coli. differential diagnosis this condition must be differentiated from proliferative and inflammatory intestinal disease caused by lawsonia intracellularis, c. rodentium, or enterotropic mouse hepatitis virus, especially in immunodeficient mice. colibacillosis provides an example of the morbidity associated with a nominally innocuous organism when it affects an immunocompromised host. prevention and control removal of affected animals and disinfection of caging and equipment will limit or reduce transmission. research complications clinical illness may develop in immunodeficient mice. historically, klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. most commercial vendors have excluded it from their barriers. it can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. etiology klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. epizootiology k. oxytoca also is purported to be an etiological agent of antibiotic-associated hemorrhagic colitis (aahc) in adult humans and adolescents. in animals, k. oxytoca has been isolated from apparently healthy sentinel rodents being monitored for pathogens in health surveillance programs and from utero-ovarian infections including suppurative endometritis, salpingitis, perioophoritis, and peritonitis in aged b c f mice (davis et al., ; rao et al., ) . a model of aahc has been developed in rats by administering amoxicillinclavulanate followed by orally infecting rats with a strain of k. oxytoca cultured from a patient with aahc. studies in humans suggest that k. oxytoca exerts its pathogenicity in part through a cytotoxin. recently, authors have showed that several animal isolates of k. oxytoca, including clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on hep- and hela cells, indicating the ability to produce cytotoxin. using mass spectroscopy techniques, they also confirmed tilivalline as the cytotoxin present in animal k. oxytoca strains. tilivalline may serve as a biomarker for k. oxytoca-induced cytotoxicity (darby et al., ) . clinical signs k. oxytoca has been cultured from cases of suppurative otitis media, urogenital tract infections, and pneumonia in c h/hej and nmri-foxn (nu) mice (bleich et al., ) . additionally, k. oxytoca was recently cultured from three breeding colonies of nod. cg-prkdc scid il rg tm wjl /szj (nsg) mice with chronic renal inflammation and ascending urinary tract infections (foreman et al., ) . differential diagnosis other bacterial infections capable of causing suppurative lesions, including staphylococci, streptococci, pasteurella sp., and e. coli, among others are considered a differential diagnosis. research complications morbidity and mortality from spontaneous infections can affect ongoing research. etiology clostridium difficile was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans and currently a considerable cause of morbidity in hospitalized patients who acquire nosocomial infections. in the early s, an increased interest in c. difficile infection (cdi) resulted from the emergence of a hyper-virulent strain (nap /bi/ ) associated with frequent recurrences and more severe clinical disease (abou chakra et al., ; mcfarland, ; kuijper et al., ) . c. difficile has also been implicated in antibioticassociated colitis in syrian hamsters (bartlett et al., ) , guinea pigs (lowe et al., ) , rabbits (thilsted et al., ; ryden et al., ) , prairie dogs (muller et al., ) , ostriches (frazier et al., ) , and horses (diab et al., ) . c. difficile is a rod-shaped strict anaerobe. cycloserinecefoxitin-fructose agar (ccfa) is a commonly used selective medium for c. difficile. cultures are incubated under anaerobic conditions at - °c. when grown on blood agar, c. difficile colonies are nonhemolytic and gray, and have a slightly raised umbonate profile with filamentous edges and a ground-glass appearance. colonies grown on blood agar have fluorescence under ultraviolet light. c. difficile forms acid from glucose and fructose, but is negative on lactose, maltose, and sucrose. two closely related exotoxins, toxin a and toxin b, are produced by c. difficile. recent taxonomic classification support placement of c. difficile and its close relatives within the family peptostreptococcaceae. the authors suggested renaming it peptoclostridium difficile (yutin and galperin, ) . epizootiology it is estimated that c. difficile spores germinate and establish infection less than h after ingestion. spores rapidly transit through the upper gastrointestinal tract and colonize the colon and cecum. spore shedding begins less than h postingestion. when c bl mice were challenged with cfu of c. difficile spores, severe cdi signs developed and all mice were clinically affected by h postchallenge (chen et al., ) . specific methods to control and prevent c. difficile infections in mice have not been described. given the method of transmission of c. difficile and c. perfringens are via ingestion or spores, these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, robust sanitation, and use of autoclaved feed, bedding, cages, and cage accessories. sudden dietary changes should be avoided and antibiotics should be used judiciously to minimize disruption of the normal gut microbiota of mice. diagnosis of c. difficile-associated disease is generally based on detection of cytotoxin using a tissue culture cytotoxicity assay. pcr assays for detection of both c. difficile and its cytotoxins have been developed (eastwood et al., ) . there are no published regimens specifically for the treatment of natural c. difficile infections in mice. oral doses given twice daily of mg vancomycin for days to experimentally infected gnotobiotic mice caused a -to -log decrease in vegetative bacterial cell count and no detectable cytotoxin. bacterial counts and cytotoxin levels returned to previous levels after treatment was discontinued. clinical signs untreated mice are relatively resistant to infection with c. difficile and do not develop fatal infections, although these mice can become asymptomatic carriers that persistently shed low numbers of spores (lawley et al., ) . susceptibility of mice to infection must be induced by disrupting the microbiota through antibiotic treatment. brief exposure to environmental laboratory animal medicine spore contamination is sufficient for transmission of c. difficile to naïve but susceptible mice. the cdi transmission model has been used to demonstrate that clindamycin treatment of asymptomatic carriers of c. difficile can inadvertently trigger the excretion of high levels of spores (lawley et al., ) . a c bl mouse model of recurrence/relapse cdi has been reported (sun et al., ) . the primary bout of cdi induced little or no protective antibody response against c. difficile toxins and mice continued shedding c. difficile spores. antibiotic treatment of surviving mice induced a second episode of diarrhea. a simultaneous reexposure of mice to c. difficile bacteria or spores elicited a full clinical spectrum of cdi similar to that of the primary infection. immunosuppressive agents resulted in more severe and fulminant recurrent disease. vancomycin treatment only delayed disease recurrence; however, neutralizing polysera against both tcda and tcdb completely protected mice against cdi relapse (sun et al., ) . a recent study in c bl mice demonstrated that antibiotic-mediated alteration of the gut microbiome favors a global metabolic profile, and therefore increases susceptibility to c. difficile clinical diseases (theriot et al., ) . c. difficile is not tissue invasive and only toxigenic strains are associated with disease. experimental c. difficile infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria, inflammation, pseudomembrane formation, and death. differential diagnosis c. difficile-induced diarrhea is most often associated with antibiotic treatment. other clostridial diseases in mice must be ruled out as well as other enteric pathogens in mice causing diarrhea and mortality. salmonella spp. and c. rodentium should be considered in the differential diagnosis. etiology clostridium perfringens is associated with a number of diseases in domestic animals and humans. c. perfringens is a nonmotile, rod-shaped, encapsulated, anaerobic bacterium measuring - µm in length and . - . µm in diameter (murray et al., ) . c. perfringens grow rapidly on blood agar, and colonies are smooth, round, and grayish in color, and are surrounded by a double zone of hemolysis. c. perfringens is grouped into five types based on the production and secretion of four major toxins. c. perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. the most significant of these is probably enterotoxin, released with the bacterial spore after cell lysis. epizootiology c. perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. the organism can be a member of the normal microbiota in human and domestic animals. factors that have been associated with the proliferation of the organism of these species include poor husbandry and sudden dietary changes (quinn et al., ) . methods to control and prevent c. perfringens infections have not been evaluated in mice. because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (quinn et al., ) . clinical signs only a few reports in the literature exist describing clinical disease associated with c. perfringens infection in mice (matsushita and matsumoto, ; rozengurt and sanchez) . disease has been observed in mice of both sexes, from to days old, and in female mice of breeding age. clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. sudden death without premonitory signs has also been reported. the toxin types of c. perfringens isolated from these cases were reported to be non-type a (matsushita and matsumoto, ) , type b (rozengurt and sanchez, ) , and type d (clapp and graham, ) . mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent c. perfringens infections. differential diagnosis c. perfringens produces a number of major and minor toxins. different types of the bacterium produce different toxins which account for different disease outcomes. c. perfringens type a is a constituent of the normal microbiota of the intestine of humans and other animal species. bacterial culture should be obtained from live or recently dead animals, and placed in anaerobic transfer medium for transport to a microbiology laboratory and should be cultured soon after their arrival. a presumptive diagnosis for c. perfringens can be based on the presence of large grampositive rods in fecal smears or in histologic sections of intestines (quinn et al., ) . definitive diagnosis is based on toxin identification. mice treated with chlortetracycline hydrochloride in drinking water at a level of mg/l for weeks have eliminated c. perfringens-associated disease (matsushita and matsumoto, ) . penicillin g in the diet or changing the diet has also been reported to be effective in disease remission. c. perfringens treatments in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (marks, ; mcgorum et al., ) . commercially available bacterins for use in mice were not effective in controlling the disease (clapp and graham, ) . research complications clostridia are large, rodshaped, gram-positive anaerobic bacteria. naturally occurring clostridial infection in mice is rare. epizootics of c. perfringens type d infection with high mortality laboratory animal medicine have been reported in a barrier colony where heavy mortality occurred in -to -week-old suckling mice. clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. however, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. c. perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice. clostridium piliforme -tyzzer's disease (fujiwara and ganaway, ; ganaway, ; ganaway et al., ; percy and barthold, ) etiology tyzzer's disease is named for ernest tyzzer, who first described it in a colony of japanese waltzing mice. the causative organism, c. piliforme (formerly bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. it has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, in select cell lines, the yolk sac of embryonated eggs, or hepatocyte cell cultures obtained from mice (ganaway et al., ; kawamura et al., ) . clinical signs clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. although outbreaks can be explosive and mortality is usually high, morbidity varies. additionally, subclinical infections can occur, accompanied by the development of antibodies to c. piliforme. stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, and young age, may predispose mice to tyzzer's disease. susceptibility and resistance also are influenced by host genotype. it has been shown, e.g., that c bl/ mice are more resistant than dba/ mice to tyzzer's disease (waggie et al., ) . resistance to severe infection appears to be due, in part, to b-lymphocyte function. the role of t cells in resistance is not clear, because susceptibility among athymic mice appears to vary (livingston et al., ) . however, the involvement of t cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. depletion of neutrophils or nk cells also increases susceptibility to infection. epizootiology current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species. some strains appear capable of cross-infecting mice, rats, and hamsters, whereas others have a more restricted host range (franklin et al., ) . therefore, the risks for cross-infection depend on the strain causing a given outbreak. although the vegetative form of c. piliforme is unstable, spores can retain infectivity at room temperature for at least year and should be viewed as the primary means of spread. natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. feces-contaminated food and soiled bedding are the most likely sources of environmental contamination. prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. pathology infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. the lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. in the liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. myocardial necrosis is sporadic in natural infection. diagnosis tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (fig. . ) and intestine. they are found more easily during early stages of infection. organisms in tissue sections do not stain well with hematoxylin-eosin stain. silver stains, giemsa stains, or periodic acid-schiff stains are usually required for visualization of the organism. pcr and serologic assays are readily available at diagnostic laboratories. older supplemental procedures included inoculation of cortisonized mice or embryonated eggs laboratory animal medicine with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. differential diagnosis the histological detection of organisms is essential for differentiating tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. it also is important not to misconstrue extracellular rods as c. piliforme. prevention and control barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of tyzzer's disease. if infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. husbandry supplies should be stored in vermin-proof quarters, and the option of heat sterilization of food and bedding should be considered. research complications research complications stem from clinical morbidity and mortality. mice with immune dysfunction are at increased risk. there is recent evidence that infection causes elevations in selected cytokines (van andel et al., ) . etiology two mycobacteria are known to be pathogenic for laboratory mice: mycobacterium avium-intracellulare and m. lepraemurium. both are acid-fast, obligate intracellular bacteria. epizootiology mycobacteria are widespread in water and soil. their presence in laboratory mice would indicate a significant break in husbandry practices. infection with m. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of c bl/ mice . the source of the outbreak was presumed to be drinking water. mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. it is highly unlikely to encounter this infection in a modern, well-managed mouse colony. clinical signs m. avium-intracellulare infection is typically subclinical but mice have developed granulomatous pneumonia . pathology lesions are classically a chronic granulomatous disease with granulomas, langhans giant cells, and concurrent presence of acid-fast bacteria in various organs including the lungs, liver, spleen and lymph nodes. m. lepraemurium may cause alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. disease can lead to death or clinical recovery. gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). lesions can also occur in the lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. the histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epitheloid macrophages (lepra cells) packed with acid-fast bacilli. diagnosis acid-fast bacilli in lesions are the hallmark of presumptive diagnosis of mycobacteriosis. definitive diagnosis results from positive culture which takes days to weeks to rule out or positive pcr assays which are more time-efficient but require associated expertise. differential diagnosis other bacterial species that cause granulomatous lesions in mice. research complications natural infection is very rare. etiology proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts of normal mice (percy and barthold, ) . epizootiology proteus mirabilis colonizes the intestinal tract of most humans and is commonly found in research mice unless specifically excluded. clinical signs clinical disease can occur following stress or induced immunosuppression. immunodeficient mice have a heightened susceptibility to pathogenic infection. pathology proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. infected animals lose weight, develop diarrhea, and die within several weeks. if septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. proteus pyelonephritis is characterized by abscessation and scarring. ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. proteus mirabilis and pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. pulmonary lesions include edema and macrophage activation. septic thrombi can occur, however, in many tissues. diagnosis culture recovery of proteus mirabilis as a predominant or single isolate confirms an opportunistic local or systemic infection. differential diagnosis gram-negative bacterial infections. research complications natural infections are typically isolated cases. etiology leptospirosis remains one of the most common zoonoses transmissible from rodents (desvars et al., ) but is exceedingly rare in laboratory mice. infection with leptospira interrogans serovar ballum has been reported on several occasions (see chapter ). epizootiology leptospira are gram-negative organisms that, after a septicemic phase, establish persistent infection in the renal tubules and are periodically excreted in the urine. clinical signs natural infection is subclinical and causes no significant lesions. experimental infections can result in severe vascular, hepatic and renal lesions dependent on serovar, mouse strain and immunocompetency. diagnosis diagnosis requires isolation of organisms in kidney culture. serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. histologic examination of kidney using silver stains can also be attempted. pcr assays are reliable for preliminary diagnosis. differential diagnosis not applicable in research colonies. research complications persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore, infected mice should be culled. elimination of infection from highly valuable mice requires rederivation. (percy and barthold, ) etiology chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). c. trachomatis causes ocular and urogenital disease in humans. however, at least one strain historically referred to as the 'nigg agent' after clara nigg, is most recently classified as chlamydia muridarum and is used experimentally to model human chlamydia infection. epizootiology mice are susceptible to natural infection and experimental infection with c. trachomatis and chlamydophila psittaci, especially immunodeficient mouse strains. clinical signs natural infections are typically subclinical but persistent. pathology c. muridarum is also known as the 'mouse pneumonitis agent' due to severe acute infection which is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and death in h. mice with more chronic infections may develop progressive emaciation and cyanosis of the ears and tail. experimental infections to model human venereal chlamydia infections will develop hydrosalpinx, cervical, and vaginal infections in female mice and urethritis in male mice. diagnosis chlamydia can be diagnosed by impression smears stained with giemsa or macchiavello stains, cell culture, or inoculation of embryonated eggs. pcr and sequencing can be used to speciate the type of chlamydia. differential diagnosis c. muridarum, c. trachomatis, and c. psittaci are included in the differential diagnosis. research complications chlamydia is a rare spontaneous infection in research mice; its potential significance is low. etiology pneumocystis murina (pm) is a common opportunistic organism of laboratory mice and other mammals. when first described by chagas in , p. carinii was misidentified as trypanosoma cruzi and was considered a protozoan (chagas, ) . it was renamed as a new species, p. carinii, when observed in a rat in (delanoë, p. and delanoë, m. ) . p. carinii, however, has now been grouped taxonomically with the fungi based on dna analysis and the homology of p. murina housekeeping genes with those found in fungi (edman et al., ; stringer et al., ; wakefield et al., ) . these dna studies and apparent differences of host susceptibility prompted a new name, p. jiroveci, for pneumocystis isolated from humans (stringer et al., ; frenkel, ) . p. carinii is now used to name the organism in rats and p. murina, the organism in mice. clinical signs pm infection is subclinical in immunocompetent mice. however, it can be clinically severe in immunodeficient mice, because an adequate complement of functional t lymphocytes is required to suppress infection (roths et al., ; shultz and sidman, ; walzer et al., ; weir et al., ) . b cells have also been shown to be critical to clearance of infection and the mechanism appears only partially related to igg and has a more important role in promoting activation and expansion of t cells (lund et al., ) . b cells may also protect early hematopoietic progenitor activity during systemic responses to pneumocystis infection (hoyt et al., ) . infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. primary signs include dyspnea and hunched posture, which may laboratory animal medicine be accompanied by wasting and scaly skin. severe cases, such as those that occur in advanced disease in scid mice, may be fatal. epizootiology pm is known to infect a number of mammalian hosts, including ferrets, rats, mice, and humans. pm is a ubiquitous organism that is often present as a latent infection. although firm prevalence data are not available, because detection methods are not simple to apply, infection is assumed to be present in mouse colonies unless ruled out by extensive surveillance. although these organisms appear morphologically similar, there are antigenic and genetic differences among p. murina isolated from different hosts (weinberg and durant, ; cushion, ) . furthermore, studies indicate that p. carinii isolated from one host species is unable to survive and replicate after inoculation into a different immunodeficient host species (gigliotti et al., b) . pm infection also occurs in human beings, but transmission between rodents and human beings has not been documented. pm is transmitted by aerosol and establishes persistent, quiescent infection in the lungs of immunocompetent mice. prenatal infection has not been demonstrated. pathology pm is normally not pathogenic but can be activated by intercurrent immunosuppression. activation fills the lung with trophic and cystic forms. gross lesions occur in the lungs, which are often rubbery and fail to deflate (fig. . ). histopathological changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (fig. . ) (roths et al., ) . alveolar spaces may contain vacuolated eosinophilic material and macrophages. special stains are required to visualize pm. silver-based stains reveal round or partially flattened -to -mm cysts in affected parenchyma (fig. . ) . in florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. disease can be especially severe when subclinically infected immunodeficient mice are reconstituted with competent immune cells that subsequently promote pneumonitis. diagnosis respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. pathologic examination of the lung, including silver laboratory animal medicine methenamine staining, is essential to confirm a presumptive clinical diagnosis. past infections of immunocompetent mice also can be detected by elisa (furuta et al., ) . pcr can be used to detect active infection (gigliotti et al., a; reddy et al., ) and is particularly useful for screening immunodeficient mice. differential diagnosis pneumocystosis must be differentiated from viral pneumonias of mice. it is worth noting, in this regard, that pneumonia virus of mice has been shown to accelerate the development of pneumocystosis in scid mice (bray et al., ; roths et al., ) . prevention and control pm infection is a significant disease threat to immunodeficient mice. its widespread distribution strongly suggests that susceptible mice should be protected by microbarrier combined, where possible, with macrobarrier housing. husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of hepa-filtered change stations. infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero. research complications pneumonia in immunodeficient mice is the major complication of pm infection. trichophyton mentagrophytes is the most common fungal agent of mice. however, infection rarely causes clinical disease. clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. invasion of hair shafts is not characteristic. diagnosis depends on effective specimen collection. hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. t. mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. histological sections should be stained with a silver stain or schiff's reagent to reveal organisms. trichophyton also can be cultured on sabouraud agar. plates are incubated at room temperature ( - °c), and growth is observed at - days. ringworm is not easily eradicated from laboratory mice. the use of antifungal agents to treat individual mice is time-consuming, expensive, and variably effective. rederivation is a more prudent course. cages and equipment should be sterilized before reuse. concurrent infection with ectoparasites also must be considered during eradication steps. candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. etiology giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. it inhabits the duodenum of young and adult mice, rats, and hamsters. clinical signs infection is often subclinical, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. additionally, immunodeficient mice may die during heavy infestation. epizootiology the contemporary prevalence of affected mouse colonies is not well documented, but surveys during the s found the rates exceeding %. transmission occurs by the fecal-oral route. crossinfection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (kunstyr et al., ) . c h/he mice are particularly susceptible to giardiasis, whereas balb/c and c bl/ mice are more resistant. additionally, female mice appear to be more resistant to infection than male mice (daniels and belosevic, ) . c bl/ females, e.g., have lower trophozoite burdens and for a shorter interval than male mice. females also shed cysts later than male mice. these differences may be related to a more potent humoral immune response to giardia in female mice. pathology gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. the crypt/villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells. diagnosis diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. ellipsoidal cysts with four nuclei also may be detected in feces. infection also can be detected by serology (daniels and belosevic, ) and by pcr (mahbubani et al., ) . treatment, prevention, and control murine giardiasis can be treated by the addition of . % dimetridazole to drinking water for days. prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. research complications accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. the potential for severe or lethal infection in immunodeficient mice was noted previously. etiology spironucleus muris is an elongated, pearshaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of lieberkühn. it is smaller than giardia muris and lacks an anterior sucking disk. clinical signs s. muris infection is usually subclinical in normal adult mice. it is more pathogenic, however, for young, stressed, or immunocompromised mice (kunstyr et al., ) . additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism. clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. mice at - weeks of age are at notably higher risk for clinically evident infection. they can develop dehydration, hunched posture, abdominal distension, and diarrhea. severe infections can be lethal. epizootiology transmission occurs by the fecaloral route and can occur between hamsters and mice as well as between mice. it does not appear to be transmitted between mice and rats (schagemann et al., ) . the most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded % among domestic mouse colonies in the mid- s. there is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (baker et al., ; brett and cox, ) . pathology gross findings associated with infection include watery, red-brown, gaseous intestinal contents. however, it is essential to rule out primary or coinfection by other organisms before attributing these lesions to spironucleosis. microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. organisms can be visualized more easily with periodic acid-schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and occasional intracryptal inflammatory exudate. diagnosis diagnosis is based on identification of trophozoites in the intestinal tract. they can be distinguished from giardia muris and tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. pcr-based detection also is available (rozario et al., ) . it is not clear whether duodenitis is a primary pathogenic effect of s. muris or represents opportunism secondary to a primary bacterial or viral enteritis. therefore, it is prudent to search for underlying or predisposing infections. treatment, prevention, and control treatment consists of adding . % dimetridazole to drinking water for days, as described for giardiasis. prevention and control require good husbandry and sanitation. research complications as with giardiasis, infection can accelerate enterocytic turnover in the small intestine. there is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. additionally, infected mice also have increased sensitivity to irradiation. such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections. tritrichomoniasis t. muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. no cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. it can be detected by microscopy or by pcr (viscogliosi et al., ) . coccidiosis eimeria falciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. it was common in european mice historically but is seldom observed in the united states. heavy infection may cause diarrhea and catarrhal enteritis. klosiella muris causes renal coccidioisis in wild mice but is rare in laboratory mice. mice are infected by ingestion of sporulated sporocysts. sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. mature schizonts rupture into bowman's capsule to release merozoites into the lumen of renal tubules. merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. infection is usually nonpathogenic and subclinical. gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. destruction of tubular epithelium may impair renal physiology. diagnosis is based on detection of organisms in tissues. prevention and control require proper sanitation and management techniques. there is no effective treatment. cryptosporidiosis cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. it is uncommon in laboratory mice and is only slightly pathogenic. cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (ozkul and aydin, ; taylor et al., ) . athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract. entamoebiasis entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. organisms live in the lumen, where they feed on particles of food and bacteria. they are considered nonpathogenic. encephalitozoonosis encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. infection is extremely rare among laboratory mice. the life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. spore cells are disseminated in the blood to the brain and other sites. infection can last more than year, and spores shed in the urine serve as a source of infection. vertical transmission has not been confirmed in mice. e. cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. organisms proliferate in peritoneal macrophages by asexual binary fission. they have a capsule that accepts giemsa and goodpasture stains but is poorly stained by hematoxylin. fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. in contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. infection is diagnosed by cytological examination of ascitic fluid smears, histopathologic examination of brain tissues stained with goodpasture stain, and elisa serology. no effective treatment has been reported. prevention and control require rigid testing and elimination of infected colonies and cell lines. pcr-based assays may also be useful. toxoplasmosis toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. however, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross-infection. toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. diagnosis is based on elisa serology, histopathology, and pcr. control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore, thorough cleaning of infected environments is required. b. cestodiasis baker, ) etiology hymenolepis (rodentolepis) nana, the dwarf tapeworm, infects mice, rats, and humans although the zoonotic risk has been questioned (macnish et al., ) . adults are extremely small ( - mm) and have eggs with prominent polar filaments and rostellar hooks (fig. . ) . clinical signs young adult mice are most frequently infected. signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. epizootiology the life cycle may be direct or indirect (r. nana is the only cestode known that does not require an intermediate host). the indirect cycle utilizes arthropods as intermediate hosts. liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen - days later. the scolex attaches to the intestinal mucosa, where the worm grows to adult size in weeks. the cycle from ingestion to patency takes - days. pathology cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. inflammation is not a feature of infection. diagnosis infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. r. nana can be differentiated from another species of rodent tapeworm, h. diminuta, by the fact that r. nana has rostellar hooks and eggs with polar filaments. however, h. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. treatment, prevention, and control drugs recommended for treatment and elimination include praziquantel ( . % in the diet for days), albendazole, mebendazole, and thiabendazole. although the benzimidazoles have an excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. the potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. because r. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents (see chapter ). hymenolepis microstoma is found in the bile ducts of rodents and could be confused with r. nana in the mouse. however, the location of the adult as well as the large size of h. microstoma eggs compared with those of r. nana make differential diagnosis relatively simple. the mouse and the rat are intermediate hosts of the cestode taenia taeniaformis. the definitive host is the cat. this parasite should not be found in laboratory mice housed separately from cats. c. nematodiasis (wescott, ) syphacia obvelata (mouse pinworm) infestation etiology syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. the rat, gerbil, and hamster are also occasionally infected. female worms range from . to . mm in length, and male worms are smaller ( . - . mm). eggs are flattened on one side and have pointed ends (fig. . ). the nucleus fills the shell and is frequently at a larval stage when eggs are laid. clinical signs infestation is usually subclinical, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. epizootiology pinworm infestation is one of the most commonly encountered problems in laboratory mice. a national survey revealed that more than % of barrier colonies and about % of conventional colonies were affected (jacoby and lindsey, ; carty, ) . syphacia obvelata infestation can occur unexpectedly in commercial barrier murine colonies, resulting in widespread dissemination of the parasite into academic mouse colonies. the epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. the life-cycle is direct and completed in - days. females deposit their eggs on the skin and hairs of the perianal region. ingested eggs liberate larvae in the small intestine and they migrate to the cecum within h. worms remain in the cecum for - days, where they mature and mate. the females then migrate to the large intestine to deposit their eggs as they leave the host. there is unconfirmed speculation that larvae may reenter the rectum. infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. syphacia infestation often occurs in combination with aspiculuris tetraptera. because the life cycle of syphacia is much shorter than that of aspiculuris, the number of mice that are apt to be infected with s. obvelata is correspondingly greater. there is evidence that resistance to infestation may be mouse strain-specific (derothe et al., ) . pathology gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. diagnosis infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. four-to -weekold mice should be examined because the prevalence is higher in this age group than in older mice. because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. eggs are usually detected by pressing cellophane tape to the perineal area and then to a glass slide that is examined by microscopy. aspiculuris tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of s. obvelata (see below). adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. they are readily observed with the naked eye or with a dissecting microscope. an elisa also is available to detect serum antibodies to s. obvelata somatic antigens (sato et al., ) . pcr assays are increasingly being used to augment traditional diagnostic methods and to discriminate between pinworm species (dole et al., ) . pcr panels for pinworm detection using fecal pellets are available from commercial diagnostic laboratories. treatment, prevention, and control pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (klement et al., ; le blanc et al., ; lipman et al., ; flynn et al., ; wescott, ; zenner, ) . because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (davis et al., ; skopets et al., ; toth et al., ) . fenbendazole diets can be fed with week on/ week off rotation with normal chow although the potential impact on experimental data must be considered (duan et al., ; gadad et al., ; landin et al., ) . prevention of reinfestation requires strict isolation because syphacia eggs become infective as soon as h after they are laid, and they survive for weeks, even in dry conditions. strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. the use of microbarrier cages can reduce the spread of infective eggs. syphacia muris is the common rat pinworm. it can potentially infest mice but is not found in well-managed colonies. it can be differentiated from s. obvelata because s. muris eggs are smaller. treatment is the same as for pinworms of mice. etiology aspiculuris tetraptera is the other major oxyurid of the mouse and may coinfect mice carrying s. obvelata. females are . - . mm long, and males are slightly smaller. the eggs are ellipsoidal (fig. . ) . clinical signs ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for - days. they move to the proximal colon about weeks after infection of the host. because the life cycle is - days longer than in s. obvelata, infestations appear in somewhat older mice; heaviest infestation is expected in - weeks after initial exposure. infection is usually subclinical, but heavy loads can produce signs similar to those discussed for s. obvelata. light to moderate loads do not produce clinical disease. epizootiology as noted under s. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. the life cycle is direct and takes approximately - days. mature females inhabit the large intestine, where they survive from to days and lay their eggs. the eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. they require - days at °c to become infective and can survive for weeks outside the host. pathology see s. obvelata (section iii, a, ,c). diagnosis aspiculuris tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. eggs are not deposited in the perianal area; therefore, cellophane tape techniques are not useful. measures for treatment, prevention, and control are similar to those described for s. obvelata. because a. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult parasites are more amenable to treatment by frequent cage rotations. immune expulsion of parasites and resistance to reinfection are hallmarks of a. tetraptera infection. research complications see s. obvelata (section iii, a, ,c). several species of mites infest laboratory mice. they include myobia musculi, radfordia affinis, myocoptes musculinus, and, less commonly, psorergates simplex. the common murine mites are described below, while less frequently encountered species are listed in table . . these include the mouse mite trichoecius romboutsi, which resembles myocoptes and ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. characteristics of specific infestations are described after a general introductory section. clinical signs mites generally favor the dorsal anterior regions of the body, particularly the top of (jacoby and lindsey, ; carty, ) reported mite infestations in % of colonies. acarids spend their entire lives on the host. populations are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. inherited resistance and susceptibility also affect clinical expression of acariasis. mite populations, e.g., vary widely among different stocks and strains of mice housed under similar conditions. pathology gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often results in secondary bacterial infections. lesions resemble allergic acariasis in other species and are associated with mast cell accumulations in the dermis. diagnosis classic methods of detection include direct observation of the hair and skin of dead or anesthetized mice. hairs are parted with pins or sticks and examined with a dissecting microscope. examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. as the carcass cools, parasites will vacate the pelage and crawl onto the paper. sealed petri dishes can also be used. cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. skin scrapings made with a scalpel blade can be macerated in % koh/glycerin or immersion oil and examined microscopically. this method has the disadvantage of missing highly motile species and low-level populations of slower moving immature forms. it is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. detecting mites in sentinels exposed to dirty bedding from colony animals has been reported to be unreliable (lindstrom et al., ) . thus, pcr assays offered by commercial diagnostic laboratories are increasingly being used to augment traditional diagnostic methods and to test individual animals or equipment using a swabbing technique; samples can be pooled to decrease cost (jensen et al., ) . gross anatomical features facilitate differentiation of intact mites. myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (fig. . ) . myobia and radfordia have a similar elongated profile, with bulges between the legs. myobia has a single tarsal claw on the second pair of legs (fig. . ) , whereas radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (fig. . ) . histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with psorergates simplex infestation. treatment, prevention, and control ivermectin can be used topically, in drinking water or as a medicated feed and often is the first-choice approach for attempting eradication although cost and potential toxicity are concerns. because of potential differences in laboratory animal medicine blood-brain barrier permeability to ivermectin, pilot treatments should be evaluated. for large facilities, ivermectin medicated feed may be an attractive option (ricart arbona et al., ) . for valuable lines of mice, rederivation may be cost-and time-effective. control and prevention programs should be carried out on a colony-wide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. research complications hypersensitivity dermatitis has the potential to confound immunological studies (jungmann et al., ) , especially those involving skin, and has been shown to elevate serum ige (morita et al., ) . heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis. myocoptes musculinus this is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with myobia musculi. the life cycle includes egg, larva, protonymph, tridonymph, and adult stages. eggs hatch in days and are usually attached to the middle third of the hair shaft. the life cycle may range from to days. transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. bedding does not seem to serve as a vector. neonates may become infested within - days of birth, and parasites may live for - days on dead hosts. myocoptes appears to inhabit larger areas of the body than myobia and tends to displace myobia during heavy infestations. it has some predilection for the skin of the inguinal region, abdominal skin, and back, but it will also infest the head and neck. it is a surface dweller that feeds on superficial epidermis. infestation can cause patchy thinning of the hair, alopecia, or erythema. lesions can be pruritic, but ulceration has not been reported. chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis. myobia musculi this is a common mite of laboratory mice. the life cycle of myobia can be completed in days and includes an egg stage, first and second larval stages, protonymph, deutonymph, and adult. eggs attach at the base of hair shafts and hatch in - days. larval forms last about days, followed by nymphal forms on day . adults appear by day and lay eggs within h. myobia are thought to feed on skin secretions and interstitial fluid but not on blood. they are transmitted primarily by contact. mite populations increase during new infestations, followed by a decrease to equilibrium in - weeks. the equilibrated population can be carried in colonies for long periods (up to years). population fluctuations may represent waves of egg hatchings. because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to days. infestation may result in hypersensitivity dermatitis, to which c bl mice are highly susceptible. clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. therefore, lesions can be exacerbated by self-inflicted trauma. radfordia affinis radfordia is thought to be common in laboratory mice, but it closely resembles myobia and may occur as a mixed infestation. therefore, its true prevalence is conjectural. additionally, its life cycle has not been described. it does not appear to cause clinical morbidity. psorergates simplex this species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. transmission is by direct contact. invasion of hair follicles leads to development of cyst-like nodules, which appear as small white nodules in the subcutis. histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. there is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic exam. mesostigmoid mites rarely, blood-sucking ornithonyssus bacoti and laelaps echidnina, normally limited to wild rodents, can also infect laboratory rodent colonies (watson, ; fox, ) . these mites may also transiently bite humans and can transmit zoonotic infections (see chapter ). unlike the more common rodent fur mites, mesostigmoid mites live off the host and can travel a long distance in search of a blood meal. they access research colonies via contaminated supplies or wild rats and mice gaining access to the facility. polyplax serrata, the mouse louse, is encountered in wild mice but no longer is a significant issue in research colonies. eggs are deposited at the base of hair shafts and nymph stages and adults can be found principally on the dorsum. p. serrata causes pruritus with associated dermatitis, anemia and debilitation and historically is the vector for mycoplasma coccoides. amyloidosis is caused by the deposition of insoluble (polymerized), mis-folded amyloid protein fibrils in organs and/or tissues. primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. secondary amyloidosis is associated with antecedent and often chronic inflammation. it results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (falk and skinner, ) . primary amyloidosis is common among aging mice (lipman et al., ) but also may occur in young mice of highly susceptible strains such as a and sjl or somewhat older c bl mice. other strains, such as balb/c and c h are highly resistant to amyloidosis (percy and barthold, ) . secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. it can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. in reactive amyloid a (aa) amyloidosis, serum aa (saa) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. aa amyloid fibrils are abnormal β-sheet-rich forms of the serum precursor saa, with conformational changes that promote fibril formation. similar to prion diseases, recent findings suggest that aa amyloidosis could be transmissible in mice and other species (murakami et al., ) . amyloid fibrils induce a seeding-nucleation process that may lead to development of aa amyloidosis. amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. amyloid appears histologically as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. however, it is birefringent after staining with congo red when viewed with polarized light. deposition patterns vary with mouse strain and amyloid type. although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (fig. . ) . naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at necropsy in some inbred strains of mice. although this condition is usually an incidental finding at necropsy, interference with organ function such as the heart cannot be ruled out if lesions are severe. it occurs in balb/c, c h, and especially dba mice (eaton et al., ; brownstein, ; brunnert et al., ) . it is found in the myocardium of the left ventricle ( fig. . ) , in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. dba mice also can develop mineralization in the tongue and cornea. dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. ectopic mineralization is associated clinically with skin and vascular connective tissue conditions in humans and mouse models have been developed to study metastatic and dystrophic tissue mineralization (li and uitto, ) . pseudoxanthoma elasticum (pxe), a heritable ectopic mineralization disorder in humans, is caused by mutations in the abcc gene. knockout abcc −/− mice model the histopathologic and ultrastructural features of pxe, notably with mineralization of the vibrissae dermal sheath, serving as a biomarker of tissue mineralization (benga et al., ) . other inbred mouse strains, including kk/hlj and s /svimj, also develop vibrissae dermal mineralization and have an snp (rs ) in the abcc gene associated with low levels of abcc protein expression in the liver. dba/ j and c h/hej mice have the same polymorphism and low abcc protein levels; however, these mice only develop tissue mineralization when fed an experimental diet enriched in phosphate and low in magnesium. a reye's-like syndrome has been reported in balb/ cbyj mice (brownstein et al., ) . the etiology is unknown; however, antecedent viral infection may be involved. affected mice rapidly become lethargic and then comatose. they also tend to hyperventilate. high mortality ensues within - h, but some mice may recover. lesions are characterized grossly by swollen, pale liver and kidneys. the major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. hepatic lesions resembling changes in reye's syndrome have been reported in scid mice infected with madv- (pirofski et al., ) . deficiencies (tobin et al., ) vitamin deficiencies in mice have not been thoroughly described. unfortunately, much of the information that does exist reflects work carried out - years ago; thus, the reliability and specificity of some of these syndromes is questionable. vitamin a deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. vitamin a is recognized for its importance in development of the immune system (ross, ) and knockout mouse models have been used to demonstrate genetic polymorphisms in humans that negatively regulate intestinal β-carotene absorption and conversion to retinoids in response to vitamin a requirements for growth and reproduction (von lintig, ) . vitamin e deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. two knockout mouse models of severe vitamin e deficiency were independently developed and lack α-tocopherol transfer protein (α-ttp), a gene that controls plasma and tissue α-tocopherol concentrations by exporting α-tocopherol from the liver. ttpa −/− mice have very low to undetectable levels of α-tocopherol and are infertile. the phenotype includes neuronal degeneration associated with progressive ataxia and age-related behavioral defects (yu and schellhorn, ) . deficiency of b complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption, poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. the gulonolactone oxidase knockout mouse (gulo −/− ) on the c bl/ background requires vitamin c supplementation although the plasma ascorbate concentration of gulo −/− mice fed a vitamin c-deficient diet is maintained at % of wild-type concentrations, suggesting an uncharacterized pathway to generate a small amount of ascorbate (yu and schellhorn, ) . the gulo −/− mouse has become the model of choice in studying the role of vitamin c in complex diseases. vitamin c production has been successfully restored in gulo −/− mice using adenovirus vectors, making it possible to robustly manipulate physiological ascorbate concentrations in an inbred mouse. mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. for example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. infertility has also been associated with this syndrome. mice have an absolute requirement for a dietary source of linoleic and/or arachidonic acid. (sundberg, ; ward, ) the significant syndrome of ulcerative dermatitis (ud) is a common idiopathic skin lesion that causes morbidity and early euthanasia losses in c bl/ and related lines of mice. significant pruritus leads to skin trauma associated with opportunistic bacterial infection and deep dermal ulcerations. initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (fig. . ) . progressive inflammation can be halted, sometimes reversed, by nail trimming and therapy with a wide spectrum of topical or systemic antibiotics, steroids, and other drugs such as vitamin e and aloe, all of which speak to the frustrating search for a primary etiology. treatment should be based on microbiological culture and sensitivity and screening for ectoparasites as hypersensitivity to acariasis has been proposed. seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. the incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. there is some evidence that incidence is related to dietary fat with mice on high fat or ad libitum diets being more susceptible than those on restricted diets (neuhaus et al., ) . ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (kunstyr, ) . this disorder has been described as acute intestinal pseudo-obstruction (ipo) in c bl/ mice free of known pathogens (feinstein et al., ) . lactating mice are either found dead or becoming moribund. segments of the small intestine become distended with fluid contents and histologically there is apoptosis of the villus epithelium of the small intestine and superficial epithelial cells of the large intestine. the enteric nervous system appears morphologically normal but necrotic enterocytes, mucosal erosions, and acute mucosal inflammation are commonly observed. there is no strong evidence for metabolic issues such as hypocalcemia or low blood glucose. the direct cause is unknown but death probably results from sepsis secondary to loss of barrier function reflected in apoptosis of the gut epithelium during peak lactation. environmental variables can affect responses of mice in experimental situations. changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed. mice do not easily acclimatize to sudden and dramatic changes in temperature. therefore, they are susceptible to both hypothermia and hyperthermia. mice also are susceptible to dehydration. poorly functioning automatic watering system valves or water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are a significant cause of husbandry-related morbidity. shipping mice between facilities, irrespective of distance, warrants institutional guidelines to minimize exposure to temperature extremes. reheat coils should be designed to fail in the closed position to avoid overheating holding rooms. ringtail is a condition associated with low relative humidity. clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene ( fig. . ). it should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. corneal opacities can result from acute or chronic keratitis, injury (unilateral) and developmental defects; the latter may occur in combination with inherited microphthalmia in c black mice (koch and gowen, ) . there is some evidence that the buildup of ammonia in mouse cages may contribute to inflammatory keratitis, because it can be controlled by increasing the frequency of cage cleaning. corneal opacities and anterior polar cataracts are a developmental defect in inbred c black mice (pierro and spiggle, ) . corneal opacity may be associated with keratolenticular adhesions involving a persistent epithelial stalk of the lens vesicle, which normally disappears around day of gestation (koch and gowen, ) . typically noted in runted or cachectic mice soon after weaning, malocclusion of the open-rooted, continually growing incisor teeth is an inherited trait expressed as poorly aligned incisors, especially of the lower incisors causing osteomyelitis, soft tissue abscesses, or necrosis in the lips or oral cavity. the incidence of inherited malocclusion varies with mouse strain (petznek et al., ) . malocclusion in older mice may be the result of trauma or oral neoplasia. overgrown molar teeth have been associated with trauma to developing tooth buds. skin lesions can be caused by fighting, tail biting, and overgrooming such as whisker chewing. barbering of facial hair and whiskers in subordinate mice by a dominant cagemate is common and may be solved by removing the dominant, normally haired mouse. hair or whisker chewing (barbering) has long been interpreted to be a manifestation of social dominance. apparent dominant animals retain whiskers, whereas cagemates have 'shaved faces' (fig. . ). chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. once chewing has ceased, many mice regrow previously lost hair in several weeks. both sexes may engage in this activity, and sometimes females may be dominant. barbering of whiskers and fur-plucking behavior in mice has been suggested to model human trichotillomania (compulsive hair plucking) because of similarities including elevated serotonin levels (dufour et al., ) , 'barbers' predominately pluck hair from the scalp and around the eyes and the genitals; the behavior is female biased, and begins during puberty and is impacted by genetic background (garner et al., ) . fighting is more common in male mice and more aggressive in some strains (sjl, fvb, balb/c) with bite wounds typically located on the head, neck, shoulders, perineal area, and tail. often one mouse in the cage is free of lesions and is the likely aggressor. removal of the unaffected male may end the fighting or simply reorder the dominance order. removing males for breeding and then regrouping them often results in fighting. for programs that produce sentinel mice in-house, castration is an option to reduce aggression in group-housed male sentinels (lofgren et al., ) . regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. improperly diluted disinfectants may also cause regional hair loss. ear tags used for identification may cause pruritis and self-induced trauma. hair removal products or clipping prior to imaging or application of experimental compounds to the skin may cause pruritus and can augment lesions that interfere with test results. dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia. (burek et al., ; percy and barthold, ) common idiopathic lesions in aging mice include cardiomyopathy (with or without mineralization or arteritis), chronic nephropathy (frequently with mineralization), myelofibrosis (fibrotic change in the bone marrow) especially in female mice, melanosis in the meninges, ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular or stromal hyperplasia, cystic endometrial hyperplasia, testicular tubular degeneration or mineralization, prostate atypical epithelial hyperplasia, gastric glandular epithelial hyperplasia, pancreatic islet cell hyperplasia, dental dysplasia of incisor teeth, pituitary hyperplasia of pars intermedia and pars distalis, cataracts, increased extramedullary hematopoiesis in spleen, and lymphocytic infiltrates or other inflammatory changes in various tissues, including harderian gland, salivary gland, kidney, liver, gall bladder, nasal, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. lymphoma is also very common . spontaneous atrial thrombosis is rare in mice (< % in -year-old mice) and appears to be strain-related, with a high prevalence in rfm mice. it also is more common in aged mice affected by kidney disease and amyloidosis. organizing thrombi will be found usually in an enlarged, hyperemic left atrium and auricle and may be accompanied by amyloidosis. affected mice may display signs of heart failure, particularly severe dyspnea. induction of atrial thrombosis in b c f mice has been used to assess cardiovascular risk of chemical exposures (yoshizawa et al., ) . myocardial and epicardial mineralization is described above (section iii,b, ). periarteritis, also known as arteritis, polyarteritis, or systemic arteritis, impacts older mice and lesions may be observed in multiple tissues, including the spleen, heart, tongue, uterus, testes, kidney, and urinary bladder. the media of the affected vessels is homogenous and intensely eosinophilic with hematoxylin and eosin stain. fibrosis and mononuclear cells infiltrate the vessel wall. experimental coronary arteritis with cardiac hypertrophy has been model in dba/ and other strains by intraperitoneal administration of mannoprotein-beta-glucan complex isolated from c. albicans (nagi-miura et al., ) . hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. pulmonary histiocytosis, acidophilic macrophage pneumonia, and acidophilic crystalline pneumonia are synonymous morphologic descriptions of an idiopathic lung lesion that can be incidental or the cause of significant morbidity. incidence varies with mouse strain or stock, with c bl, s /svjae and swiss mice and older mice in general particularly susceptible. histologically, alveoli and bronchioles are filled with varying quantities of macrophages containing eosinophilic crystalline material . the crystalline material consists of ym and/or ym chitanases and can be found in other tissues including the upper respiratory tract, stomach, gall bladder, and bone marrow where it is described as hyalinosis (nio et al., ) . gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. gastric ulcers have been induced by cold stress, food restriction (rehm et al., ) , chemical injury (yadav et al., ) , and gastritis and gastric tumors by helicobacter infection (fox et al., ) . germfree mice have reduced muscle tone in the intestinal tract. cecal volvulus is a common cause of death in germfree mice and is caused by rotation of the large, thin-walled cecum. age-associated lesions are common in the livers of mice. cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by months. mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. extramedullary hematopoiesis occurs in young mice and in response to anemia. exocrine pancreatic insufficiency has been reported in cba/j mice. acinar cell atrophy is common but is strain-and sex-dependent. blood-filled mesenteric lymph nodes may occur in aged mice, especially c h mice. this condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. these nodules should not be mistaken for lymphosarcomas. grossly observable black pigmentation in the spleen of c bl/ is normal and is melanosis caused by melanin deposition (weissman, ) . the spleen is subject to amyloidosis and hemosiderin deposition. lipofuscin deposition is common, especially in older mice. the thymus undergoes age-associated atrophy. a variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more importantly, fails to develop a thymus and thus has a severe deficit of t-cellmediated immune function. additionally, scid mice, which lack both t and b lymphocytes, are used widely and are highly susceptible to opportunistic agents such as pneumocystis murina. specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (table . ). age-associated osteoporosis or senile osteodystrophy can occur in some mice. it is not associated with severe renal disease or parathyroid hyperplasia. nearly all strains of mice develop some form of osteoarthrosis. it is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration. glomerulonephritis is a common kidney lesion of mice. it is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. its prevalence in some strains approaches %. nzb and nzb × nzw f hybrid mice, e.g., develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in nzb mice (nzb mice have a high incidence of autoimmune hemolytic anemia). renal changes occur as early as months of age, but clinical signs and severe disease are not present until - months. the disease is associated with wasting and proteinuria, and lesions progress until death intervenes. histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. later, tubular atrophy and proteinaceous casts occur throughout the kidney. immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. mice infected with lcmv or with retroviruses can also develop immune complex glomerulonephritis. mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by pas-positive material that does not stain for amyloid. this lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. advanced cases may lead to renal insufficiency or failure. interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. if renal insufficiency or failure ensues, it can lead to ascites. some strains of mice, such as balb/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. urinary tract obstruction occurs as an acute or chronic condition in male mice. clinical signs usually include wetting of the perineum from incontinence. in severe or chronic cases, wetting predisposes to cellulitis and ulceration. at necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. in chronic cases the urine may be cloudy, and calculi may develop in the bladder. additionally, cystitis, urethritis, prostatitis, laboratory animal medicine balanoposthitis, and hydronephrosis may develop. this condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. hydronephrosis also may occur without urinary tract obstruction. ascending pyelitis occurs in mice secondary to urinary tract infection. parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. amyloidosis is also common in the ovaries of old mice. cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. in some strains, the prevalence in mice older than months is %. endometrial hyperplasia is often associated with ovarian atrophy. mucometra is relatively common in adult female mice. the primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands were described in control b c f mice from national toxicology program -year carcinogenicity and toxicity studies conducted in one of four different laboratories (suwa et al., ) . lymphocytic infiltration, inflammation, edema, epithelial hyperplasia, mucinous cyst, mucinous metaplasia, adenoma, adenocarcinoma, granular cell tumor, and glandular atrophy were variously observed in accessory sex glands. accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. these nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. thyroid cysts lined by stratified squamous epithelium and generally of ultimo-branchial origin may be seen in old mice. amyloid can be deposited in the thyroid and parathyroid glands as well as in the adrenal glands. spontaneous diabetes mellitus occurs in outbred swiss mice and genetic variants of several strains such as nod mice (lemke et al., ) . high levels of estrogen in pregnancy may influence postpartum hair shedding. various endocrine effects on hair growth have also been described. abdominal and thoracic alopecia have been reported in b c f mice. symmetrical mineral deposits commonly occur in the thalamus of aged mice. they may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in a/j mice. lipofuscin accumulates in the neurons of old mice. age-associated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in c bl/ mice. deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. a number of neurologically mutant mice have been described. they commonly have correlative anatomical malformations or inborn errors of metabolism. a seizure syndrome in fvb mice has been described (goelz et al., ) and can be spontaneous or associated with tail tattooing, fur clipping, and fire alarms. mice are most often female with a mean age of . months (range, - months) and can exhibit facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that may progress to tonic convulsions and death. ischemic neuronal necrosis was consistently observed in these mice and is consistent with status epilepticus in humans. unilateral and bilateral microphthalmia and anophthalmia are frequent (as high as %) developmental defects in inbred and congenic strains of c bl mice, especially impacting the right eye and female mice. these conditions may first be recognized due to ocular infections, secondary to inadequate tear drainage. other common findings include central corneal opacities, iridocorneal and corneal-lenticular adhesions, abnormal formation of the iris and ciliary body, cataracts, extrusion of lens cortical material with dispersion throughout the eye, failure of vitreous development, and retinal folding. these syndromes can be reproduced by exposure to alcohol at critical stages of embryogenesis when the optic cup and lens vesicle are developing and impacting normal development of other ocular structures, including the iris, ciliary body, vitreous, and retina . retinal degeneration can occur as either an environmental or a genetic disorder (chang et al., ) in mice. nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. mouse genetics have laboratory animal medicine been shown to be more important than potential light associated tissue injury (serfilippi et al., a) . other strains such as c h, cba, and fvb are genetically predisposed to retinal degeneration because they carry the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (farber and danciger, ) . presence of the rd gene in some mouse strains highlights that impaired vision must be a consideration when selecting strains for behavioral assays that rely on visual clues (garcia et al., ) . blindness does not interfere with health or reproduction and blind mice cannot be distinguished from non-blind mice housed in standard caging. cataracts can occur in old mice and have a higher prevalence in certain mutant strains. vestibular syndrome associated with head tilt, circling, or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and medium-sized arteries in the vicinity of the middle and inner ears. (jones et al., (jones et al., - maronpot et al., ; percy and barthold, ) neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of - %. there are, however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. leukemogenesis in mice may involve viruses and chemical or physical agents. viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family retroviridae (type c oncornaviruses) and contain rna-dependent dna polymerase (reverse transcriptase). these viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genome. although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as akr mice. their phenotypic expression is controlled by mouse genotype. endogenous retroviruses are transmitted vertically through the germ line. horizontal transmission is inefficient but can occur by intrauterine infection or through saliva, sputum, urine, feces, or milk. the leukemia induced by a given endogenous virus is usually of a single histopathological type. loss of function in nucleic acid-recognizing, tlr , tlr , and tlr can result in spontaneous retroviral viremia and acute t-cell lymphoblastic leukemia (yu et al., ) . chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines, and physical agents such as x-irradiation can also induce hematological malignancies in mice. the most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. clinical signs include dyspnea and ocular protrusion. the latter sign is due to compression of venous blood returning from the head. tumor cells spill into the circulation late in disease. most of these tumors originate from t lymphocytes or lymphoblasts, but there are leukemias of b-lymphocyte or null cell lineage. in the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal. reticulum cell sarcomas are common in older mice, especially in inbred strains such as c bl/ and sjl. primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. histiocytic sarcomas correspond to the older dunn classification as type a sarcomas and are composed primarily of reticulum cells. the tumor typically causes splenomegaly and nodular lesions in other organs, including the liver, lung, kidney, and the female reproductive tract. follicular center cell lymphomas correspond to dunn type b sarcomas. they originate from b-cell regions (germinal centers) of peripheral lymphoid tissues, including the spleen, lymph nodes, and peyer's patches. typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. tumors also often contain small lymphocytes. type c reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. they consist of reticulum cells with a prominent component of well-differentiated lymphocytes. myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. disease begins in the spleen, resulting in marked splenomegaly, but leukemic spread results in involvement of many tissues including the liver, lung, and bone marrow. leukemic cells in various stages of differentiation can be found in peripheral blood. in older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. the green hue fades on contact with air. affected mice are often clinically anemic and dyspneic. erythroleukemia is rare in mice. the major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. hepatomegaly can follow, but there is little change in the thymus or lymph nodes. erythroleukemia can be experimentally induced in mice by friend spleen focus-forming virus (sffv) which initially activates the erythropoietin (epo) receptor and the receptor tyrosine kinase sf-stk in erythroid cells, resulting in proliferation, differentiation, and survival. in a second stage, sffv activates the myeloid transcription factor pu. , blocking erythroid cell differentiation, and in conjunction with the loss of p tumor suppressor activity, results in the outgrowth of malignant cells (cmarik and ruscetti, ). mast cell tumors are also very rare in mice. they are found almost exclusively in old mice and grow slowly. they should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or x-irradiation. natural plasma cell tumors are infrequent in the mouse. they can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils, particularly in balb/c mice. mineral oil-induced plasmacytomas in balb/c mice produce large amounts of endogenous retroelements such as ecotropic and polytropic murine leukemia virus and intracisternal a particles. associated inflammation may promote retroelement insertion into cancer genes, thereby promoting tumors (knittel et al., ) . similar to other spontaneous cancers, plasmacytoma development in mice is inhibited by innate immune responses of nk cells which when activated by viruses will release γinf (thirion et al., ) . mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. certain inbred strains of mice, such as c h, a, and dba/ , have a high natural prevalence of mammary tumors. other strains, such as balb/c, c bl, and akr, have a low prevalence. among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. several major variants are known. the primary tumor virus mmtv-s (bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. metastases go primarily to the lung. some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. ovariectomy will dramatically reduce the incidence of mammary tumors in c h mice. if surgery is done in adult mice - months of age, mammary tumors will develop, but at a later age than normal. grossly, mammary tumors may occur anywhere in the mammary chain. they present as one or more firm, welldelineated masses, which are often lobular and maybe cystic (fig. . ) . histologically, mammary tumors have been categorized into three major groups: carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. the carcinomas are divided into adenocarcinoma types a, b, c, y, l, and p. most tumors are type a or b. type a consists of adenomas, tubular carcinomas, and alveolar carcinomas. type b tumors have a variable pattern with both well-differentiated and poorly differentiated regions. they may consist of regular cords or sheets of cells or papillomatous areas. these two types are locally invasive and may metastasize to the lungs. type c tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. type y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. adenocarcinomas with a lacelike morphology (types l and p) are hormone dependent and have a branching tubular structure. the control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally, whereas others are transmitted vertically. although horizontally transmitted virus such as mmtv-s can be determined by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. fortunately, these latter tumor viruses have generally low oncogenicity relative to the bittner virus. mammary tumors are increased in frequency in c bl apc +/− female mice infected with h. hepaticus (rao et al., ) . mice develop an assortment of liver changes as they age, including proliferative lesions which can progress from hyperplastic foci to hepatomas to hepatocellular carcinomas. almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency and h. hepaticus infections in susceptible strains of mice such as the a/jcr male mouse ward et al., ) . the prevalence of spontaneous liver tumors in b c f hybrids is increased by feeding choline-deficient diets or when infected with h. hepaticus (hailey et al., ) . tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic (hoenerhoff et al., ) . spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated darkred masses. they are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to trabecular. large carcinomas also may contain hemorrhage and necrosis. carcinomas also may metastasize to the lungs. primary respiratory tumors of mice occur in relatively high frequency. it has been estimated that more than % of these tumors are pulmonary adenomas that arise either from type pneumocytes or from clara cells lining terminal bronchioles. pulmonary adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. they invade pulmonary parenchyma and are prone to metastasize. the prevalence of spontaneous respiratory tumors is mouse straindependent. for example, the prevalence is high in aging a strain mice but low in aging c bl mice. the number of tumors per lung is also higher in susceptible mice. pulmonary tumors often occur as well-defined gray nodules. microscopically, adenomas of alveolar origin consist of dense ribbons of cuboidal to columnar cells with sparse stroma. adenomas of clara cell origin are usually associated with bronchioles. they have a tubular to papillary architecture consisting of columnar cells with basal nuclei. pulmonary adenocarcinomas, though comparatively rare, are locally invasive. they often form papillary structures and have considerable cellular pleomorphism. given the rapid development of mouse strains genetically predisposed to neoplasia, the mouse tumor biology database maintained by jackson laboratory is a valuable centralized resource for the most current tumor descriptions. the database contains information on more than strains and substrains, tissues and organs, over , tumor frequency records, and nearly histopathological images and descriptions. risk factors for recurrence, complications and mortality in clostridium difficile infection: a systematic review detection of 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astroviruses chemical-induced atrial thrombosis in ntp rodent studies nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors recent applications of engineered animal antioxidant deficiency models in human nutrition and chronic disease dissecting the effects of mtdna variations on complex traits using mouse conplastic strains a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia effective eradication of pinworms (syphacia muris, syphacia obvelata and aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy possible allelic structure of igg a and igg c in mice one-step generation of different immunodeficient mice with multiple gene modifications by crispr/cas mediated genome engineering key: cord- -w h rir authors: nan title: abstracts cont. date: - - journal: clin microbiol infect doi: . /j. - . . c.x sha: doc_id: cord_uid: w h rir nan objectives: in this study we wanted to examine the prevalence of caga, vaca and baba status in helicobacter pylori (hp) isolates from patients with gastritis or peptic ulcer; to compare them and to know if there were any relationships between those virulence factors in each group. methods: gastric biopsy specimens from hp positive patients with peptic ulcer ( cases) and gastritis ( cases) were studied. dna was extracted and pcr performed to detect caga, vaca s / s alleles and baba gene. results: gastritis: in % of strains, the expected caga fragment was amplified by pcr; % carried the s -allele and % the s allele; baba gene was detected in % of strains. peptic ulcer: % of strains were caga+; % were vaca s -allele and % were vaca s -allele; baba gene was detected in % of strains. no significant differences in the prevalence of caga, vaca or baba were found in both groups. neither of them showed relationship between the presence of baba gene and caga gene or vaca s /s -alleles. conclusions: although the risk of developing more serious gastric lesions increased as the number of virulence factor genes are accumulated in a given hp strain, we did not find any significant differences or relationship in the caga, vaca or baba status between the hp isolates from patients with gastritis or peptic ulcer in this study. a low percentage of baba gene was found in both groups. helicobacter pylori cells in clinical and wastewater samples p. piqueres, y. moreno, a. jimenez, j. hernández, m. ferrú s valencia, e introduction: the presence of viable but non-cultivable helicobacter pylori cells in environmental samples may underestimate the importance of this way for its transmission. the determination of resistance to antibiotics in these strains is important to a better understanding of the epidemiology of the infection. objectives: we have evaluated the use of a fluorescent in situ hybridisation (fish) assay directly from biopsies and wastewater to detect h. pylori and simultaneously its macrolide resistance genotype. methods: a total of gastric biopsies samples from ulcerpatients were homogenised in ml of selective broth, and a ll aliquot was used for fish detection. twenty-nine wastewater samples collected from different treatment plants were centrifuged and subsequently fixed with % paraformaldehyde solution for h at c and then washed with % pbs buffer. hpy probe, a s rrna targeted fitc-labelled oligonucleotide sequence was used for the detection of all h. pylori strains. in addition to cla - , a set of three cy -labelled probes was used for the detection of s rrna mutations associated with resistance to clarithromycin. hybridisation was performed with % formamide at c for h. results: fish allowed the detection of h. pylori in out of clinical samples and samples were positive in wastewater. the % of the positive biopsies showed the presence of clarithromycin resistant strains and . % of the positive wastewater samples yielded resistance genotype to this macrolide. by using a double filter set we could observe directly the clarithromicyn resistant h. pylori organisms in the samples and its morphology in the different types of environments. the predominant cells' morphology in both clinical and wastewater samples was of helicoidal form. conclusions: the fish is a specific and rapid culture-independent method to determine directly the presence of clarithromycinresistant h. pylori cells in clinical and environmental samples. results showed the presence of macrolide resistant cells in water and, therefore, water must be considered a potential route of h. pylori transmission. acknowledgement: this work was supported by ministerio españ ol de ciencia y tecnología, project agl - -c - . objectives: helicobacter pylori is a leading cause of various gastrointestinal diseases such as atrophic gastritis and gastroduodenal ulcer. the caga gene product caga is directly injected into the bacteria-attached host cells and deregulates intracellular signalling pathways and thereby initiates pathogenesis. caga gene is located on pathoginicty island but the function of other genes on the island is unknown. the goal of the work was to evaluate the impact of cag island genotype on the outcome of the therapy. materials and methods: three groups of patients each with total number of patients were investigated. first group was taking a typical antibiotic therapy (amoxicillin, claritromycin, rabeprasol), patients in the second group were treated with the same antibiotics together with probiotic laminolact (e. faecium strain l- in the form of bon-bons together with pectin, soy bean amino acids and sea weed), and the third group was taking only laminolact without any antibiotic. the genotype was determined by pcr with dna primers against three h. pylori genes ureb, caga and cagh. cagh was used as a marker cag island integrity and ureb was a marker of h. pylori presence. five different genotypes were determined: ureb+,cagaÀcaghÀ, ure+,cagaÀcagh+, ureb+,caga+cagh+, ureb+,cagaÀcagh+ and ureb+,caga+caghÀ . treatment with antibiotics alone was leading to % of eradication. the best eradication percentage ( %) took place in the second group where classical antibiotic treatment was taken together with probiotics. interestingly, probiotic treatment alone was giving % of eradication. results of the therapy were highly consistent with cag genotype. patients were found to be statistically less susceptible (p < . ) to the therapy in case when the entire cag regulon was present regardless of the therapy used. this fact suggests immunosuppressant function of caga or other proteins encoded by the genes on cag pathogenicity island. conclusion: the effect of h. pylori eradication depends on cag pathogenicity island genotype. probiotics including e. faecium l- might significantly improve the anti-h. pylori treatment. screening of p. aeruginosa isolates. this study was done to determine if the p. aeruginosa strains isolated from the cultures of patients hospitalised in the infectious diseases unit were from an individual strain. this technique was preferred because it is cheap and provides a rapid detection opportunity. methods: samples obtained from the clinical specimens of the patients and from the hands of the staff of the infectious diseases unit were cultured. of the samples, were isolated from blood, six from sputum, from drainage and from the hands of the medical staff. p. aeruginosa identification was made by api ne system. dna was extracted from the culture material by phenol-chloroform extraction method. ap-pcr was performed by using the primer ¢-gtt gcg atcc- ¢, and subjected % page. band patterns were visualised by silver staining. results: in none of the isolates of the hospital staff p. aeruginosa was cultured. out of the clinical samples of the patients, different genotypes were determined. conclusions: the p. aeruginosa strains of the patients were individual strains, neither related to the staff of the department nor to a specific patient. objectives: pseudomonas aeruginosa is a leading cause of nosocomial infections, particularly pneumonia or sepsis, on intensive care units. its high intrinsic antibiotic resistance and the ability to develop multidrug resistance pose, especially for critically ill patients, serious therapeutic problems. since, culture based techniques for pathogen identification and resistance determination requires at least days, a calculated antibiotic therapy may harbour the risk of an increase in antibiotic resistance and therapy failure. therefore, the development of a fast and reliable identification and antimicrobial susceptibility test is essential for the improvement of the therapy. the aim of the present study was to develop an oligonucleotide-array for a quick, genotypic test of antibiotic susceptibility combined with the determination of relevant virulence factors. methods: dna from different clinical specimen was isolated with a modified qiamp dna blood mini kit. template dna was amplified and simultaneously labelled with cy during multiplex pcr. oligonucleotide capture probes ( - mer), containing a poly-t( )-spacer at the ¢-end, were spotted on epoxy-slides to build an array covering regulatory genes of multidrug efflux pumps (mexr, mext, nfxb), alginate synthesis (muca), metallo-beta-lactamases (bla-vim, bla-imp), aminoglycoside modifying enzymes (aac, aad, aph) and virulence factors (exou, exos, exot) . results: of clinical p. aeruginosa isolates could be correctly genotyped. three isolates displayed a hybridisation pattern that could be assigned neither to wild-type nor to known mutations. a sequence analysis of these isolates revealed an unknown mutation in mexr and nfxb. hybridisation with dna from other non-fermenter or enterobacteriacea showed no crossreactivity. genotypic resistance profile of p. aeruginosa deduced from the array data correlated fully with the susceptibility pattern obtained by standard tests. the sensitivity of the array was genome equivalent even with an exp -fold excess of non-pseudomonas dna. the whole analysis, including dna processing, array hybridisation and data evaluation could be performed in less than h. conclusions: due to the good correlation with standard procedures, the pseudomonas-array may be used for a rapid susceptibility test even directly from clinical samples. combined analysis of antibiotic resistance and virulence factors may improve the outcome of an antimicrobial therapy. objectives: because of a high prevalence of pseudomonas aeruginosa infections in cystic fibrosis (cf) patients, we conducted a study to assess p. aeruginosa isolates collected over years from the sputa of cf adult patients attending an italian cf centre. some phenotypic characters of bacteria (o-serotype, motility, production of enzymes and resistance to antibiotics) and their pfge genotypic patterns were evaluated to analyse for the presence of epidemic strains. moreover some sequential isolates collected from cf patients were investigated to look for the chronicisation of the infection. the strains were identified biochemically. the o-serotype was determined by slide agglutination; the production of enzymes (protease, elastase, gelatinase, haemolysin, betalactamase) and motility were detected using specific techniques. the antibiotic susceptibility was analysed by the vitek ams system and disc diffusion method. pfge was used to discriminate the genotypes of p. aeruginosa. results: in our hands, o serotyping failed to identify . % of isolates, considering the bacteria collected at the onset of colonisation; the most frequent serotypes were o: , o: and o: . moreover, the percentages of protease, haemolysin, gelatinase and elastase production were respectively . , . , . and . , whereas . % of the microorganisms were non-motile. pfge allowed the typing of all strains except one. the heterogeneity of isolates indicated that cross-infection is unusual; we also observed in several strains isolated in the last years a predominant pattern. some cf patients were harbouring the same p. aeruginosa genotype in sequential isolates and the susceptibility of bacteria to antibiotics tested varies greatly, also in strains belonging to the same pfge profile. conclusion: our results indicate no relationship between genotype and phenotype suggesting that the phenotypic variability is due to an adaptation of the microorganism to the host. moreover, the presence of several strains with the same genotypic profile suggests a possible cross-colonisation in cf patients due to the circulation of a transmissible strain. for ser/thr protein kinases and phosphoprotein phosphatase of pseudomonas aeruginosa and analysis of their properties j. nedvedova, p. lnenicka, k. hercik, p. branny prague, cz objectives: pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. three genetic loci of p. aeruginosa which encodes ser/thr protein kinases were identified. two of them, ppka and stk , were also characterised but little is known about their function in cell signalling. gene stp localised upstream of stk encodes stk cognate phosphoprotein phosphatase. a possible relationship between quorum sensing and protein phosphorylation in gram-negative bacteria has already been described. the aim of this work was to prepare unmarked deletion mutants in ppka, stk and stp genes and to find out if the linkage between quorum sensing and protein phosphorylation in p. aeruginosa exists. to prepare the unmarked deletion mutants an improved method for gene replacement in p. aeruginosa which employs a broad-host-range flp-frt recombination system for site-specific excision of chromosomally located dna sequences was used. the phosphorprotein pattern and biochemical properties of the mutants were examined. double mutant in homoserine lactone synthase genes (lasi and rhli) was also subjected to phosphoprotein pattern analysis. results and conclusion: stk , stp and a double mutant stk /stp were prepared. no differences were found in either biochemical properties or phosphoprotein pattern. deletion of ppka gene failed due to the integration of vector into the unknown, but specific site of p. aeruginosa genome. the comparison of phosphoprotein patterns of lasi, rhli double mutant and wild type strain showed important differences. this result suggested that phosphorylation circuit operating in p. aeruginosa is related to quorum sensing system(s). objectives: botulism is a rare but potentially fatal disease generally caused by the neurotoxin produced by clostridium botulinum. symptoms of the disease include paralysis which is due to bont inhibiting neuro-transmitter release. laboratory diagnosis of botulism relies on detecting bont in clinical or food specimens using in vivo tests. diagnosis also includes isolation and identification of the bacterium which again relies on in vivo tests for detection of toxin production from the bacterium growing in vitro. we previously described the development of real-time pcr assays for bonta, b and e gene fragments, and here presented further evaluation data. methods: dna was extracted from faeces, enrichment cultures of naturally contaminated food and clinical samples and from colonies growing on agar plates. taqman-based assays for bonta, b and e gene fragments were performed using a sequence detector (applied biosystems). the assays were performed as a duplex reaction for bonta and b using fam and vic labelled probes, respectively, and as a monoplex for bonte using a single fam labelled probe. all samples were tested by using the conventional bio-assay and results were compared with real-time pcr assay results. results: pcr and bio-assay were found to be consistent in all samples except those that contained 'silent b' neurotoxin genes in addition to bonta genes. the samples tested comprised: direct examination of six faecal samples, enrichment cultures for six clinical and foods and pure culture growing in vitro. conclusion: this study is the first to report the successful identification of different c. botulinum toxin types for wild type bonta, b and e by using taq-man real-time pcr assay. this assay has already provided a useful adjunct to in vivo tests for the rapid identification of bacteria containing bont genes in wild type c. botulinum. (vntrs) polymorphisms for genotyping 'rickettsia conorii complex' strains l. vitorino, r. de sousa, l. zé-zé, f. bacellar, r. tenreiro lisbon, p introduction: mediterranean spotted fever (msf) is an acute, febrile tick transmitted rickettsiosis caused by strains of rickettsia conorii complex. msf is endemic in portugal and is an obligatory notifiable disease. during - the annual incidence rate of the disease was . / inhabitants. in portugal, msf is caused by two strains: r. conorii malish and israeli tick typhus (itt). this strain was isolated, for the first time in , from a patient. moreover, data from the national institute of health points out that half of msf cases occurring in portugal are caused by itt, showing a similar prevalence of infection as r. conorii malish. objective: in this work we present a pcr-based method to detect vntr sequences that enables amplicon size differentiation between r. conorii malish and itt human isolate. vntrs have a high discriminatory capacity not only because they contain greater diversity but also because they often vary in copy, therefore they are being used for molecular typing of many bacteria species. methods: human strains were isolated by shell-vial technique from patient's total blood. dna from tick isolates and reference strains were also used for comparative purposes. vntr loci were identified by the tandem repeats finder software within the r. conorii genome. the vntr locus was selected based on the following criteria: repeat units nucleotides in length, % nucleotide sequence identity between individual repeat units, and two or more copies of the repeat unit. primers flanking this sequence were designed to enable vntr-pcr amplification. the clinical isolates' identification was also confirmed by ompa gene sequencing. results: the vntr sequence chosen was highly informative since it possesses different repeats of the consensus pattern among the strains tested, namely r. conorii malish and itt. the former contains five tandem repeats and the later only have three repeats of the bp motif unit, which can be easily detected by agarose gel electrophoresis. therefore, the polymorphism observed enabled discrimination between these two strains. these results are in agreement with ompa gene sequence. conclusion: this pcr-based method provides a useful and rapid way for genotyping r. conorii malish and itt isolates (r. conorii complex) which are responsible for msf in portugal. ompa and glta gene amplification are currently the most widely rickettsiae detection method used. however, strain identification is only accomplished by sequencing. p monitoring the ability of the human intestinal microflora to become re-established after antibiotic treatment using t-rflp c. jernberg, Å . sullivan, c. edlund, j. jansson huddinge, stockholm, s objectives: to study the composition of the human normal faecal microflora during administration of clindamycin and a probiotic or placebo product. since only a small portion of the faecal flora is cultivable the samples were primarily analysed using a culture independent molecular fingerprinting technique, terminal-restriction fragment length polymorphism (t-rflp). methods: the study included eight healthy volunteers. all subjects received clindamycin orally for days. four subjects received a probiotic yoghurt concomitantly containing exp cfu/ml of the strains lactobacillus f , lactobacillus acidophilus ncfb and bifidobacterium lactis bb . the placebo group received ordinary yoghurt. faecal samples were taken before the administration (day ), the last day of administration (day ) and days after the administration (day ). the samples were analysed both by conventional cultivation and by t-rflp. both universal bacterial primers and lactobacillus specific primers were used when analysing the samples using t-rflp. the areas of the different terminal restriction fragments (trfs), each of which theoretically corresponds to one or a group of closely related species, were used to calculate the relative abundance values for the trfs. these values were used for principal components analyses (pca) and upgma analyses to compare the microbial flora at the three different time points. results and conclusions: in the group ingesting the probiotic, the microflora in three out of four subjects became re-established close to their original compositions weeks after antibiotic treatment ceased. by contrast, only one subject out of four in the placebo group had an intestinal microflora that showed tendencies towards normalisation during the same time period. these findings were in accordance with the results from the culture-based analysis. t-rflp was also used to monitor specific bacterial populations that were either positively or negatively impacted by clindamycin. for example, one of the dominating populations, belonging to the clostridium coccoides subgroup, was highly negatively impacted by clindamycin administration in all subjects. when using lactobacilli specific primers, l. acidophilus and lactobacillus f were the two dominating populations in the group receiving the probiotic. t-rflp was shown to be a reproducible technique for analyses of antibiotic and probiotic induced alterations in the normal intestinal microflora. campylobacter species using maldi-tof mass spectrometry d. dare, h. sutton, c. keys, h. shah, m. lunt, g. wells manchester, london, uk laser interrogation of bacteria by matrix assisted laser desorption/ ionisation time of flight (maldi-tof) mass spectrometry (ms) reveals unique fingerprint patterns of biomarkers. these patterns are reproducible for a given set of conditions and can be used as the basis for bacterial identification against a database of known bacterial spectra. manchester metropolitan university in collaboration with the health protection agency (uk) and waters corporation have created a maldi-tof mass spectral database of clinical, environmental and food borne pathogens. these pathogens are all supplied from the uk national collection of type cultures (nctc). the database spectra are therefore representative of organisms from a world-renowned collection. this database has continued to grow over the last years from the initial to currently over spectral entries covering $ different genera. bacterial identification using this database is often conclusive with the top five matches suggesting the same genera/species. however for identification to be robust, the strains within the database must be well characterised and their identity well established. for campylobacter the number of representative strains in the database has increased significantly from around to over years. the species covered within this taxa are: c. coli, c. consicus, c. curvus, c. fetus, c. gracilis, c. helveticus, c. hyolei, c. hyointestinalis, c. jejuni, c. lari, c. rectus, c. sputorum and c. upsaliensis. this study presents the results of analysing the same datasets for campylobacter strains against the expanding databases containing , , and > mass spectral entries respectively. the results demonstrate a significant improvement (i.e. - %) in the number of campylobacter sp. correctly identified as the number of representative strains increase. therefore maldi-tof ms provides a potential rapid identification system for campylobacter sp. p application of s- s intergenic spacer sequencing for the detection and molecular differentiation of legionella species f. grattard, c. ginevra, s. riffard, a. ros, j. etienne, b. pozzetto saint-etienne, f objectives: among the more than species of legionella that have been identified so far, have been reported to be pathogenic for humans. by now, the precise identification of clinical isolates in reference laboratories needs the use of monoclonal antibodies or of molecular markers such as s rrna-, mip-, rpob-or dota-gene sequencing. we developed a rapid and convenient technique based on the sequencing of the s- s intergenic spacer using nondegenerated primers specific for legionella spp. methods: we tested legionella species (reference and clinical isolates), including serogroups of l. pneumophila subsp. pneumophila. the amplification step was performed by using a real-time pcr (lightcycler, roche diagnostics) and sequencing was performed on the ceq sequencer (beckman). the comparative analysis of the sequences was done with the computer program mega and the dendrograms obtained by the neighbour-joining method. results: the phylogenic tree of the s- s intergenic spacer sequences was found able to clearly differentiate all legionella species at the subspecies level. actually three subspecies of l. pneumophila (subsp. pneumophila, subsp. fraseri and subsp. pascullei) were clearly distinguished. species sharing the same autofluorescence properties and ubiquinone and fatty acid composition were shown to be phylogenetically related. in addition to rpob sequen-cing that was shown previously to exhibit similar results, our technique was found able to detect and identify strains present in clinical or environmental specimens that could not be cultured on agar medium. although this tool was not discriminatory enough to differentiate all strains of l. pneumophila subsp. pneumophila at the serogroup level, it was used in two different outbreaks to demonstrate rapidly the identity of the sequences between strains responsible for severe human infection and those isolated in the hot water reservoir, suggesting a common origin. conclusion: the s- s intergenic spacer sequencing was found to be suitable for rapid detection and powerful identification of legionella species in clinical settings. whipple's disease (wd) is a rare multisystemic bacterial infection, with variable clinical manifestations occasionally involving the central nervous system. as the cultivation of the aetiologic agent, tropheryma whippelii, is difficult, the laboratory diagnosis is usually based on histological methods. in the last few years, molecular detection of the bacterial srrns genes by the polymerase chain reaction (pcr) with two primer sets, has greatly contributed to the diagnosis. we present a cerebral case of wd in a -year-old male, successfully diagnosed by pcr of t. whippelii in the blood and the faeces. as far as we know this is the first case reported from greece. for the diagnosis of wd histological examination of duodenum biopsy for diastase resistant, non-acid fast, periodic acid schiff (pas)-positive inclusions in macrophages, and molecular detection of the srrna genes of by pcr in csf, blood and faeces were performed. the histological detection was negative. pcr was positive in the blood and the faeces of the patient and negative in the csf. seven months after the onset of antimicrobial therapy, pcr was negative in all three clinical specimens. in conclusion, the application of pcr proved to be an invaluable tool for the recognition, the differential diagnosis and the early start of the antimicrobial therapy of wd, a generally fatal disease, if it remains untreated. results: only one clinical isolate had the same api code profile as the reference strain. fifteen per cent of clinical strains were tested urease positive, as was the reference strain. by fatty acid analysis, clinical isolates could be separated in four different groups (i-iv), containing , , and isolates, respectively. atcc was grouped to group ii. sequences were obtained from three strains of groups i and ii, respectively, and from one strain of groups iii and iv, respectively. comparison of the determined eight sequences with public databases showed the greatest similarity score with c. asperum (x . ) with values between . and %. c. asperum and c. amycolatum are considered as synonyms, because they exhibit a level of dna-dna relatedness greater than % (ruimy r et al. int j syst bacteriol ; : ) . homology with c. amycolatum atcc (x . ) was only between . and . %. sequencing of the c. amycolatum reference strain yielded % homology with the published sequence (x . ). conclusions: our data confirm the hypothesis that atcc is atypical for clinical c. amycolatum strains. furthermore, our data are in concordance with the observation, that by pyrolysis-gasliquid chromatography c. amycolatum isolates can be separated in two different groups (voisin s et al. res microbiol ; : ) . p detection of mutations associated with resistance to tetracycline and clarithromycin in helicobacter pylori using the pyrosequencer a. lawson, c. arnold, r. owen london, uk objectives: clarithromycin and tetracycline are key components of h. pylori eradication therapy. resistance to clarithromycin occurs due to single nucleotide mutations in s rdna and an assay to detect these was amongst the first to be developed for the pyrosequencer. recently it has been shown that resistance and reduced susceptibility to tetracycline occur due to single, double or triple mutations in s rdna. the aim of this study was to develop a single multiplex assay using the pyrosequencer to determine susceptibility to clarithromycin and tetracycline from h. pylori isolates and direct from gastric biopsy samples. methods: pyrosequencer assays to detect mutations conferring tetracycline and clarithromycin resistance were designed to work singly and in multiplex. the assays were evaluated using isolates with fully characterised s and s rdna sequences. subsequently, dna extracts from clinical isolates and h. pyloripositive human gastric biopsies -all of unknown antibiotic susceptibility -were examined and the results compared with those achieved by conventional culture-based techniques, namely antibiotic disc diffusion and etest. results: the pyrosquencer multiplex assay correctly determined the s and s rdna sequences of the characterised control isolates. when applied to dna extracted from clinical isolates and gastric biopsy samples, the pyrosequencer assay was in agreement with the clarithromycin and tetracycline susceptibilities determined by culture-based analysis. conclusion: the pyrosequencer assay allowed rapid determination of clarithromycin and tetracycline susceptibility from both h. pylori isolates and gastric biopsy samples. the sequence data obtained for the mutations occurring in each strain may provide useful epidemiological information and guide patient management. diseases. the aim of this prospective pilot study was to detect iga, igg and anti-caga antibody status and to evaluate the correlation with anti-h. pylori iga, igg western blot and elisa tests in adult dyspeptic patients. methods: upper gastrointestinal endoscopy, two from gastric antrum and two from corpus, was performed in patients (mean age ae . ) with dyspeptic symptoms. h. pylori was assessed by rapid urease test and by histopathologic examination in these biopsy specimens. patients' sera were tested by anti-h. pylori iga, igg western blot, iga, igg elisa and anti-caga-iga, igg elisa (euroimmun medizinische labordiagnostika, lü beck) tests. results: a total of patients were evaluated and h. pylori infection was diagnosed in ( . %) patients by rapid urease test and/or histopathology. serological anti-h. pylori test results were shown as below (table ) . twenty-eight ( %) of adult dyspeptic patients sera were positive for anti-caga-igg elisa and ( . %) were positive for anti-caga-iga elisa. conclusion: infection with h. pylori results in the production of local and systemic antibodies. cag a is the important pathologic marker with high immunogenic power. a set of serological tests may give more accurate determination of h. pylori infection than one test detecting specific antibody or bacterial antigen. it seems that there is a good correlation with western blot and elisa test results and gold standards. acknowledgement: this work was supported by euroimmun medizinische labordiagnostika, lü beck, germany. p susceptibility of helicobacter pylori isolates to the anti-adhesion activity of a high-molecular-weight constituent of cranberry h. shmuely, o. burger, i. neeman, j. yahav, z. samra, y. niv, n. sharon, e. weiss, m. tabak, a. athamna, i. ofek petach tiqva, haifa, rehovot, jerusalem, kfar qaraa, tel aviv, il background: previous studies have shown that a high molecular mass non-dialysable constituent derived from cranberry juice inhibited the adhesion of helicobacter pylori to human gastric mucus and to human erythrocytes. the aim of the present study was to determine the sensitivity of a large number of both antibiotic-resistant and susceptible clinical isolates of h. pylori to the anti-adhesion effect of the cranberry constituent. material and methods: confluent monolayer of gastric cell line in wells of a microtitre plate was exposed to bacterial suspensions prepared from h. pylori clinical isolates, including from patients after treatment failure. adhesion was estimated by the urease assay to calculate the percent inhibition of adhesion by the non-dialysable material. antibiotic susceptibility of h. pylori isolates to metronidazole, tetracycline and amoxicillin were tested by the etest. results: in two-thirds of the isolates, adhesion to the gastric cells was inhibited by . mg/ml of the non-dialysable material. all isolates were susceptible to amoxicillin and tetracycline and isolates ( %) were resistant to metronidazole. there was no relationship between the anti-adhesion effect of the cranberry material and the resistance to metronidazole in isolates from either the antibiotic-treated or untreated patients. most important, only isolates ( %) were resistant to both non-dialysable material and metronidazole and isolates ( %) were resistant to the non-dialysable material alone. no cross-resistance of the isolates to cranberry constituent and metronidazole was found. conclusions: the data suggest that a combination of antibiotics and a cranberry preparation may improve the eradication of h. pylori. methods: for the seroprevalence study a total of people of different states from the country were evaluated: symptomatic and asymptomatic adults; symptomatic and asymptomatic children. the determination of specific igg antibodies was made by commercial elisa. the presence of the gene caga was evaluated in patients of the metropolitan area and the center of gastric cancer control of san cristó bal (endemic zone of gastric cancer). the detection of vaca was determined in biopsy from patients of san cristó bal and biopsy from patients of the metropolitan area. the biopsies were analysed by different methods for diagnosis of h. pylori: culture, urease test, polymerase chain reaction and rapds for genotyping the h. pylori isolates. results: the percentage of asymptomatic children with values of specific igg antibodies anti-h. pylori (over u) varies from to % (metropolitan area vs. san cristó bal). in symptomatic adults groups, the seroprevalence was between and % according to the studied geographic area. a decreased title of igg antibodies anti-h. pylori was observed in patients with diffuse antral gastritis associated with metaplasia type ii. in the group of endemic cancer area the titles of igg anti-hp were elevated in patients with antral diffuse gastritis. the caga gene was detected in % of patients of the metropolitan area unlike the group of patients of san cristó bal a smaller frequency was observed ( . %) (p < . ). a high incidence of s a and m genotype was observed in the h. pylori isolated from the patients of endemic gastric cancer area ( %), unlike what we observed in the metropolitan h. pylori isolates where an elevated prevalence of s b and m genotypes was found. samples from gastric antro. no current resident in our area and patients treated previously with eradicated therapy have been excluded. samples were homogenised and cultured in blood-agar, chocolate-agar, pylori-agar and tioglicolate broth. it was incubated to c in microaerofile atmosphere during - days. we studied the susceptibility to: amoxicillin (am), claritromicin (ch), metronidazole (mz), tetracycline (te) and ciprofloxacin (cp) by detection of imc by e-test (biodiskâ). we have followed nccls criteria for antibiogram lecture. results: from samples, were males and females. we found the follow primary resistance; ch ( . %), mz ( . %), te ( . %), am ( . %), cp ( . %) and samples ( . %) with a mix resistance to ch and mz. ch and mz resistance are more common in females, but the difference is only statistically significant for mz (p: . ). conclusions: there is a progressive increased antibiotic resistance in h. pylori in our area. this may be related with a raised used of antibiotics for other indications. ch resistance data agree with other spanish and multicentre european studies, which show a foremost rate in the mediterranean area. the mz resistance is higher than other spanish works. our high prevalence of resistance supports the idea of avoiding imidazol therapy as primary choice treatment. p the prevalence and consequences of antibiotic resistance in danish h. pylori strains isolated with an interval of years objectives and background: the treatment of h. pylori (hp) infections is complex and the use of combination therapy is imperative. the choice of the antibiotics is often made exclusively on empirical basis although resistance to many therapeutically relevant antibiotics has been described. the mainstay of hp treatment in denmark is various combinations of normally two of the following antibiotics: metronidazole, amoxicillin, tetracycline and clarithromycin. to clarify whether these compounds were to remain the drugs of choice we decided to determine the susceptibilities of metronidazole, clarithromycin, tetracycline, and amoxicillin against hp strains recently isolated from patients with duodenal ulcer. the results were compared with results previously obtained by us in using a similar methodology. over a period of years only the development of resistance to metronidazole appears to constitute a problem. otherwise hp has remained remarkably susceptible to these therapeutically relevant antibiotics. on the basis of our results we recommend that surveillance of especially metronidazole resistance in denmark is markedly intensified, e.g. by increasing the use of diagnostic methods of hp infections that allow susceptibility testing. in cases where treatment with metronidazole is considered, susceptibility testing is of course of major importance, if not downright necessary. objectives: helicobacter pylori is the main causative agent of peptic ulcer disease. clarithromycin resistance of h. pylori is the common reason of failure of the eradication therapy, which includes amoxicillin-clarithromycin and proton pomp inhibitor. the aim of this study was to determine the prevalence of clarithromycin resistance among h. pylori strains isolated from gastric biopsies obtained during routine endoscopies at the baskent university medical faculty in ankara, turkey. methods: h. pylori strains were isolated from antral biopsy specimens taken from dyspeptic patients. antibiotic susceptibilities of the isolates to clarithromycin were performed using the nccls approved agar dilution and the e test methods. results: h. pylori isolates were included in the study. clarithromycin resistance was found in ( . %) of the isolates. the resistance rates were similar by the e test and agar dilution methods. conclusion: the percentage of the clarithromycin resistance among h. pylori strains in our population is significantly high. this information is important to monitoring the eradication therapy and defines regional treatment policies. introduction: helicobacter pylori has been the subject of many studies that contributed to a better understanding of its epidemiology and its clinical importance in the pathology of the upper gastrointestinal tract, being an important cause of duodenal, gastric ulcers and a definite cause of gastric adenocarcinoma in human. objectives: to determine the seroprevalence of h. pylori among population living in rural community, in relation to the epidemiological aspect, and to study the seroprevalence of anti-caga as a virulence factor in a step that might be helpful in studying the magnitude of h. pylori infection. also, to determine a cut-off value among the population in this community. subjects and methods: this is a community based, field study which was performed on randomly chosen subjects representing villagers of eight villages in giza governorate egypt. serological testing for anti-h. pylori and anti-cag a were performed by elisa. results: the overall seroprevalence of anti-h. pylori igg was . % with different degrees of positivity: . % mild, . % moderate and . % high. anti-cag a was present in . %. there was a significant agreement between the presence of the two antibodies; however, on studying the relation of anti-h. pylori igg level with anti-caga no statistically significant relation was found denoting that the level of infection even if mild does not rule out the possible association of virulent strain of h. pylori. no age or sex difference was noted as regards anti-h. pylori seropositivity but subjects seropositive for anti-cag a had a statistically significant higher mean age. when relating the seroprevalence of anti-h. pylori to type of community, it was found to be the same in semi-rural communities and rural ones and when investigating the respective conditions in both communities it was found that the prevalence is rather related to pattern of life, socioeconomic status and to other possible vehicle of transmission as animals or flies than faecaly contaminated water which is not considered the only vehicle for h. pylori transmission in our study. conclusion: h. pylori is holoendemic in egypt; however, infection by virulent strains is not common. objectives: extended-spectrum beta lactamases (esbls) are an increasing cause of resistance in enterobacteriaceae. unfortunately, the laboratory detection of esbls can be complex and, at times, misleading. the aim of this study was to determine whether routine methods performed in a clinical microbiology laboratory of a tertiary care hospital, are adequate for detecting emerging esbl producing clinical isolates. methods: to evaluate the esbl confirmation protocol, we collected enterobacteriacae strains, isolated in our laboratory. each isolate met the nccls screening criteria for potential esbl producers (ceftazidime or cefotaxime mics were ! for all isolates). we tested kl. pneumoniae, five ent. cloacae, two ent. aerogenes, five e. coli and four pr. mirabilis strains, by methods routinely used in our laboratory. initially, the isolates were tested for clavulanic acid effect by disk diffusion method and all were analysed by the vitek automated system (biomerieux, france), which performs a susceptibility testing, by determining the mic breakpoints. the advanced expert system (aes) of vitek was set on the phenotypic resistance knowledge-based system and the panel gn was used. in parallel, the isolates were tested by the esbl e-test with ceftazidime and cefotaxime plus beta lactamase inhibitor (ab, biodisk, sweden). in order to confirm the esbl production, all strains were tested by isoelectric focusing (ief) followed by pcr for blatem, blashv, blaoxa, blaibc and blactx genes. results: twenty-one out of isolates proved to produce esbls by molecular methods. all enterobacter strains and one proteus mirabilis were not esbl producers. no blaoxa or blaibc genes were detected. the pcr detection of esbl genes results were compared with the double disk diffusion, vitek and esbl e-test to estimate the sensitivity, specificity and the predictive value of the methods tested. the sensitivity of the methods was . , . and . %, respectively, the specificity . , . and . %, respectively, and the predictive value . , . and %, respectively. discussion: given the increasing incidence of esbl producing clinical isolates, it is important that esbl screening is incorporated into routine diagnostic testing. the backup of the simple disk diffusion method by the automated vitek system increases the possibility of identifying esbl activity of clinical strains in the hospital microbiology laboratory setting. objectives: extended-spectrum beta-lactamases (esbl) are plasmid-mediated beta-lactamases and most of them are mutant of tem or shv beta-lactamases. esbls have been associated with clinical failures due to serious interpretive problems of standard laboratory tests. detection of esbls remains a challenge for the laboratory, since routine tests for monitoring a susceptibility to oxyimino-cehalosporins and aztreonam have not been sensitive enough to detect esbl strains and require up to days. we describe an oligonucleotide array for rapid identification of single nucleotide polymorphisms (snps) of the esbl tem beta-lactamases. methods: plasmid dna was amplified and cy labelled during pcr with consensus primer pair flanking the blatem gene. oligonucleotide arrays were constructed with oligonucleotide capture probes. the probes were designed with the snp at the central base of the probe sequence for maximum perfect match/ mismatch discrimination. results: of snp positions were correctly identified. the signal intensity values ranged up to for the perfect match probes. the discriminatory power of the array expressed as relative intensity of mismatches (rimm) remained for % of the mismatches below . . a perfect match was considered as correctly identified, if rimm did not exceed . . analysis of the array reproducibility revealed that in analysed blatem- samples all snp positions could be identified. the mean rimm values varied, but % remained below . . in dna isolated from clinical samples all mismatches in blatem were identified without ambiguity, and % of them remained below the rimm limit. since the reduction of the array-hybridisation time to min had no influence on rimm (rimm limit less than . for % mismatch positions), the assay may be performed within . h while keeping its discriminatory power. conclusion: the blatem gene variants could be amplified by the use of a single consensus primer pair. using dna-array we were able to discriminate snps in of the tem variants. snp mismatches could be analysed by array within . h enabling the identification of the corresponding esbls or inhibitor resistant tems. the nccls recommendations. the production of extended-spectrum beta-lactamases (esbl) was detected by double diffusion test. the presence of blatem gene was determined by pcr method. transferability of resistance determinants was studied by bacterial conjugation. results: . % of the clinical isolates were resistant to ampicillin (ampi); . % to cefoxitine (cfox); . % to cefotaxime (ctax); . % to ceftazidime (ctaz); . % to ceftriaxone (ciax); . % to cefepime (cepi); . % to azthreonam (aztr); . % to meropenem (merp); . % to gentamicin (gen); . % to tobramycin (tob); . % to netilmicin (net); . % to amikacin (ami); . % to isepamicin (ise); . % to ciprofloxacin (cip). a total of . % of clinical isolates were identified as esbl producers. the presence of blatem gene coding for tem-type beta-lactamases was detected in . % of clinical isolates tested. resistance determinants to all antibiotics tested, with only one exception of merp, were transferable by bacterial conjugation to the recipient strain escherichia coli k- . frequency of transfer ranged from .  À to .  À . conclusions: the occurrence of resistance to beta-lactam antibiotics was very high. the most efficient beta-lactams were the carbapenem meropenem and the fourth-generation cephalosporin cefepime. aminoglycoside antibiotics netilmicin, amikacin and isepamicin had high efficiency, too. on the other hand, more than one half of the clinical isolates tested were resistant to the fluoroquinolone ciprofloxacin. beta-lactam resistance was due to the production of esbl and to the presence of the bla-tem gene in the majority of clinical isolates. transferability of beta-lactam and aminoglycoside resistance determinants by bacterial conjugation is important from the epidemiological point of view. objectives: today there are very few significant data on the effectiveness of cephalosporin antibiotics in nosocomial infections caused by the microorganisms producing esbl. methods: the cases of nosocomial infections caused by enterobacteriaceae with a proved esbl production were analysed. esbl producing enterobacteriaceae strains were assayed for susceptibility to different antimicrobials and mics were determined by a broth microdilution method. to determine molecular typing of esbl genes polymerase chain reactions and sequencing reactions were used. patients received initial empiric intravenous antibacterial therapy with third-generation cephalosporin (cefotaxime). in case of failure cefepime g a day was prescribed. results of those infections treatment with third-and fourth-generation cephalosporins were assessed depending on mic. results: esbl production with specific shv and ctx oligonucleotids was proved for six strains of enterobacteriaceae in four patients with nosocomial pneumonia (in two cases mixed infection took place), among them four strains were klebsiella spp. and two strains e. coli. the analysis of the dependence of mic on the results of the treatment gave the following results (table ) . it may be stated that with the proved enterobacteriaceae esbl production mic values for third-generation cephalosporins of the majority of strains were within the resistance range (more than lg/ml), and these antibiotics were not effective in all cases. as for cefepime, mic showed intermediate sensitivity ( lg/ml) to the drug only in . % cases; the rest of the strains (mic ¼ - lg/ml) were sensitive. the therapy with cefepime was effective in three of four patients. , À / a (n ¼ ), À (n ¼ ), À /À (n ¼ ) and À / (n ¼ ). conclusions: (i) using mic breakpoint > mg/l for reduced susceptibility to third-generation cephalosporins we detected esblproducing e. coli and k. pneumoniae with low mic-values ( . - mg/l). (ii) cefotaxime-hydrolysis was the dominating profile in esbl-positive e. coli strains whereas ceftazidime was the most sensitive substrate for detection of esbl-production in k. pneumoniae. (iii) the different methods showed almost the same sensitivity in detecting esbl production assuming that more than one substrate was used, i.e. both cefotaxime and ceftazidime. (iv) ctx-m was the dominating esbl-type. objectives: during , the srmd received isolates of escherichia coli for confirmation of esbl production with a phenotype implying a ctx-m-type beta-lactamase, i.e. cefotaxime (ctx) mics fourfold greater than ceftazidime (ctz) mics. isolates were from hospital patients and, in some instances, from community patients with little or no recent hospital contact. tem-and shv-type esbls are largely confined to nosocomial isolates, so the apparent spread of ctx-m enzymes in the community is cause for concern. we compared the isolates and investigated the genetic basis of their ctx-m phenotype. methods: isolates were compared by pfge of xbai-digested genomic dna and data were analysed using bionumerics software. mics were determined by etest or agar dilution, and interpreted using bsac breakpoints. isolates with a ctx-m phenotype were tested for blactx-m alleles by pcr, initially with universal primers, and then with primers specific for various blactx-m groups. selected amplicons were sequenced, either directly or after cloning into pcr . . transfer of ctx-m to e. coli j was attempted in broth and on agar plates. results: over ctx-m-producing e. coli were obtained from more than uk centres. these isolates represented multiple strains, although clusters of related isolates (> % similarity) were observed, some including isolates from more than one centre. sequencing confirmed that e. coli from different centres all produced ctx-m- . most isolates had substantial resistance to ctx (mics > mg/l) and ctz (mics > mg/l), consistent with ctx-m- . isolates (n ¼ ) associated with a large community cluster produced atypically large amplicons with group i ctx-m primers, as did two related isolates from another centre. these isolates were less resistant to ctx (mics - mg/l) and ctz (mics - mg/l), and susceptible to gentamicin; sequencing of a representative isolate identified is within the terminal inverted repeat of the isecpi element upstream of blactx-m- , separating the allele from its usual promoter, and the spacer between isecpi and blactx-m- had a t/c polymorphism not seen in other sequenced isolates. we studied also antimicrobial sensitivity of the strains, co-resistance to non-beta-lactam antimicrobials and relationship with antibiotic use. methods: data of monthly non-duplicate ebsl-ec and antibiotic use (hospital: ddd/ pat-day and community: ddd/ inhabitants-day) were collected for january to october . time series dynamic regression models were adjusted to evaluate the relationship between the use of antimicrobials and the emergence of the bacteria. sensitivity testing was determined by microdilution with gram-negative and urine panels (microscanâ). esbl producing strains were initially selected by screening with microscanâgram-negative and urine panels (mic > lg/ml for cefotaxime, ceftazidime or aztreonam, and/or a difference of three or more dilutions between ceftazidime and ceftazidime with lg/ml of clavulanic acid . on univariate analysis only connective tissue disease (p < . ), genitourinary pathology (p < . ), infections in the past year (p < . ) and previous exposure to second-generation cephalosporins (p < . ) were factors associated with ca infection due to esbl ec. in our regression model, only previous exposure to secondgeneration cephalosporins was strongly associated (or . , . conclusions: in the last years there has been a marked increase in infections due to esbl ec, especially from the community. only previous exposure to second-generation cephalosporins (not to ciprofloxacin, third-generation cephalosporins or aminoglycosides) was predictive of an esbl ec ca infection. strikingly, neither comorbidity nor previous contact with the healthcare system was risk factors for esbl ec. enzymes of the ctx-m family are currently classified as extended-spectrum beta-lactamases (esbls). over the last decade, ctx-m-type enzymes have been increasingly reported from several countries in europe. the aim of this study was to search for ctx-m-type enzymes in escherichia coli isolates obtained at our institution (varese, northern italy). methods: we studied consecutive e. coli isolates recovered over a -year period ( ) ( ) ( ) . stains suspected of producing esbls (according to nccls criteria) were further investigated. the double-disk synergy test and etest esbl strips (ab biodisk, solna, sweden) were used to confirm esbl production. the etest method was also used to evaluate mics of amikacin, gentamicin, ciprofloxacin, and beta-lactams (including last-generation cephalosporins, carbapenems, and aztreonam). esbl-positive isolates were evaluated for the presence of ctx-m-type genes using specific dna probes. patient records were examined to assess risk factors for infections and underlying clinical conditions. results: a total of consecutive e. coli isolates were studied. overall, out of esbl-positive strains were found to carry a ctx-m-type gene and to produce a ctx-m-type enzyme. most isolates ( / ) showed high mic values for cefotaxime (> mg/l) and borderline values for ceftazidime ( - mg/l). the remaining five isolates had also high mics for ceftazidime. ctx-m-positive isolates were obtained both from inpatients (n ¼ ) and outpatients (n ¼ ). epidemiological analysis showed that most strains were isolated from urinary tract infections, even though some isolates were recovered from the lower respiratory tract, wounds and blood. most patients ( / ) were treated with immunosuppressive therapy. recurrent urinary infections occurred in five outpatients. conclusions: ctx-m-type enzymes appear to be emerging among e. coli isolates in both the hospital and community environments. the analysis of clinical records demonstrated that these microorganisms can cause severe and persistent infections. therefore, despite the currently low prevalence of ctx-m phenotype, we suggest that a monitoring of this resistance phenotype should be established to avoid the spreading of resistance traits. background and objectives: class c beta-lactamases (cbls) are enzymes that confer broad-spectrum beta-lactam resistance (including penicillin, expanded-spectrum cephalosporins, and cephamycins) and are poorly or not susceptible to commercially available beta-lactamase inhibitors. in strains with reduced outer membrane permeability, they can also provide resistance to carbapenems. a number of these enzymes are chromosomally encoded, but plasmid-mediated cbls are also known as a cause of acquired resistance to expanded-spectrum cephalosporins and cephamycins in clinical isolates of enterobacteriaceae. in italy, only the fox- acquired cbl has previously been reported, in klebsiella spp. in this work we report the first detection of acquired cbls of the cmy-lat lineage in escherichia coli and klebsiella pneumoniae clinical isolates from an italian hospital. methods: ten consecutive non-replicate clinical isolates of e. coli (eight) and k. pneumoniae (two) resistant to expanded-spectrum cephalosporins and cephamycins were collected, during , at the laboratory of microbiology of the s. matteo hospital of pavia (northern italy). in vitro susceptibility testing was determined by a microdilution method according to nccls. beta-lactamase production was investigated by analytical isoelectric focusing (ief) coupled with a bio-assay. molecular characterisation of beta-lactamase genes was carried out by a multiplex pcr approach designed for detection of all major lineages of acquired cbls genes, and by sequencing. transferability of resistance genes was tested by mating assays in liquid medium. results: two isolates, one of e. coli and one of k. pneumoniae, were found to be resistant to expanded-spectrum cephalosporins, except for cefepime, and cephamycins (cefoxitin mics > mg/l). both isolates produced a beta-lactamase of pi > . that showed hydrolytic activity against cefoxitin, cefotaxime and ceftazidime. molecular characterisation revealed, in both cases, the presence of an acquired cbl gene of the cmy-lat lineage, which was compatible with blacmy- /lat- (the leader peptide-encoding region was not sequenced). the cbl determinant was transferable by conjugation from the e. coli isolate, while conjugal transfer was not detected from the k. pneumoniae isolate. conclusions: these findings reveal that acquired cbls of the cmy-lat lineage, which are the most common acquired cbls, can also be encountered in nosocomial settings from northern italy. enteropathogens p dissemination of sulphonamide resistance genes: first sul found in salmonella from portugal p. antunes, j. machado, j.c. sousa, l. peixe porto, lisbon, p objectives: the purpose of this study was to determine the distribution of sulphonamide resistance genes sul , sul and sul and class integrons in portuguese salmonella isolates collected during - , from human and nonhuman sources. methods: eight hundred and seventy-five isolates were tested for resistance to antimicrobial agents by the agar dilution method. sulphonamide resistant isolates were screened for resistance genes sul , sul , and sul and class integrons by pcr assays. results: resistance was found in % and multiresistance in % of the isolates. in ( %) sulphonamide-resistant isolates (mics mg/l), ( %) sul genes, ( %) sul genes and nine ( %) sul genes were detected. in isolates, more than one gene encoding sulphonamide resistance was present: sul and sul in , sul and sul in three and sul , sul and sul in four. class integrons were found in % of those isolates. among the isolates carrying class integrons, presented sul gene, found alone ( isolates) or simultaneously with sul ( ) or sul ( ) and with sul and sul ( ). the two strains with class integrons, which lacked the qaced and sul genes, carried a sul gene. of the sul -positive isolates, harboured class integrons. conclusion: class integrons and sulphonamide resistance genes are widespread among salmonella. the newly described sul gene has now been identified in nine salmonella isolates collected from human and nonhuman sources in portugal. salmonella from portugal p. antunes, j. machado, j.c. sousa, l. peixe porto, lisbon, p objectives: the aim of this study was the characterisation of betalactamase production in portuguese salmonella isolates collected during - , from human and non-human sources. methods: eight hundred and seventy-five isolates were tested for resistance to antimicrobial agents by the agar dilution method. a double-disk synergy test for the detection of extended-spectrum beta-lactamase production was performed by disk diffusion method. the identification of beta-lactamases was done in ampicillin resistant isolates by ief and pcr assays, with primers, which detects genes encoding tem, pse- and oxa group iii enzymes. to evaluate the association of beta-lactamase genes to class integrons, cs- cs primers were used in a pcr assay. pcr products were purified and both strands sequenced. results: in total, % of the isolates exhibited resistance to ampicillin, with mics mg/l. resistance to ampicillin was conferred by a tem- beta-lactamase in ( %) of the isolates, pse- in ( %) and oxa- in nine isolates. it is to be noted that there is the detection of the extended-spectrum beta-lactamase (esbl) tem- in one isolate. the tem-type beta-lactamases was not associated with class integrons. in contrast, all the blapse- and blaoxa- genes were inserted in and bp class integrons, respectively. conclusion: a considerable percentage of portuguese salmonella were resistant to beta-lactams, mostly due to the production of tem- like beta-lactamase and pse- inserted in integrons. the detection of an isolate that produce an esbl, such as tem- , and nine isolates carrying a class integron with oxa- , are causes of concern due to the possible therapeutic failures with broad-spectrum beta-lactams. p increasing incidence of salmonella typhii with reduced susceptibility to ciprofloxacin in kuwait a.a. dashti, p.w.j. west, d. panigrahi suleibikhat, kwt objectives: to determine the current incidence of reduced ciprofloxacin susceptibility in salmonella typhi, to compare with previous data and to investigate the mechanism responsible. methods: isolates of s. typhi collected in - were tested for susceptibility to ciprofloxacin and other antibiotics using the vitek and e-test. isolates showing reduced ciprofloxacin susceptibility were subjected to pcr to determine if a mutation of the gyra gene was responsible. pcr was carried out using two primers (atgagcgaccttgcgagagaaattacaccg) and (ttcc-atcagcccttcaatgctgatgtcttc). results were compared with those for isolates collected from - . results: out of ( %) of the isolates were resistant to multiple antibiotics, including ampicillin, chloramphenicol tetracycline and trimethoprim. of these ( %) showed resistance to nalidixic acid and reduced susceptibility to ciprofloxacin (mic . - . mg/l). of the susceptible isolates, seven ( %) showed reduced ciprofloxacin susceptibility. isolates from to showed % of multi-resistant strains, but none of susceptible isolates with reduced ciprofloxacin susceptibility. pcr results showed mutations of the gyra gene. conclusion: reduced susceptibility to ciprofloxacin in multi-resistant s. typhi has increased from % in - to % in - and from to % in susceptible strains. mutation of gyra is the mechanism responsible. p comparison of antimicrobial resistance in diarrhoeagenic escherichia coli isolates causing traveller's diarrhoea between two periods, - and - e. mendez arancibia, j. ruiz, r. cabrera, j. gascon, j. vila barcelona, e objectives: to compare the antimicrobial resistance levels in escherichia coli clinical isolates causing traveller's diarrhoea in two periods, - and - . material and methods: presence of enteroaggregative (eaec) and enterotoxigenic e. coli (etec) was established by pcr among those isolated from travellers with diarrhoea during the periods - and - . susceptibility to ampicillin (amp), amoxicillin plus clavulanic acid (amc), tetracycline (tet), chloramphenicol (chl), cotrimoxazole (sxt), nalidixic acid (nal) and ciprofloxacin (cip) was determined by disk diffusion. results: one hundred thirty-two ( eaec, etec) and ( eaec, etec) diarrhoeagenic e. coli were recovered during two periods, - and - , respectively . the levels of resistance of eaec to all tested antibacterial agents increased in the second period: amp from to %, amc from to %, tet from to %, sxt from to %, nal from to % and cip from to % (p < . ), whereas the leaves of resistance to chl showed a slight decrease ( - %) but not statistically significant. in etec strains resistance to amp, nal, cip and amc increased from to %; to %; to %; to %, respectively, while resistance to chl decreased from to %. the levels of resistance to tet and sxt did not present greater differences, but suggested a slight increase in the resistance ( - % and - % respectively). conclusions: a trend to an increase in the resistance of eaec and etec to amp, amc, nal, and cip has been detected, and the decrease of resistance to cip is worthy of note due to the fact that this antimocrobial agent is considered a first choice treatment for traveller's diarrhoea. a.j. hakanen, s. pitkänen, a. siitonen, p. kotilainen, j. jalava, p. huovinen turku, helsinki, fin objectives: quinolone-resistant salmonella isolates emerged in finland in the mid- s. the main origin of these strains is travellers returning from southeast asia. this study was performed to evaluate the incidence and changes of fluoroquinolone resistance in salmonella isolates between and in finland. methods: we collected a total of salmonella enterica isolates which were considered to be epidemiologically unrelated. the isolates were divided into two groups (finnish and foreign isolates) on the basis of travel history. the collection was performed in four phases: each year in , , and starting in january, we consecutively collected finnish and foreign isolates. mics for nalidixic acid, ciprofloxacin and additional fluoroquinolones were determined by the standard agar dilution method (nccls). results: during the study period, the number of isolates with decreased ciprofloxacin susceptibility (mic of ciprofloxacin > . lg/ml) increased from to % of all isolates (p < . ). a similar trend could be seen both among the isolates of foreign and finnish origin. in addition, within the non-susceptible population the mic values were increasing. mic of ciprofloxacin increased from . to . lg/ml among the isolates with decreased ciprofloxacin susceptibility between and . the respective figures for mic were . and lg/ml. all isolates with decreased ciprofloxacin susceptibility had also increased mics to additional fluoroquinolones. conclusion: the number of salmonella isolates with decreased ciprofloxacin susceptibility continues to grow in finland. moreover, the mic levels of these isolates have increased. this phenomenon might have serious clinical implications. p bacteraemia caused by esbl-producing salmonella enterica serovar. virchow . :r: , -a cause for concern a. guleri, g.d. corcoran, s.r. alcock, d.j. brown glasgow, uk background: antibiotic resistance in salmonellae is now common. in developed countries such strains are largely zoonotic and acquire resistance in the animal host before transmission to humans in food. we present our first case of bacteraemic illness with multi-resistant, extended spectrum beta lactamase (esbl) producing non-typhoidal salmonella. case summary: a -year-old male, with a history of recent foreign travel was admitted to hospital with a - day history of gastrointestinal symptoms/fever. on admission he was febrile and splenomegaly was detected. physical examination was otherwise normal. biochemistry revealed mildly deranged liver function. salmonella enterica serovar. virchow . :r: , was isolated from blood culture. it was sensitive in vitro (nccls disk test) to ciprofloxacin and gentamicin but resistant to ampicillin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, co-trimoxazole, nalidixic acid and streptomycin. mic of ciprofloxacin was . mg/l. antibiotic treatment was with ciprofloxacin, to which he responded well. esbl detection: the isolate was identified as salmonella enterica serovar. virchow . :r: , [api identification system (bio-mérieux), serogrouping, serotyping and phagetyping]. the isolate, resistant in vitro (nccls) to cefotaxime and ceftazidime, was tested for extended-spectrum beta lactamase/ampc production by phenotypic methods. ab biodisk esbl e-tests (cefepime, ceftazidime and cefotaxime, each ae clavulanic acid) and oxoid esbl combination disks (cefpodoxime, ceftazidime, cefotaxime and cefpirome, each ae clavulanic acid) and cefoxitin alone were used based on modified nccls/manufacturer's guidelines. the isolate tested positive for esbl production by both esbl e-tests and combination disks. molecular typing of the esbl is awaited. conclusion: invasive infection with salmonella virchow is uncommon. the source of infection in this case appears to have been undercooked chicken. the emergence of resistance to antimicrobial agents within the salmonellae is a worldwide problem that has been associated with the use of antibiotics in livestock. invasive infection with s. virchow, resistant to broad-spectrum beta-lactams, is a cause for concern. if antimicrobial therapy is indicated for travellers with a history of recent foreign travel, physicians should be aware of the possibility of treatment failures and in such cases mics of third-generation cephalosporins and ciprofloxacin should be determined. the aim of the present study was to assess the distribution and the antibiotic resistance rates (arr) of the various nontyphoidal salmonella serotypes originated from non-human sources in greece, during a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . material and methods: a total of isolates, belonging to different serotypes, were selected from the collection of national reference center for salmonella and shigella (nrcss), in order to reflect the prevalence of these serotypes during the mentioned period. the sample consisted of isolates from animals, isolates from foods, and environmental isolates. susceptibilities to antibiotics of various classes were determined using mics broth micro-dilution method. results and conclusions: the highest arr and also the higher incidence of multiresistance have been observed for s. virhow, followed by s. hadar and s. typhimurium. the vast part of s. typhimurium isolates was resistant at least to ampicillin, tetracycline and chloramphenicol, while the main resistance phenotype of s. enteritidis isolates was the monoresistance to ampicillin (table) . the arr and the phenotypes of resistance for the isolates of the above four serotypes were similar with the corresponding ones of human isolates as resulted from a recent greek study derived also from nrcss (eur j epidemiol ; : - ) a fact consistent with possible transfer of antibiotic resistant strains from animals to humans through the food chain. the incidence of resistance for the rest of serotypes was very low. all the examined isolates were susceptible to ceftriaxone and ciprofloxacin. interestingly, almost all the examined isolates belonged to animals bred at a non-industrial scale (e.g. pigeons) and the environmental isolates were sensitive to all tested antimicrobials, possibly because of the reduced antibiotic pressure in these isolates. - and - . the isolation rates of s. enteritidis, s. typhimurium and the others were . , . and . %, respectively, in the first period. in the second period isolation rates were found . , and . %, respectively. antimicrobial resistance for (amp and tmp-sxt) in s. enteritidis, s. typhimurium and others were found ( . / . %), ( . the laboratory data were analysed in shigella spp. isolated from stool materials of adult patients over a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) retrospectively. shigella isolates were identified by standard biochemical reactions and using specific antisera. antimicrobial susceptibility for ampicillin (amp), trimethoprim-sulfamethoxazole (tmp-sxt) and ciprofloxacin (cip) were determined by the disk diffusion method according to national committee for clinical laboratory standards. results: in order to show the differences in epidemiology and antimicrobial resistance, the study was divided into two periods. the first period was from to , and the second period was from to . a total of shigella spp. isolates were obtained in the first period and isolates in the second period. isolation rates of the strains in the first and second periods were, respectively, for shigella flexneri . and . %; for shigella sonnei . and . %; for shigella dysenteria . and . %; and for shigella boydii . and . %. the rates of resistance to (amp) in the first and second periods were respectively in s. flexneri . and . %; s. sonnei . and . %, s. dysenteria . and . %; s. boydii . and . %. the rates of resistance to tmp-sxt in s. flexneri . and . %; s. sonnei . and . %; s. dysenteria . and . %; s. boydii . and . %. all strains were susceptible to ciprofloksasin. conclusions: s. flexneri was the most common species isolated in the first period and s. sonnei was the common species in the second period. in ankara, the data showed an increase in the resistance to the commonly used antimicrobial agents are ampicillin and trimethoprim-sulfamethoxazole. ciprofloxacin seemed to be the best choice for the treatment of shigelliosis. to determine the antimicrobial resistance in salmonella and shigella strains isolated from stool specimens during a -year period, from patients admitted to our clinics with a diagnosis of diarrhoea. methods: the identification and susceptibility testing was done by vitec (biomerieux, fr) automated system. the antibiotics tested for the study were ampicillin, ampicillin-sulbactam, cefotaxime, cefepim, ciprofloxacin, ofloxacin, and trimethoprim-sulfametaxazole. results: nineteen salmonella and seven shigella isolates obtained between january and november were tested for their susceptibilities to seven antimicrobial agents. the total numbers of isolates during - (including the year of big marmara earthquake) were . five of six shigella isolates were s. sonnei, one was s. flexneri. thirteen of salmonella isolates were s. typhimurium, three were s. enteritidis, two were identified as salmonella spp., one was s. arizonae. although all of the isolates were found susceptible to the therapeutic agents, ampicillin susceptibility was decreased to % from % and trimethoprimsulfametaxazole susceptibility was decreased to % from % in salmonella strains during a -year period. only one strain was resistant to cefotaxime. no resistance was found against ofloxacin and ciprofloxacin. all of the shigella isolates were susceptible to all tested antibiotics. conclusions: ( ) the incidence of salmonella and shigella infections seemed to decrease significantly over a -year period. ( ) s. typhimurium and shigella sonnei are the most commonly identified serotypes. ( ) there is no significant change in resistance to 'old' and 'new' antibiotics. ( ) all of the isolates showed a very good sensitivity all the antimicrobials tested. ( ) a careful rotational use of antibiotics might be the best policy to make old drugs again active, and abuse of new agents. objectives: since the incidence of human campylobacteriosis has shown a significant increase in austria. consumption of contaminated poultry products is a well known risk factor for human infections. during the slaughter process meat products can become contaminated with intestinal organisms. furthermore antibiotic resistance is increasing in humans and animals. the aim of the study was to determine the resistance patterns and the transmission routes of campylobacter sp. on the chicken-carcasses along the slaughter line. to compare the frequency of isolation and occurrence of antimicrobial resistance among c. jejuni and c. coli isolated in humans, retail poultry meat and farm broilers in . methods: fifty-three human, retail poultry meat and campylobacter spp. isolates from broiler cloacal swab were investigated for antibiotic susceptibility to antimicrobials by disk-diffusion method. mics were further determined for erythromycin-and ciprofloxacin-resistant isolates by etest. to confirm ciprofloxacinresistance we used a mismatch amplification mutation assay (mama) pcr to detect the gyra mutation. species were determined by multiplex pcr and genetic diversity by pfge typing. results: c. coli isolated in significant proportion in all three sources, . , . and . %, respectively. resistance to one or more antibiotic tested was . , . , and . % and multiresistance . , . and . % in human, retail poultry and farm isolates, respectively. no significant difference was found in the overall resistance rates, and for each antibiotic tested between c. jejuni and c. coli isolates from all three sources, which is unusual finding. moreover, they were higher in c. jejuni. given that after the war population in this region were mostly the muslim, c. coli in humans originated from other sources than pigs. thus, it may suggest that c. coli resistance is origin-related. erythromycin-and ciprofloxacin-resistance was high and almost equal in all three sources ( . , . , . %, and . , . , . %, respectively) .imported retail poultry meat (from five countries) had higher resistance rates for erythromycin than domestic one, . vs. . %, but ciprofloxacin resistance was higher in domestic one, . vs. . %. conclusion: the distribution of c. jejuni and c. coli species and drug resistance in isolates from chicken and farm animals were similar to that seen in humans, even in the absence of antibiotic pressure ( % of patients were under years of age, and growth promoters ban in bosnia and herzegovina), suggesting that poultry may play a role in human infections. when pfge patterns were considered, they were remarkably diverse, suggesting considerable genetic heterogeneity. it may support the hypothesis that campylobacter spp. from food animals and humans may not be represented by discrete populations but rather, form part of a common population shared by food animals and humans, suggesting related sources of infection. objectives: this investigation was designed to study the potential usefulness and economic benefits of oral linezolid as an alternative to outpatient parenteral antibiotic therapy (opat) in the treatment of primary cellulitis. methods: patients with moderately severe cellulitis referred to an infusion centre for antibiotic treatment were enrolled into an open, non-randomised, pilot study. after informed written consent, patients were treated with oral linezolid, mg q h, in place of their prescribed parenteral antibiotic. patients were followed with clinic visits and lab monitoring. results: a total of patients, five males and five females (mean age, years), were enrolled. seven were obese (mean weight, kg; range, - kg), six had lower extremity cellulitis, one had lymphedema, and two were smokers. the average length of linezolid therapy was days (range, - days). all were compliant with the treatment regimen and had a clinical cure of their infection. mild side-effects (nausea, loose stools, headache, metallic taste) were reported by four patients. none developed thrombocytopenia or prematurely discontinued therapy. a -day course of linezolid therapy (drug costs, clinic visits, and lab monitoring) was found to be less expensive than days of vancomycin treatment ( g q h) in the infusion centre. conclusions: in this study, we found that oral linezolid was safe and effective in the treatment of moderately severe cellulitis and could be a suitable replacement for opat. furthermore, oral linezolid has the potential to improve patient satisfaction as well as lower overall treatment costs when compared with opat. objective: vancomycin (v) in combination with rifampin (r) and gentamicin (g) has been the recommended regimen for the treatment of pve caused by mrsa but intolerance to these agents and emergence of mrsa strains with reduced susceptibility to the glycopeptides create the need for alternative agents. we describe the case of a patient with mrsa tricuspid pve who was successfully treated with linezolid (l) after failure of glycopeptides. case: a -year-old man was admitted for persistent mrsa bacteraemia. he had been treated with v, g, r, and trimethoprim/ sulfamethoxazole (t/s) for mrsa pve of the tricuspid valve which had recurred after a -day course of v and a -day course of t/s. due to acute renal failure g was discontinued and v was changed to teicoplanin (t). he was transferred to our department because of persistent bacteraemia of days duration despite adequate blood levels of t. blood cultures revealed a mrsa strain with mics of v, t, and l of , , and . mg/l, respectively. he was started on l ( mg bid) and r ( mg tid) and bacteraemia cleared the seventh day of treatment. he completed a -week course of l and a -week course of r. during his treatment he developed anaemia which was managed with blood transfusions and erythropoetin, mild leucopenia and mild thrombocytopenia. he was discharged afebrile with sterile blood cultures and a tee showing reduction in the size of the vegetation. the patient remained well and blood cultures were sterile one month later while pancytopenia fully recovered. the mrsa isolate was investigated for heteroresistance to glycopeptides with ( ) a simplified and ( ) a detailed population analysis profile method. ( ) . ml of a cfu/ml bacterial suspension was plated on bhi agar with v ( mg/l). subclones that grew after h were submitted to mic determination. ( ) tenfold serial diluents of an inoculum of cfu/ml were plated on bhi agar plates with increasing concentrations of v or t alone or with % nacl. viable colonies were counted at h and plotted against the antibiotic concentration. subclones with v mic - mg/l were identified, suggesting that the isolate had heterogeneously reduced susceptibility to v which probably explained the failure of v treatment. the population curve showed that % of the original inoculum survived on t concentration ! mg/l suggesting heteroresistance to t. in all three studies, patients treated with lnz had shorter intravenous antibiotic treatment (ivat) duration than patients in comparator groups, which results in increased rates of early patient discharges and reduced use of resources. in two of the studies, patients had significantly shorter mean los and greater odds of early discharge from hospital. indeed, compared with tei, treatment with lnz had % greater odds of early discharge (p ¼ . ); a similar early discharge potential was also seen when lnz was compared with van (p ¼ . ). in select patient populations, such as those with cssti due to suspected/confirmed mrsa, reduction in los may be even more pronounced in lnz-vs. van-treated patients. for the cost comparison in the third study, total mean adjusted cost was also reduced by us $ (p > . ) in the lnz group compared with the tei group in patients from south america and mexico. conclusions: across multiple studies, there is consistent evidence of significant reductions in los and ivat associated with lnz treatment, with significant differences in the rate of early patient discharge. therapy with lnz shows pharmacoeconomic advantages that have the potential to reduce total costs of treatment. in vitro activity against mrsa and its oral administration represents an excellent alternative to iv vancomycin, which is currently recommended for cf patients colonised with mrsa. material and methods: oral lnz ( mg/ bid) was administrated in two male cf patients ( and years) during and days, respectively. s. aureus isolates were cultured from different sputum samples recovered before, during, and after lnz treatment. antibiotic susceptibility was performed by nccls microdilution method using the wider system (fco. soria melguizo, s.a., madrid, spain). pfge-smai was applied to analyse genetic relatedness of s. aureus isolates. results: in the first patient, a total of isolates were analysed during the studied period; six of them recovered in the previous year to lnz administration, two isolates during lnz administration period, and two isolates and months after the end of treatment. with the exception of one isolate that was methicillinsusceptible recovered during lnz treatment period, all isolates were mrsa and presented homogeneous antibiotic susceptibility pattern. a single clone, with a subtype variant that included two isolates, was identified in all isolates, except in the meticillin-susceptible one. in the second patient, two mrsa and one meticillinsusceptible isolates were recovered during months before lnz therapy. another methicillin-susceptible isolate was recovered after the lnz therapy and no s. aureus were identified in the following cf controls during months. mrsa isolates shared the same pfge and antibiotic susceptibility pattern, whereas meticillin-susceptible isolates corresponded to two different clones unrelated with the mrsa clone. independently of microbiology results, patients' pulmonary function remains unchanged after lnz administration. conclusion: oral lnz treatment in cf may affect population dynamics of s. aureus colonisation, being effective in mrsa eradication. despite this fact and assuming the brief follow-up period, maintenance or eradication of mrsa colonisation after lnz treatment seems not to affect pulmonary function, which may be related to the uncertain role of this pathogen in cf patients. p in vitro spectrum of linezolid and other agents against clinical isolates of anaerobes k. aldridge, c. manders, s. broyles new orleans, usa objectives: linezolid is an oxazolidinone antimicrobial with established in vitro and in vivo activity against aerobic gram-positive cocci. in infections such as wounds these gram-positive pathogens may be mixed with other pathogens including anaerobes. the role of linezolid as an anti-anaerobe agent has yet to be determined. this study was performed to establish the in vitro activity of linezolid and comparative agents against recently isolated anaerobes. methods: approximately anaerobes were tested for susceptibility to linezolid (lzd), ceftriaxone (axo), cefoxitin (fox), clindamycin (cl), and metronidazole (mrd) using twofold dilutions ( . - mg/l) of each agent using the nccls-recommended broth microdilution method. the sources of test isolates included wounds, abscesses, body fluids, and tissues. results: against all test isolates lzd had an mic range of . - mg/l, a mode mic of mg/l, and mic and mic values of and mg/l, respectively. lzd activity was judged by percentage of isolates inhibited at and mg/l. overall lzd inhibited and % of isolates at and mg/l, respectively; at and mg/l, respectively, lzd inhibited and % of bacteroides fragilis group; and % of clostridium isolates; and % of prevotella isolates; % of fusobacterium isolates; and % of peptostreptococcus isolates. by comparison of mic values lzd was -to -fold more active than axo, -to -fold more active than fox, and -to -fold more active than cl against these same groups of isolates. lzd and mrd had virtually equal in vitro activity. interestingly, all isolates with mics of mg/l or higher to lzd had mics of mg/l or less to mrd, while isolates with mics of mg/l or higher to mrd had mics of mg/l or less to lzd. conclusions: based on these results and arbitrary use of nccls breakpoints for gram-positive isolates, we conclude that lzd is highly active against anaerobe pathogens, but this needs to be verified by pharmacokinetic and clinical studies. results: on comparing the mics from the current study with the results from years ago, an unmistakable and very alarming decline in susceptibility was noted for all the antimicrobial agents tested. the greatest difference in susceptibility was noted for cefoxitin (from to %), metronidazole (from to %), piperacillin (from to %) and amoxicillin (from to %). the antimicrobial agents for which < % decrease in susceptibility was found, included meropenem (from to %), clindamycin ( to %) and ciprofloxacin (from to %). a great concern, however, was an % decrease found in the susceptibility for imipenem (from to %). conclusions: a decade ago, most anaerobic bacteria were susceptible to antimicrobial agents usually used for infections caused by these bacteria. the results from this study, however, indicate a situation that has undergone some dramatic changes in a relatively short period. it is of concern that the agents most frequently used in the empirical treatment of anaerobic infections, such as metronidazole and the b-lactams such as cefoxitin and piperacillin have shown the most alarming decrease in susceptibility. there is now, more than ever before, a definite need for continuous susceptibility testing of anaerobes and a serious restructuring of the treatment regimes for anaerobic infections. a. camarda, d. pennelli, p. battista, m. corrente, i. alloggio valenzano, i objectives: escherichia coli isolated from fattening-rabbits dead for enteritis were biotypised, tested with pcr for the presence of virulence genes eae and afr coding for intimin and the fimbrial adhesin af/r and investigated for antimicrobial resistance. methods: fifty-six strains of e. coli isolated in farms were biotypised using the fermentation of sorbose, dulcitol, raffinose, sucrose and l l-rhamnose. detection of drug resistance was determined using the method of kirby-bauer on mueller-hinton agar with antibiotic disks containing gentamicin (gm ), amikacin (an ), tetracycline (te ), erythromicin (e ), spiramicin (sp ), enrofloxacin (enr ), flumequine (ar ), trimethoprim/ sulphametoxazole (sxt), amoxicilline (amx ), apramycin (apr ), difloxacine (dfx ), marbofloxacine (mar ), nalidixic acid (na ), neomycin (n ), colistin (cl ), streptomycin (s ). results: biotypes (b , b , b , b , b , b , b , b , b , b ) were detected: biotypes and were predominant in rabbitries. eae and afr genes were almost observed in e. coli strains belonging to them. results of antibiograms have shown that all the isolates ( %) were e resistant. high rate of resistance were also found towards sp ( . %), sxt ( . %), te ( . %), s ( . %), gm ( . %), n ( . %). about % of e. coli tested showed the same susceptibility rate ( . %) to mar and cl . susceptibility to dfx , enr , ar , na , was exhibited by the . , . , , . %, of the strains, respectively. sensitivity against amx was quite high ( . %). multiple antibiotic resistance was expressed by all e. coli tested. the most prevalent resistotypes were resistant to te -e sp -sxt, detected in strains ( . %), te -e -sp -sxt-s , which accounted for over % and te -e -sp -sxt-gm detected in . % of isolates. conclusions: no significant correlation was observed between enteropathogenic e. coli (eae+ and af/r +) and pattern of antibiotic resistance. quinolones have shown very good activity; in particular mar , which has been recently adopted in veterinary medicine seems to possess high efficacy. on the other hand, e. coli strains exhibited high-level of resistance to antimicrobials. like human e. coli, rabbit strains revealed different patterns of multiresistance, which could make disease control difficult in rabbits and also promote dissemination and increasing of antimicrobial resistance in human strains. objective: to determine the frequency and susceptibility patterns of bacterial pathogens isolated from bloodstream (bsi) of haematology-oncology patients hospitalised at latin american medical centres. material and methods: as part of the sentry antimicrobial surveillance program, a total of bsi isolates were recovered from haematology-oncology patients from to . the isolates were susceptibility tested to > antimicrobial agents in a central laboratory using nccls broth microdilution method. results: the most frequent isolated pathogen was coagulase-negative staphylococci (cons; . %), followed by escherichia coli ( . %), staphylococcus aureus ( . %), klebsiella pneumoniae ( . %), pseudomonas aeruginosa ( . %), enterobacter spp. ( . %), acinetobacter spp. ( . %), and enterococcus spp. ( . %). oxacillinresistance rates were . and . % among s. aureus and cons, respectively, isolates. the prevalence of esbl-producing strains ranged from . % for e. coli to . % for k. pneumoniae. for enterobacter spp., susceptibility rates were . and . % to ceftazi-dime and cefepime, respectively. all enterobacteriaceae isolates tested were susceptible to carbapenems. the susceptibility of p. aeruginosa to imipenem and meropenem was . and . %, respectively; . and . % of the gram-negative bacilli were susceptible to cefepime and meropenem, respectively. only . % of the enterococcus spp. isolates were resistant to vancomycin. conclusions: in contrast to american and european reports, gram-negative bacilli represented the major cause of bsi among haematology-oncology patients in the latin american hospitals evaluated. the antimicrobial agents with the best covered against such pathogens were the carbapenems and cefepime. however, none of the evaluated antimicrobial agents inhibited the growth of . % of the gram-negative bacilli. thus, continued monitoring by surveillance programs is necessary to determine if the observed trends would continue to be recorded. objective: we attempted to verify if the frequency of occurrence (fo) and antimicrobial susceptibility profile (asp) of bacterial isolates responsible for causing bloodstream infections (bsi) in paediatric patients varied along the years and age categories. methods: a total of bloodstream isolates were collected from paediatric patients hospitalised in latin american hospitals through the sentry program between and . the asp to various antimicrobials was determined by the nccls broth microdilution method. the fo and asp were studied according to age categories (ac): year, - years, and - years. results: overall, s. aureus (sa) was the most frequently isolated pathogen among children year ( . %) and - years ( . %) followed by coagulase negative staphylococci (cons). among children year and - years, s. pneumoniae (spn) ranked among the top five pathogens. in contrast, it has caused less than . % of bsi among children - years. curiously, in this age group, acinetobacter spp. and p. aeruginosa ( . %) assumed the fifth position in the rank order of frequency. in general, among sa, the oxacillin resistance (or) rates were lower in the - -year-old ac ( . %; p . ) than in children year ( . %) and - years ( . %). in contrast, among the cons, elevated rates of or were noticed in all acs ( year, . %; - years, . %; - years, . %; p > . ). esbl-producing k. pneumoniae were more frequently detected in the ac year ( . % of the k. pneumoniae isolates) and - years ( . %) than - years ( . %). on the contrary, esbl-producing e. coli isolates were less frequently encountered among children year ( . %) than children ! years ( . %). however, these differences did not reach statistical significance (p > . ). spn isolates showing reduced susceptibility to penicillin were detected more frequently in the ac of year ( . %) and - years ( . %) than in - years ( . %; p . ). conclusions: although only slight differences in the fo of bsi pathogens was noticed along the years and ac, important differences were observed on the asp of the bsi pathogens according to the age categories, especially for spn and sa isolates. objectives: the aim of this prospective, multicentric study was to assess incidence of gram-positive bacteria in bloodstream infections (bsi) and characteristics of their resistance to antibiotics in the czech republic. methods: the study was done in sites in the czech republic from january to april . consecutive gram-positive strains isolated from blood were assessed and their clinical significance was evaluated. results: the strains of staphylococcus aureus ( %), coagulase-negative staphylococci ( %), streptococcus pneumoniae ( %) and enterococcus spp. ( %) were identified as the etiologic agent of gram-positive bsi. the frequency of oxacillin-resistant strains was in staphylococcus aureus and in coagulase-negative staphylococci and %, respectively. all streptococcus pneumoniae strains were susceptible to penicillin and chloramphenicol. no strains resistant to glycopeptides were found in enterococci. clinical significance of isolated gram-positive bacteria was significantly conditioned by bacterial species (p ¼ . ) and reached % in streptococcus pneumoniae, % in staphylococcus aureus, % in enterococcus spp. strains and % in coagulase-negative staphylococci. production of bacterial biofilm was shown in % staphylococcus aureus strains and in % coagulase-negative staphylococci. bsi was the immediate cause of death of the patient in %. conclusion: we could confirm that presence of artificial material means significant risk factor for bsi. catheter-related infections were present in % of cases. forty-six per cent of bsi can be characterised as secondary and pneumonias, git infections and urinary tract infections were the most common sources. the frequency of staphylococcus spp. with positive finding of biofilm was % in this study; this finding supports its clinical significance. methods: a total of s. aureus positive sample isolates between january and december from the laboratory were analysed. the susceptibility to antibiotics was assessed by antibiogram based on the api system (biomérieux Ò ) according to the french guidelines (ca-sfm). the sa strains were classified as methicillin susceptible and gentamicin susceptible (gentas-mssa), methicillin resistant and gentamicin susceptible (gentas-mrsa), methicillin susceptible and gentamicin resistant (gentar-mssa) or methicillin resistant and gentamicin resistant (gentar-mrsa). the number of isolates was calculated for admissions. means per year were compared using kruskal wallis test. the spearman coefficient (r) was used to calculate the correlation between the proportion of isolates (for each antibiotic resistance profile) and months. results: the overall proportion of sa positive samples for admissions during the study period was: . , . , . , . , . and . for , , , , and , respectively (r ¼ À . ; p ¼ . ). the percentage of mssa was . ( . for gentas, . for gentar) and the percentage of mssa was . ( . for gentas, . for gentar) for the total period. patients with mrsa were older ( . years) compared with patients with mssa (mean age . , p < . ) but patients with gentar-mrsa were younger ( . years) compared with patients with gentas-mrsa (mean age . , p ¼ . ). the proportion of gentas-mssa for admissions was similar by time ( . , . , . , . , . , . , p ¼ . ) (r ¼ À . ; p ¼ . ). however, the proportion of gentas-mrsa strains increased significantly ( . , . , . , . , . , . , p ¼ . ) (r ¼ . ; p < . ) while the proportion of gentar-mrsa strains decreased significantly during the period ( . , . , conclusion: although the proportion of sa positive samples for admissions remains constant during the last years, there is a continuous increasing trend of isolates with gentas-mrsa and a decreasing trend of isolates with gentar-mrsa. the age difference between these two sub-groups should be explored. greek region -corfu island e. gatsouli, m. ovrenovits, a. pasxali, a. tzanavari corfu, gr background: in order to assess the regional trends of microbiological resistance pattern, all cultured bacteria isolated in in our laboratory were reviewed as to specimen source and susceptibility profile. materials and methods: in , samples were cultured, % ( ) of hospitalised patients and % ( ) from ambulatory patients. the samples were: urine, blood cultures, lesions and samples of other secretions. classic culture methods, vitek system and nccls breakpoints were used. results: cultivations were positive in % ( , adults and children samples). the distribution of bacteria differed according to the types of specimens. the distribution of gram(À) was enterobacteriaceae and nonfermentative bacilli. there were gram(+) cocci and yeasts, too. e. coli predominated in enterobacteriaceae ( %), followed by klebsiella sp., p. aeruginosa in non-fermentative bacilli ( %) and a. baumanii ( %). among the gram(+) s. aureus was the most frequent ( . %), followed by cns. ampicillin inhibited growth of % for e. coli. ttime/sulfa combination could inhibit less than % and the second-generation cephalosporins less than %, while fluoroquinolons were very effective against enterobacteriaceae strains (more than %). piperacillin inhibited growth of % of p. aeruginosa and quinolons less than %. enterococcus strains were highly sensitive to teicoplanin ( %) and nitrofurantoin ( . %). mrsa were % but gisa were %. a. baumanii and gisa were in icu. conclusion: a permanent surveillance of frequency and sensitivity levels of the most common pathogens responsible for infectious enables to identify local antimicrobial activity and plays a key role in starting empiric therapy pending bacterial identification and in vitro assay. objectives: the biochemistry and genetics of antibiotic resistance are well documented; however, information regarding the medical and social factors that influence its occurrence remains lacking. the aim of this study was to elucidate these latter relationships and to examine the dynamics of their effects. methods: antibiotic resistance data for bacterial isolates obtained from the community was collected from all microbiology laboratories in wales from to . antibiotic prescribing data, practice demographics, deprivation indices, general practitioner demographics, and details of sampling behaviour was also obtained for the same period for all general practices in wales. initial analyses exploring the nature of these data and the relationships of the various components were undertaken using excel and spss. results: preliminary analyses indicate that both antibiotic resistance and prescribing varied between practices. for coliform utis, there was a clear association between high prescribing and higher levels of resistance, with prescribing accounting for - % of variation in resistance. the correlation between prescribing and resistance was not confined to the urinary coliforms but seen throughout a range of pathogens including those responsible for respiratory and skin infections. there was an association between resistance and social deprivation exceeding that expected from high prescribing in deprived areas and an apparent association between resistance and the number of practitioners in a practice and the practice list size. resistance was more common in infections in the young (< years), the aged (> years) and for some pathogens resistance was significantly greater in males. multilevel modelling, regression analysis and time series analysis of this complex data set is in progress. conclusions: antibiotic usage appears to affect resistance at practice level and the dynamics of this selection process are currently being investigated. it is hoped that these studies will assist in the design of interventions to limit the future impact of resistance and contribute to our ability to predict their outcomes. objectives: data about the prevalence of antimicrobial resistance in indonesia are limited. the amrin study measured the prevalence of antimicrobial resistance in the indonesian population inside and outside hospitals. methods: individuals were targeted to be screened constituting four different populations in each of two cities: patients admitted to hospital, patients discharged from hospital, patients visiting primary health centres, and relatives of patients admitted to hospital. nasal swabs and rectal swabs were taken and cultured using phenol red mannitol agar for the isolation of staphylococcus aureus, and chrom agar orientation medium for escherichia coli. susceptibility testing was performed by disk diffusion method recommended by nccls. results: individuals were included in the study between july and october in surabaya and between january and may in semarang equally distributed over the four groups and two cities. s. aureus isolates (n ¼ ) were frequently resistant to tetracycline ( %) and oxacillin ( %) without obvious differences between the four populations. none of the oxacillin resistant strains of s. aureus harboured mec a gene. e. coli isolates (n ¼ ) showed considerable levels of resistance against a number of commonly used antibiotics. the highest levels of resistance to ampicillin ( . %), chloramphenicol ( . %), gentamicin ( . %), cefotaxim ( . %), ciprofloxacin ( . %), and cotrimoxazole ( . %) were among e. coli isolated from patients on the day of discharge from hospitals. resistance rates were consistently lowest among e. coli from relatives of patients on admission to hospital and among patients visiting primary health care centres. conclusions: the results show that antimicrobial resistance among common bacterial pathogens has emerged in indonesia. among e. coli the prevalence of resistance to ciprofloxacin and other antibiotics is remarkable high, especially in individuals after hospitalisation. although the prevalence of mrsa is low, tetracycline resistance is common among s. aureus and not associated with hospital stay. methods: isolates from patients with invasive diseases caused by haemophilus influenzae (hi), neisseria meningitidis (nm), group a streptococcus (gas), and group b streptococcus (gbs) were forwarded to reference laboratories in alaska ( alaska ( - , canada ( canada ( - , and greenland ( greenland ( - for confirmation and serotyping. chart reviews were conducted on confirmed cases to verify illness episode information. data reported for are preliminary. results: the total numbers of reported cases were hi, nm, gas, and gbs. crude annual rates of invasive disease per population varied by country and organism [hi conclusion: native peoples of ak and n can have high rates of invasive bacterial disease caused by hi, nm, gas and gbs. overall rates of nm disease are higher in gn than ak and n can. cases of invasive hib disease continue to occur in children < years of age. rates of hia appear to be elevated in n can and increasing in ak, however, caution needs to be used when interpreting rates due to the small number of cases. this trend merits further surveillance. elevated case fatality rates in ak for hi and nm also warrant further investigation. objectives: for tertiary care hospitals, knowing the local patterns of spectrum and susceptibility at the referring institutes can add significantly to the selection of appropriate antimicrobial therapy. our objective was to get information regarding the region specificity, frequency of occurrence and pattern of antimicrobial susceptibility of common bacterial infections in central illinois. methods: we used hospital antibiogram data to assess predominant pathogens and pattern of in vitro antimicrobial susceptibility of bacterial infections in the four regions (west, southwest, central and south) of central illinois from january to june . results: gram-negative bacteria were predominant in four regions ( , , and % respectively). in all regions, e. coli was the most common organism ( , , , and %) followed by s. aureus ( , , , and %). e. faecalis, p. aeruginosa, and k. pneumoniae were also among the five most frequently reported species. on the other hand, the frequency of occurrence of s. pneumoniae was - % in the four regions. the pattern of methicillin-resistant s. aureus was different in the four regions ( , , , and %) with only . % of the total number of s. aureus showing intermediate resistance to vancomycin. e. faecalis, , , and %, respectively, were susceptible to vancomycin. susceptibility of s. pneumoniae to penicillin was almost the same in the four regions ( , , , and %). it was not surprising that p. aeruginosa was the least susceptible species among gram-negative bacteria, and this species showed decreased susceptibility to gentamicin ( , , , and %) and to ciprofloxacin ( , , , and %). conclusions: our data show that different communities in central illinois have variable occurrence and pattern of antimicrobial susceptibility of common bacterial infections. we plan to formulate a regional antibiogram, distribute it to all hospitals in the area, and follow the patterns prospectively with renewal of the antibiogram once a year. p antimicrobial resistance surveillance of gramnegative anaerobic bacteria isolated in six greek hospitals j. papaparaskevas, n.j. legakis, a. katsandri, a. avlamis -the hellenic study group for gram-negative anaerobic bacteria objectives: the antimicrobial resistance surveillance of gram-negative anaerobic bacteria isolated in six greek hospitals. methods: a total of gram-negative anaerobic clinical strains ( bacteroides fragilis group, other bacteroides spp. non-fragilis, prevotella spp., fusobacterium spp. and miscellaneous) isolated during the period november to november were tested using the etest method on brucella blood agar plates. incubation in a chellab . anaerobic chamber was performed for h and interpretation was according to nccls guidelines. results: overall gram-negative non-susceptible (intermediate and fully resistant) rates to penicillin, ticarcillin + clavulanic acid, cefoxitin, tetracycline, clindamycin, metronidazole, imipenem and ertapenem were , , , , , , and %, respectively. bacteroides fragilis group rates were , , , , , , and %, respectively. prevotella spp. rates were , , , , , , and %, respectively. overall gram-negative mic s were , , , , , , . and mg/l, respectively. bacteroides fragilis group mic s were , , , , , , and , respectively. prevotella spp. mic s were , , , , , , . and . , respectively. metronidazole resistance was detected among four prevotella spp., one bacteroides spp., one porphyromonas spp. and one fusobacterium spp. isolates. additionally, a b. fragilis strain was found highly resistant (mic > mg/l) both to imipenem and ertapenem and resistant to all other antimicrobials tested except metronidazole. conclusions: carbapenems, beta-lactam + inhibitor combinations and metronidazole remain the antimicrobial agents of choice against most gram-negative anaerobes. however, metronidazole resistance seems to be an emerging problem in greece, especially among prevotella spp. isolates. in that respect species identification and periodic susceptibility surveillance is mandatory. imipenem and ertapenem activity was comparable, though ertapenem mics were slightly higher. acknowledgements: members of the hellenic study group for gram negative anaerobic bacteria are drs a. avlamis, c. koutsia-karouzou, c. kontou-kastelanou, a. pangalis, e. papafrangas and e. trika-grafakos. the value of quality control strains in susceptibility tests k. huppertz, i. noll, b. wiedemann and the genars group objectives: the goals of a quality control programme are to assist in monitoring the precision and accuracy of the susceptibility test procedure, the performance of reagents used in the test and the performance of persons who carry out the tests and read the results. they are best accomplished by the testing of quality control (qc) strains with known susceptibility to the antimicrobial agents to be tested (nccls). therefore, qc strain measurements done by laboratories taking part in the genars-project (german network for antimicrobial resistance surveillance) were used for a comparison of the performance of three different methods for mic determination. methods: in the genars-project two commercial mic test systems and one manual microdilution system according to nccls are used for the determination of antimicrobial susceptibility. the commercial systems are the vitek (biomérieux) and the micronaut system (merlin diagnostics) with -well microtitre-plates. qc strains measured by all test systems were evaluated for those antibiotics where a range of ae dilution step of the modal value of the respective qc strain is included in the range of concentrations tested. for reliable assessment of the test quality the distance of the modal value from the lowest and highest concentration tested has to be two or more dilution steps. results: from a multitude of antibiotics tested only few drugs are tested with a range of concentrations which meets the above mentioned requirements. table indicates the number of test combinations available for evaluation. the vitek system offers the shortest ranges of concentrations. however, from the range of concentrations only few are tested, while the others are calculated, e.g. for gentamicin the range includes six concentrations while only three are measured (ast-p ). conclusions: an evaluation of qc strain measurements should be possible for all antibiotics tested. however, due to the concentrations chosen and the short ranges of concentrations available in the different test-systems only few antimicrobial agents can be used for a comparison of the performance of the test methods. therefore, either the range of concentrations has to be extended, or more suitable qc strains have to be implemented in a way that their mics fall into the range of concentrations which are sufficient in clinical terms. p lack of evidence for dna in antibiotic preparations as a source of antibiotic resistance genes s.k.p. lau, p.c.y. woo, a.p.c. to, a.t.k. lau, k.y. yuen hong kong, hk objective: to investigate the significance of dna encoding antibiotic resistance genes present in antibiotic preparations in the rapid development of antibiotic-induced antimicrobial resistance. methods: a comprehensive study using sequence alignments and phylogenetic analysis of genes encoding antibiotic resistance in antibiotic-producing bacteria and the corresponding ones in nonantibiotic-producing human or animal bacterial isolates [erythromycin resistant methylase (erm), aminoglycoside -phosphotransferase (aph ), aminoglycoside -phosphotransferase (aph ), aminoglycoside acetyltransferase (aac), class a beta-lactamase, tetracycline resistance efflux protein, tetracycline resistance ribosomal protection protein and vancomycin resistance proteins (vana, vanh, vanx) and bacitracin transport proteins (bcra, bcrb, bcrc)] was carried out. if dna encoding antibiotic resistance genes present in antibiotic preparations has been important in the development of antibiotic resistance, genes of almost identical amino acid sequences would be expected to be present in antibiotic-producing organisms and other human or animal bacteria, inferring that horizontal transfer of antibiotic-resistance genes had occurred from the former to the latter. results: the maximum amino acid identities of genes among different non-antibiotic-producing bacterial isolates were close to % for most genes, but those between antibiotic-producing and human or animal bacteria ranged from < to < %. therefore, recent horizontal transfer of antibiotic resistance genes has not occurred from antibiotic-producing organisms to human or animal bacteria. on the other hand, frequent horizontal transfer of antibiotic resistance genes was observed among the human or animal bacteria, even if they were phylogenetically distantly related. moreover, such transfer was particularly common among gastrointestinal tract flora or pathogens. conclusion: dna encoding antibiotic resistance genes in antibiotic preparations has not been an important source of antibiotic resist-ance genes. dna decontamination during the process of antibiotic synthesis is probably not necessary. the human gastrointestinal tract has been an important place for bacterial gene exchange. the role of the human gut in the dissemination of antibiotic resistance should be further investigated. enterococci and other gram-positive bacteria p glycopeptide-resistant enterococci (gre) have emerged as important pathogens since the late s. an important factor associated with the appearance of gre in the community in europe has been avoparcin, a glycopeptide antimicrobial drug used for years in many european countries as a growth promoter in food-producing animals. in europe, evidence suggests that food-borne gre may cause human colonisation or infection. objectives: the objective of this study was to investigate the prevalence and to determine the genotypes of gre from different human and animal sources in styria, austria. methods: stool specimens from each patients with precedent antibiotic therapy and non-hospitalised humans without precedent antibiotic therapy, faecal cattle specimens, faecal pig specimens and faecal poultry specimens were collected in . one millilitre of diluted faeces was added to ml of enterococcosel bouillon (bd) for enrichment. after incubation, ml was subcultured on vre screen agar (bd). species identification was performed with the api strep systems and vitek (bio-mérieux). resistance to vancomycin and teicoplanin was determined by the e-test method (ab biodisk). determination of glycopeptide resistance genotypes (vana, vanb, vanc , vanc / ) was performed by pcr. results: % of the patients with precedent antibiotic therapy harboured vre. among these, two were identified as e. faecium vana, two as e. gallinarum vanc and e. casseliflavus vanc , respectively. eight per cent of the non-hospitalised human specimens contained vre (six e. gallinarum, two e. casseliflavus). a total of vre strains were isolated out of the animal samples, . % e. faecium, . % e. gallinarum, and . % e. casseliflavus strains. no resistant e. faecalis strains were detected. pcrs confirmed that all e. gallinarum were of the vanc , all the e. casseliflavus of the vanc and all the e. faecium strains of the vana genotype. about . % of all e. faecium vana strains were isolated out of the poultry samples. one strain was isolated from a cattle sample, no specimen from pigs yielded glycopeptide-resistant e. faecium. conclusion: the present study indicates that the prevalence of gre in humans and in pig and cattle husbandry appears to be low, but it reveals a high prevalence of gre (e. faecium) in styrian poultry years after the use of avoparcin was banned. glycopeptide resistant enterococci (gre) have become an increasing problem in the us and in europe. enterococci are intrinsically resistant against cephalosporins, aminoglycosides (low-level), polymixins, lincomycin and clindamycin. furthermore, enterococci are able to acquire resistance to a wide range of antibiotics. there remain concerns that antibiotic use for growth promotion, prophylaxis and therapy in animal husbandry may lead to increased resistance to antibiotics used in human medicine. objectives: the aim of this study was to evaluate the species distribution and the antibiotic resistance of gre isolated from styrian food-producing animals. methods: a total of gre strains isolated from cattle, pig and poultry faecal specimens in were collected. the strains were identified using the vitek automated methods (gpc) and the api strep systems (biomérieux). antimicrobial susceptibilities were determined by vitek p card and by disk diffusion (linezolid). the strains were studied for susceptibility to antibiotics: ampicillin (am), amoxicillin/sulbactame (amc), ciprofloxacin (cip), erythromycin (ery), gentamicin high level (ge), linezolid (li), norfloxacin (nor), penicillin (p), quinupristin/ dalfopristin (syn), streptomycin high level (str), teicoplanin (tp), tetracycline (te) and vancomycin (va). results: e. casseliflavus was the most common gre species isolated ( . %), followed by e. gallinarum ( . %) and e. faecium ( . %). all e. gallinarum and e. casseliflavus were of the vanc, all e. faecium of the vana phenotype. all investigated strains were sensitive against linezolid and gentamicin high level. p and am resistance ( . %) and reduced susceptibility to cip ( . %) was seen in e. faecium only. ery resistance for e. faecium revealed . %, for e. casseliflavus . % and for e. gallinarum . %. resistance against te for e. faecium was . %, for e. casseliflavus . % and for e. gallinarum . %. about . % of e. faecium strains were not susceptible to quinupristin/dalfopristin. conclusions: resistance phenotypes to p, am, cip, ery and te differed among enterococcus species. resistances found against tetracyclines, quinupristin/dalfopristin and erythromycin are causes of concern. high levels of antibiotic and multidrug resistance were observed among the e. faecium strains. the identification was carried out by the vitek system (biomerieux). the susceptibility test was performed either by the breakpoint system:mini api or the vitek system (biomerieux). results: ( %) and ( %) streptococci strains were isolated from outpatients' and inpatients' urine cultures, respectively. the distribution by sex was % women/ % men in outpatients and % women/ % men in inpatients. a total of ( %) of streptococci strains were enterococcus faecalis, ( %) were enterococcus faecium, ( %) were enterococcus gallinarum and ( %) were streptococci group b. the in vitro antibiotic resistance of enterococci spp. was: penicillin . % ( / ), ampicillin % ( / ), gentamicin % ( / ), nitrofurantoin . % ( / ), ciprofloxacin % ( / ), tetracyclines % ( / ), vancomycin % ( / ), linezolid %. eight vre strains were enterococcus faecium, three were enterococcus gallinarum and two were enterococcus faecalis. the in vitro antibiotic resistance of group b streptococci was: vancomycin %, nitrofurantoin . % ( / ), ampicillin . % ( / ), penicillin . % ( / ), erythromycin . % ( / ), tetracyclines % ( / ). conclusions: streptococci are responsible only for the . % of urinary tract infections. enterococcus faecalis was the most frequent pathogen ( %). enterococci spp. showed high resistance in ciprofloxacin, tetracyclines, gentamicin, penicillin, and ampicillin. objectives: enterococcal infections are becoming an increasing concern, particularly due to the emergence and spread of resistance to animicrobial agents. we have investigated the phenotypic and genotypic properties of enterococcus faecium clinical isolates, expressing resistance to the combination of quinupristin/ dalfopristin, recovered during a -year period in the university hospital of patras. methods: all isolates were characterised at species level by gram stain, catalase production and by the crystal id gram positive system (bbl). minimal inhibitory concentrations (mics) to ampicillin (amp), erythromycin (em), chloramphenicol (chl), gentamicin (gm), ciprofloxacin (cip), vancomycin (va), teicoplanin (tp), quinupristin/dalfopristin (rp) and linezolid (lin) were performed by the e-test (ab biodisk) according to nccls recommendations. the presence of vana and vanb genes was investigated by the evigene commercial kit (statens serum institut), while the presence of vga, vgb and sat genes by pcr with specific primers. clonal types were characterised by pfge of smai dna digests. results: in a collection of e. faecium, ( %) expressed mic of rp ! mg/l, and among them isolates ( %) showed mic > mg/l. high-level resistance to gm was detected in ( %) isolates, ( %) to cip, ( . %) to chl, ( %) to amp and ( %) to em. forty-three ( %) isolates were vancomycinresistant, carrying the vana gene. no isolate was found to carry vga, vgb and sat genes. pfge classified isolates to clonal type a, to type b, to type c and the remaining isolates belonged to more types. conclusions: high prevalence of low-level resistance to quinupristin/dalfopristin (mic: - mg/l) was detected in this collection of e. faecium, with strains expressing higher mic levels. this was mainly due to the dissemination of certain clones in the hospital. in a previous work, we described the dissemination of renc and renc e. faecalis multiresistant clones colonising patients of four different icus. the renc clone was frequently found in bacteraemias, suggesting a blood invasion from an intestinal origin. the aim of this study was to analyse the dynamic population evolution of enterococcal intestinal isolates and if the acquisition of epidemic hospital clones may occurs during icu admittance. material and methods: a close follow-up of four patients from the neurosurgery icu who were admitted after acute traumatism was performed. rectal swabs were collected at the admittance and daily until they were discharged from the icu. stool samples were seeded in m-enterococcus agar, eventually supplemented with selective antibiotics, and multiple colonies were analysed in each sample. pfge-smai and the phoretrix . software were applied to analyse the genetic relatedness among these isolates and the previously described hospital endemic clones. results: patient and stayed in the icu for days, and patient and for days. patient carried along the days the original e. faecalis and e. faecium clones. moreover, five e. faecalis clones, one identical to the epidemic clone renc , and one e. faecium clone were acquired during the icu stay, all of them persisting over the rest of the studied period. patient presented at admission three e. faecalis and two e. faecium clones; two e. faecalis were lost in days, and e. faecium were lost at the second day. four new e. faecalis and one e. faecium clones were found during all stays, whereas five more clones were occasionally isolated without persistence. in patient an e. faecium clone was identified along all the studied period, and two new e. faecium clones were later acquired. patient had two e. faecium clones at admission, one of them being lost after the first day; the second persisted during all days; a new e. faecium clone was acquired during the icu stay. patient and methods: the patient was a -year-old woman hysterectomised years ago. she reported four surgical interventions due to a cystocele. the last operation took place years ago and she reported no further admittances at any hospital. in the last years the patient also suffered from repeated urinary tract infections. in the present episode she consulted because of typical uti symptoms (dysuria, bladder tenesmus) and a urine sample was collected. after h of incubation, a gram positive coccus was isolated (more than ufc/ml). the identification and susceptibility were preliminarily achieved by a commercially available method following manufacturer's recommendations (microscan, dade). identification was confirmed by api rapid strep system (biomerieux). to discard enterococcus species intrinsically resistant to vancomycin the absence of motility was observed with direct microscopic detection and the absence of pigmentation was determined by culture on tsa agar. susceptibility to vancomycin, teicoplanin and ampicillin were assessed by disk diffusion, e-test and broth mcrodilution. results: the isolated microorganism was identified as enterococcus faecalis and showed high mics to vancomycin (> mg/l by broth microdilution and mm by disk diffusion) and teicoplanin ( mg/l by broth microdilution and mm by disk diffusion) but was susceptible to ampicillin ( . mg/l by broth microdilution). to characterise the resistance mechanism involved in a series of vancomycin resistant enterococcus faecium (vref) strains recovered in two spanish hospitals of the same city, and to determine their clonal relationship. methods: a surveillance programme was carried out during a -year period in ms-hospital in order to detect vref intestinal colonisation. seven vref strains were recovered from seven faecal samples which represents < % of vref intestinal colonisation. in the same period, four clinical vref strains, implicated in infectious processes were recovered in ms-hospital (n ¼ ) and rv-hospital (n ¼ ). all vref strains (n ¼ ) were recovered from unrelated patients, most of them previously treated with glycopeptides or broad spectrum antibiotics and diagnosed with severe diseases. antibiotic susceptibility testing was performed by agar dilution method and vancomycin resistance genes (vana, vanb, vanc , vanc- / , and vand) were studied by pcr. vanb amplicons were sequenced to determine the subtype and the vanb cluster of genes were also characterised. other resistance genes were studied by pcr: aph( ¢)-iiia, ant( ¢)-ia and erm(b). pfge assays were performed with smai digestion. results: nine of the vref strains (eight of ms-hospital and one of rv-hospital) showed a vanb phenotype [mic (mg/l): vancomycin ( - ) and teicoplanin ( . )]. the vanb gene was detected in these nine strains and in addition, the intergenic vansb-yb region showed the characteristic mutations of the vanb subtype. the vanb gene cluster was integrated into the tn like element in all of them, as it was demonstrated by specific pcrs and sequencing. these strains were resistant to streptomycin, kanamycin and erythromycin and ant( ¢)-ia, aph( ¢)-iiia and erm(b) genes were detected by pcr. all of them were included in the same pfge clonal type a and two closely related subtypes were distinguished: a (seven strains from both hospitals) and a (two strains from ms-hospital). both subtypes were found in clinical strains as well as in strains recovered from faecal samples. only three of them were from serious infections, the rest was isolated from carriers. all but one present vana phenotype and harbour vana gene. the only vanb harbouring strain was resistant to teicoplanin. the outbreak at haematology was at the beginning polyclonal ( pfge clones) but eventually three of them became predominant in both wards. the outbreak in cracov centre was spread to two other wards of this hospital (surgery and geriatry) with , and vrem isolated up to now, respectively. five were from serious infections, were from wounds in the surgery, the rest represents for carriers detected during infection-control measurements. all but two of them were vana phenotype/genotype ( vanb phenotype/genotype isolates). one predominant pfge clone was observed, differentiated into pfge sub-types ('hospital clone'). five other pfge clones detected seemed to be unique (one to five isolates). in both outbreaks two basic mechanisms of vre spread were detected, clonal spread of vre strains and the vana-elements horizontal transfer. conclusion: after time of vrem presenting vanb phenotype caused sporadic outbreaks two of haematology centres in poland become the stages of multi-drug-resistant vana vrem outbreaks, eventually turning into endemic. the colonisation rate was - times higher than infection in both cases. the danger of transmission to other centres and non-haematological hospitals in the country appears very high in these circumstances. material and methods: thirty-three selected vrefls from different patients at three hospitals (huc, hsa and hst) in the north and centre of portugal ( portugal ( - were studied. susceptibility to antibiotics was performed by the agar dilution method (nccls). isolates were searched for genes coding for resistance to glycopeptides, macrolides, and aminoglycosides. tn characterisation was done by an overlapping pcr strategy and sequencing when necessary. clonal relatedness was performed by smai-pfge. virulence traits (cyl, agg, gele, esp) were investigated by a multiplex pcr assay. results: all vrefls showed vana phenotype and were mostly resistant to ery, cipro, hlrgm, and hlrkm ( , , , and %, respectively). resistance genes found were vana, erm(b), aac -aph , and aph -iiia. nine pfge types were isolated: eight from eight patients and one (clone b) from patients. clone b was disseminated among the three hospitals for years giving eight pfge subtypes, each one characteristic of a specific hospital. vsefls showing pfge patterns identical to two clone b subtypes were found in hst. six variants of tn were found, five of them among isolates of clone b. tn -pp was found in all hospitals for years and predominates in huc and hst. it contains an isef insertion in the intergenic vanx-vany region. tn -pp , only found in hsa, lacks genes involved in transposition. pp and pp were variants of pp and were recovered at huc. pp was a pp- variant found in hsa. all vre but one isolate of clone b were agg+ and gel+. cyl and esp were present in % of the vre. conclusion: our findings indicate that the dissemination and establishment of successful e. faecalis clones in the hospital setting amplify particular genetic determinants in local metagenomes resistant to vancomycin, and therefore influences future evolutionary events. we also report the first tn -variant containing an isef insertion. staphylococcus spp. is widely distributed in medical and veterinary pathology and represents one of the most important causes of infection. many strains are antibiotic-resistant even for the presence of an eso-polysaccharide matrix. the aim of this work was to individuate, among different staphylococci of human and animal origin, the slime producing strains and to correlate the presence of biofilm to the resistance to eight antibiotics. a total of coagulase negative staphylococci (cns) and s. aureus isolated from different sources and identified with sceptor system, were tested for antibiotic susceptibility (kirby bauer method) and for slime production (polystyrene plates -stained with alcian blue -spectrophotometric reading at nm). the strains were classified as weak, strong and no slime-producing on the basis of od results. the results were submitted to statistical analysis using student's t-test and chi-square tests. evaluating the differences of slime production among medical and veterinary strains, we found different statistical frequencies (p > . ). no statistical differences were obtained between s. aureus and the other cns. instead, the statistical analysis on s. epidermidis vs. the other staphylococci has shown no statistical differences among average values using student's ttest (p < . ) and significant frequency differences using chi square tests (p < . ). finally in the cns, between s. epidermidis and the other strains, no statistical differences were found. the relation between slime production and the origin of strains was evaluated and no correlation was found. about the correlation between antibiotic-resistance and slime production a resistance increment of about % was obtained in strongly slime producing strains. staphylococcus spp. is often involved in nosocomial infections as complication of post-surgery wounds, catheters and orthopaedic devices. the presence of antibiotic-resistant strains interferes in the therapy successes and seems to be strictly related to biofilm production beyond that genetically acquired. human and veterinary strains have shown a similar behaviour towards biofilm production and antibiotic-resistance. the results confirm that s. epidermidis is one of the most slime-producer and introduce s. aureus as a new high slime-producer. ( ) recommendations. the macrolide resistance phenotypes were determined using the erythromycin-clindamycin double disk test. results: all the s. agalactiae isolates tested were found susceptible to penicillin g and vancomycin while the resistance rate to erythromycin was . % (seven strains). the expression (%) of the macrolide resistance phenotypes among the resistant strains as they were evaluated by the double disk test were: constitutive (cmlsb) phenotype % (four isolates) and inducible (imlsb) phenotype % (three isolates). no s. agalactiae strain was assigned to the m resistance phenotype. the overall resistance rate to clindamycin was . %. conclusions: our findings demonstrate that s. agalactiae remains fully susceptible to penicillin and vancomycin while there are relatively low resistance values to macrolides and lincosamides. the mlsb phenotype predominated among the macrolide-resistant strains, a finding that raises concern about the use of clindamycin instead of erythromycin in prophylaxis or treatment of s. agalactiae infection in patients allergic to beta lactams. however, continuing surveillance is needed to detect any change in susceptibility patterns. effect of enterovirus infection on the risk of type diabetes mellitus has been studied mainly using indirect serological evidence of past infections, or using rt-pcr detection of the virus in plasma. with respect to enterovirus biology, we decided to assess the exposure to enterovirus using real-time rt-pcr detection and quantification from stool samples. this exposure is studied in relation to signs of autoimmune process ultimately leading to type diabetes. methods: the study population comes from the norwegian 'midia' study which screens newborns from the general population for the highest hla-encoded risk of type diabetes mellitus. the high-risk babies are followed-up by questionnaires, serum samples for markers of beta-cell autoimmunity, and stool samples collected in monthly intervals from month to month . the stool samples are collected by parents and mailed to the laboratory where rna and dna is co-purified on qiagen columns together with a low quantity of exogenous control rna. enterovirus is quantified by real-time rt-pcr using armored rna as a standard. control rna is detected in late cycles in the same reaction using a differently coloured probe reporter. adenovirus quantity is simultaneously investigated as a viral exposure which has not been implicated in triggering type diabetes. here we present the results of the pilot study. objective: there are conflicting reports regarding cmv-dna positivity among healthy cmv-seropositive individuals. we aimed to determine the frequency of cmv-dna positivity among healthy subjects and to evaluate its association with physical and mental stress in a longitudinal study. subjects and methods: weekly peripheral blood samples were drawn into from healthy cmv seropositive subjects aged between and years during a -week study period. each subject rated their physical and mental stress and they also recorded their alcohol consumption and any change in their health status. cmv dna was screened in plasma and peripheral blood leukocyte samples with a nested pcr using primers targeting mie gene of cmv. results: in total, samples ( plasma and peripheral blood leukocytes, each) were screened and only one peripheral blood sample obtained during the second week of the study gave positive result. this sample belonged to the oldest subject of the study. according to our results, cmv-dna positivity among healthy cmv seropositive individuals seems to be a rare event. results: all centres reported qualitative results; four centres also reported quantitative results. all samples were correctly identified by all centres. various extraction and amplification methods were used. fourteen centres reported results of internal controls. most of the centres controlled only the amplification step and did not adjust the detection sensitivity of the internal control to the detection limit of the target. three centres failed to detect one internal control in two positive samples and one negative sample. for quantification of hcmv dna all centres used real-time quantitative pcr. cv of hcmv dna load between centres were low ( . - %) except for one sample ( %), but this could be attributed to a heterogeneous preparation of this sample by the organisers. using student's t-test, no statistically significant difference was observed between hcmv load whatever the medium or the number of added cells. conclusion: results of this external quality assessment for molecular detection and quantification of hcmv dna were excellent. almost all centres used internal control of pcr inhibition; however, control of the whole pcr process, including extraction and better adjustment of the detection sensitivity of the internal control to the sensitivity limit of the pcr target is desirable. the most accurate way to identify false negative results, e.g. those caused by pcr inhibitors, in real-time pcr assays is to spike samples with an internal control that will be co-amplified with the target (pathogen) dna. however, current internal control procedures, which usually involve the introduction of a dna fragment, are complex, time consuming and expensive. we present a novel technique for simple internal control of real-time amplification assays. methods: single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time pcr assays. mismatches were included in the probe-binding region of the internal control oligonucleotide (ico) to prevent probe-control hybridisation during the fluorescence acquisition step of the pcr. icos could be added directly to the sample material prior to dna extraction. results: to demonstrate the feasibility of the new approach, we designed icos for the following lightcycler hybridisation probe assays: mycobacterium tuberculosis complex, hepatitis b virus, herpes simplex virus and varicella zoster virus. in each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis of the pcr products. conclusion: a single-stranded oligonucleotide, which mimics the target region of the pathogen yet is clearly distinguishable from the target during analysis, can serve as a simple, cost-effective internal control for real-time amplification assays. such control oligonucleotides are easy to design and cheap. a costly second probe system is not necessary. moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications. objectives: biomérieux has developed a new nucleic acid isolation method (nuclisens magnetic extraction reagents) that uses boom chemistry in combination with magnetic silica particles. the nuclisens mini mag instrument is facilitating the washing and collection of the silica particles in a user friendly and efficient way. in principle the extraction method is generic and can be applied to a broad range of different sample types. the objective of this study was to measure the performance of this new extraction platform in terms of rna and dna recovery, purity and integrity. in addition, user aspects were also addressed in the study. methods: rna recovery was measured by spiking e. coli rna to human normal edta plasma, extracted rna was quantified by using a fluorescence dye for rna detection (sybr green ii). dna recovery was measured by spiking plasmid dna (pbr ); extracted dna was determined by a measurement. an indication of rna and dna purity was obtained by measuring a / a ratios. the integrity/intactness of the extracted nucleic acid was determined by gel analysis or by using the bioanalyzer (agilent technologies) for rna and dna, respectively. the extraction method was tested on three external test sites in order to score relevant user aspects. results: the average recovery rates were and % for rna and dna, respectively. for rna extracts an average a /a ratio of . was measured, whereas for dna this value was . . these values indicate that the purity of both preparations is high since for pure preparation the expected values are . and . for rna and dna, respectively. in addition, it was found that both rna and dna were intact recovered since no degradation products were detected. in addition, all users scored the method as labour friendly. the total amount of time needed to process samples was < min, the throughput time was improved further by using two instruments in parallel, in this way samples can be completed within min. in addition the method was also verified for a broad range of different sample types including plasma, serum, csf, sputum and stool. dna sequencing is the gold standard method for accurate genotyping of human papillomaviruses (hpv) and provides nucleic acid sequence information, which is the core of every organism. pyrosequencing method has been successfully used for hpv genotyping with sequencing of only - bases. multiple hpv infections are a common phenomenon in clinical samples with a varying rate depending on the group investigated. dna sequencing techniques cannot differentiate between different genotypes as uninterruptible sequence results are obtained when multiples infections and unspecific amplification products are present in the amplicon. to address these problems, a type-specific multiplesequencing primer dna sequencing strategy, suitable for genotyping and detection of hpv- , - , - , - , - , - and - has been developed. in the new method seven type-specific sequencing primers, combined in a pool, are added to the dna sample. the oligonucleotide hybridising to the dna sample will function as a primer during the subsequent dna sequencing procedure. the new method is especially suited for detection and typing of samples harbouring different hpv genotypes (multiple infections) and unspecific amplifications, which eliminates the need for nested pcr, stringent pcr conditions and cloning. furthermore, the method has proved to be useful for samples containing subdominant types/species, and samples with low pcr yield, which avoids re-performing 'failed' pcrs. we also introduce the sequence pattern recognition when there is a plurality of genotypes in the sample, which facilitates typing of more than one target dna in the sample. moreover, target specific sequencing primers could be easily tailored and adapted according to the desired applications or clinical settings based on regional prevalence of hpv as well as other microorganisms and viruses. as the cost for dna sequencing is dropping, a sample could be sequenced in parallel with two or three different target specific primer pools covering a broader range of genotypes. the pyrosequencing hpv detection assay is fully automated and could be used for detection and identification of different microorganisms and viruses. reagents to reference extraction methods for the isolation of rna and dna from various sample types results: by performing several extractions (up to ) of a dilution series of strain coxacksie b in csf, it was shown that the analytical sensitivity of the enterovirus rt-pcr was found to be independent of the extraction method used, whereas in very low frequency higher sensitivities were obtained in combination with magnetic extraction. as expected the higher input samples gave better reproducible results than lower input samples. after evaluation of the enterovirus pcr using csf and stool samples a % correlation between the two extraction methods was found. in addition, using a broad panel of clinical specimens for m. tuberculosis pcr, the same samples were identified as positive using the boom extraction method and magnetic extraction. however, the latter method resulted in less samples having inhibition in pcr, but this needs to be confirmed in a larger study group. methods: conjunctival scrapings were sent to our laboratory in ml of viral transport medium and were inoculated to monolayers of a- and mrc- cells in tubes, incubated at c in stationary phase, and scored daily for cytophathic effect (cpe) for days or until cpe developed. when a characteristic adenovirus cpe was observed (usually after days of culture), a passage was done to two homologous monolayers in shell vials that were incubated h at c and stained with specific fluorescent reagents to adenovirus. dna from . ml of the remaining transport medium was purified by a commercial procedure and resuspended to a final volume of ll. five microlitres of this purified dna was used for real-time amplification in a final ll reaction volume, using  fast start sybr green i master mix (roche), mgcl ( mm m), and . lm m of each primer. the region amplified belongs to the hexon gene. total processing time was less than h. results: adenovirus was isolated in of samples processed by conventional cell culture and all theses culture-positive samples were positive by real-time pcr; of samples testing negative with conventional cell culture, real-time pcr detected eight as negative and five as positive. gel electrophoresis analysis showed amplification bands of the expected molecular weight in all these real-time pcr positive, cell culture negative samples. a control group of samples from patients with bacterial conjunctivitis was tested and all of them were negative by pcr. a plasmid containing the hrv- sequence spiked into a negative synovial tissue or blood specimen was used as a positive control. extracted dna from a negative synovial tissue or blood specimen was included between every two specimens as a negative control. suitability of dna for pcr was verified using a pcr assay for beta-globin. positive specimens were subjected to bidirectional sequencing. fisher exact test (two-tailed) was used for statistical analysis. results: all specimens were positive for beta-globin (extraction control). cloned hrv- proviral dna spiked into tissue, mononuclear cells and granulocytes followed by extraction yielded an amplified product in all cases. the limit of detection of the assay was . copies/ml blood and . copies/mg tissue. two hundred tissue specimens, mononuclear cells, and of granulocyte specimens tested negative for hrv- proviral dna. two ra and two oa granulocyte specimens, however, yielded a positive signal for hrv- proviral dna. all were detected at a low copy number (quantitated by comparison to a known quantity of cloned hrv- proviral dna spiked into blood), range - copies of hrv- proviral dna/ml blood. all four showed - % identity to genbank sequence af by ncbi blast search. conclusion: we did not find an association between hrv- and ra or oa ( p ¼ . ) using a real time pcr assay. recently it has been shown that hrv- is actually rabbit endogenous retrovirus h (j virol : ; - ). we hypothesise that experimental rabbit studies ongoing in our laboratory while the granulocyte specimens were being prepared account for the low level of 'hrv- ' proviral dna detected in . % of the specimens tested. results: csf virus load ranged from to  copies per millilitre. in comparison the virus load in vesicular fluid was  copies per millilitre. the highest virus loads (  and  ) were detected in a patient with paresis of facial nerve and a young patient with relatively mild disease. the lowest virus load (  copies per millilitre) had a child with varicella meningitis and an old patient with severe herpes zoster of the trunk. quantitative pcr has good reproducibility and is useful for assessment of viral load in csf samples. however, the correlation between virus load and severity of illness remains uncertain. the purpose of this study was to develop enzyme immunoassay (eia) for the detection of igg anti-hsv- activity using two new recombinant proteins as antigenic targets, and to evaluate these eia with the aid of statistical methods. methods: fragments of glycoprotein g (gg- ), comprising residues to aa of herpes simplex virus type (hsv- ) and glycoprotein d of hsv- (gd- ( - aa)), were expressed in the e. coli as gst fusion proteins to develop an assay for the detection and hsv- type-specific antibodies. results: a new enzyme immunoassay for the detection of igg anti-hsv- (igg-eia) in sera was developed using two new recombinant proteins. the igg-eia was evaluated using serum specimens obtained from patients with culture-proven hsv- infection (cp) (n ¼ ) and from normal blood donors (bd) (n ¼ ). all specimens were additionally tested for igg anti-hsv- activity by two commercially available eias. this new igg-eia detected anti-hsv activity in all specimens from hsv infected patients. when bd were tested the overall concordance between these three assays varied between and . %, concordance between positive samples ranged from . to . %. in the absence of a gold standard the accuracy of these eias was assessed by the computer program based on a maximum likelihood approach using a 'latent class' model. this analysis estimated the igg-eia sensitivity and specificity to be within the range - % and - %, respectively. dna was detected in one csf specimen from a patient with aseptic meningitis, in three aqueous humor specimens from patients with uveitis, in one swab from a patient with herpetic vesicular skin lesions and in three conjunctival swabs from patients with conjunctivitis, and (b) vzv dna was detected in two aqueous humor specimens from patients with iridocyclitis, in two swabs from patients with vesicular skin lesions, and in the vesicle aspirate and bronchoalveolar lavage from the patient with varicella pneumonitis. the precise diagnosis of herpetic infection was available within - h, which allowed for an early initiation of adapted antiviral therapy. conclusion: the detection of the six commonest human herpesviruses in clinical specimens by the herpesvirus consensus pcr methodology allowed rapid, sensitive and specific results. objectives: bovine herpesvirus- (bhv- ) is the aetiological agent of many infections and may predispose infected animals, possibly through immunosupression, to secondary bacterial infections. immunosupression may directly be associated with the induction of programmed cell death (pcd) in some virus infected cells. nitric oxide (no) has an important mediating role against fungal, bacterial, protozoal, viral pathogens and tumours. in this study, role of no was questioned in the pcd process. methods: this study was planned in two consecutive stages. in the first stage, the morphological (with and without staurosporin) and biochemical changes caused by virus-induced pcd in mdbk cells were investigated. morphological assessment of pcd was performed using hoechst nuclear staining and fluorescence microscopy technique. in the second phase of the study, the induction of pcd with staurosporin (ss) (alone or with bhv- addition) and apoptotic route of bhv- infections (with/without staurosporin) were analysed by applying , , , caspase inhibitors (r&d, germany). results: it was interesting to see that bhv- inhibited pcd following h of poi instead of being induced by staurosporin and induced apoptosis alone between . and h of poi in mdbk cells, however, between and h of poi, pcd response has found to be decreased. these results showed similarities with those obtained from herpes simplex type- infections in human epithelial cells. following caspase , , , and inhibitors applications pcd responses decreased after h whereas no responses increased following h of infections with caspase , , , and inhibitory peptides. conclusion: in conclusion, bhv- inhibited the apoptotic response in a caspase-independent way and bhv- may modulate the no response through the apoptotic pathways. objectives: the aim of this study is the questioning the programmed cell death (pcd) process in acute phase of bhv- infection in cultured epithelial like cells' microenvironments and to investigate its relation with possible nitric oxide responses in hep- cells infected with bhv- with and without staurosporin induction. methods: this study was planned in two consecutive stages. in the first stage, the morphological (with and without staurosporin) and biochemical changes caused by virus-induced pcd in hep- cells were investigated. morphological assessment of pcd was performed using hoechst nuclear staining and fluorescence microscopy technique. in the second phase of the study, the induction of pcd with staurosporin (ss) (alone or with bhv- addition) and apoptotic route of bhv- infections (with/without staurosporin) were analysed by applying , , , , and total caspase inhibitors (r&d, germany). results: it is known that following hsv- infection of - h of poi anti-apoptotic activity is triggered in human cells. and this activity is through caspase . it is interesting to see that in these experiments following h of bhv- infection the number of apoptotic cells reduced whereas no response continuously increased following -h poi. conclusion: anti-apoptotic activity of bhv- seems to be activated through caspase like hsv- , and this inversely proportional relation between no and pcd responses seem to be related with the triggering effect of no on pcd response. this effect was explained as non-specific stimulation of the host immune system. however, direct anti-viral effect cannot be excluded. the goal of present study was to evaluate the effect of probi-otics strains derivative metabolites on the reproduction of herpes simplex virus type (hsv- ). materials and methods: probiotic strains used were: lactobacillus plantarum a-p , enterococcus faecium-l , escherichia coli m . one hundred and six vero cells were infected with - id of hsv- and then incubated with supernatants from bacteria or bacteriocin preparations applied in serial dilutions. acyclovir % (lek, slovenia) was used as anti-viral drug control. cytopathic effect of the virus was determined by light or immunoflourescence microscopy after h. results: hsv- alone or in the presence of the e. coli m extracts caused the most profound cytopathic effect. addition of acyclovir completely inactivated the effect of the virus that was taken for %. supernatants obtained from l. plantarum, and e. faecium generated dose dependant effect from to % of viral inhibi-tion. e. faecium strain l- extract was - % more active than l. plantarum. extract from the strain l- was analysed for the presence of bacteriocins. two types of peptides were determined -enterocin a and enterocin b ( . - . kd). bacteriocin preparation demonstrated similar anti-viral effect ( - % of inhibition) which allows to consider enterococcal bacteriocins as major antiviral agents in present model. conclusions: extracts of several probiotic bacterial strains express a specific activity against reproduction of hsv- in vitro. antiviral effect of e. faecium strain l- was the strongest due to the presence of enterocins a and b in the supernatant. acknowledgement: work was supported by public health service grants ai and tw from nih, grant - - from rffi and regional foundation of support to new technologies in medicine. we detected ribotypes, among toxin b producing strains (tcdaÀtcdb+) only one ribotype was detected. among non-toxigenic strains four ribotypes were detected. it seemed to be interesting to observe the dominating ribotypes. between toxigenic (tcda+tcdb+) five belonged to ribotype and four to . all strains (n ¼ ) (tcdaÀtcdb+) belonged to one ribotype - . in summary, pcr-ribotyping is a good method to discriminate c. difficile strains. we decided to continue further epidemiological study in poland. objectives: the aim of the study was to identify risk factors of c. difficile-associated diarrhoea due to adp-ribosyl transferase producing strains. materials and method: a retrospective case control study was performed. each case (patient with a diarrhoea due to an actin-specific adp-ribosyl transferase producing strain) was compared with two controls (patient with diarrhoea due to a c. difficile strain which does not produce an actin-specific adp-ribosyl transferase) matched on ward and on date of hospitalisation. cdta and cdtb genes were screened by pcr (stubbs et al., fems microbiol letters ; ; - ) . production of cdt was studied by western blot using an antiserum anti ia and ib from c. perfringens and the activity of the toxin was assessed using an adp-ribosyl transferase assay. results: twenty-six cases ( males and females) were identified in and . they were hospitalised in six different hospitals of paris and its surrounding area. all the cdt positive strains were also positive for toxins a and b. cases were compared with controls. cases and controls did not differ significantly for sex, age, previous administration of antibiotics, of chemotherapy or immunosuppressive treatment. endoscopic examination was performed in . % of cases and in . % of controls (p ¼ . ) and frequency of mucosal abnormalities was similar. diarrhoea was more often community-acquired in cases than in controls ( . vs. . %, p ¼ . ) and represented more often the cause of hospitalisation ( . vs. . %, p ¼ . ). moreover, diarrhoea from cases was more frequently associated to abdominal pain ( . vs. . %, p ¼ . ) and to liquid stools ( . vs. . %, p ¼ . ). conclusions: these results suggest that there could be a correlation between the production of binary toxin and the severity of diarrhoea. the binary toxin could induce intestinal lesion independently of toxins a and b or it may act in synergy with these toxins. methods: outbreak was detected by the c. difficile surveillance programme survey of the infection control unit. c. difficile infection was diagnosed by stool culture and by detection of toxin a with a qualitative rapid immunoassay. isolates of c. difficile were genotyped using pulsed-field gel electrophoresis. results: incidence of c. difficile-associated diarrhoea increased from cases per patient-days before to cases per patient-days during the outbreak. this outbreak involved patients of four geriatrics wards, located on two geographically distinct sites (with the same medical team). mean age was (range - ) years; sex-ratio (f/m) ¼ . ; % ( / ) of cases had received one or more antibiotics before onset of diarrhoea. about % ( / ) of cases were long-term care facilities-acquired diarrhoea, and secondary hospital transmission resulted in three clusters involving cases. serotyping and genotyping were performed on isolates from different stools; of these strains belonged to the same type a whereas three displayed profiles different from the outbreak strain. management of this outbreak consisted in reinforcement of contact isolation precautions for patients with diarrhoea, cohortage of infected patients in the same ward and in promotion of hand disinfection with an alcoholic solution. environmental disinfection with hypochlorite was introduced during the outbreak. the ward where most transmission occurred was closed during days for a completed disinfection after last patient discharge. after resolution of the outbreak, incidence for acquisition was cases per patient-days. ninety per cent ( / ) of patients were treated by metronidazole or vancomycin. relapses occurred in % ( / ) of patients. two patients died with severe colitis. mean hospital stay was (range - ) days (annual mean of length of stay in the department ¼ days). conclusion: rapid control of this nosocomial outbreak of c. difficile among geriatric patients was obtained with early implementation of cohortage and ward closure associated to reinforcement of environmental disinfection, hand hygiene and enteric isolation. introduction: toxigenic clostridium difficile is the main cause of nosocomial diarrhoea and has recently been described as involved in community acquired infections. two main toxins have been classically described as the main virulence factors although strains that lack one of them are emerging with increasing frequency. objective: we aimed to characterise toxigenic phenotypes in an institution with high prevalence of c. difficile-associated diarrhoea (cdad). materials and methods: c. difficile isolates were obtained and collected over a -month period from diarrheic stools submitted to our laboratory. specimens were cultured in ccfa plates with blood and presumptive colonies identified by standard procedures. toxin b was detected with a standard cytotoxicity assay on human fibroblast culture using both diluted samples and pure broth cultures of the microorganism. toxin a was detected by a commercial enzyme-immunoassay (cdtox a oia, biostar, finland) using colony suspensions in order to increase the sensitivity of the test. all negative results for any of both toxins were checked by pcr using previously published primers and conditions. results: a total of c. difficile isolates were obtained during the study period. one hundred and ninety-nine isolates ( . %) produced both toxins (a+b+); isolates ( . %) were classified as non-toxigenic (aÀbÀ) by phenotypic procedures; in isolates ( %) only toxin b was detected (aÀb+), while no isolates were classified as producers of toxin a exclusively (a+bÀ). all non-toxigenic strains showed pcr positive results for gene b and four of them also for gene a (six isolates were aÀb+ and four were a+b+). from all aÀb+ isolates, only five were confirmed by pcr, while in six of them, toxin a gene was also detected. conclusion: the vast majority of c. difficile isolates obtained in our laboratory were toxigenic (a+b+) by traditional approaches. we have detected, using classical methods and confirmation by pcr, the presence of aÀb+ isolates in our collection. all isolates considered as non-toxigenic by phenotypical methods were pcr-positive for one or both toxins. disagreements between results of phenotypical and genetic methods can be justified as the presence of incomplete or unexpressed genes or a lack of sensitivity of the former methods. background: it has been estimated that the extra cost to the nhs for every patient that contracts c. difficile in hospital is £ . in light of this it seemed imperative that the possible components involved in the mode of transmission of this nosocomial infection be investigated with a view to control the spread. objective: to look at the level of contamination of health care workers' hands with c. difficile after dealing with a known positive patient. methods: hands were sampled using the finger streak method on a c. difficile moxalactam norfloxacin (cdmn) agar plate. plates were incubated for h under anaerobic conditions and then examined for any possible colonies of c. difficile. these were identified using the gram stain and rapid ana ii system. hands were sampled directly after patient contact and the type of contact was also noted. hands were also sampled after the removal of gloves and after hand washing. in all, duplicate samples were taken after various contacts with colonised patients. results: % of samples taken immediately after patient contact were positive. nine per cent of samples taken after the removal of gloves were positive. no samples taken after hand washing were positive. conclusion: this study showed that hands do readily and regularly become contaminated after contact with a known positive patient and that this contamination can follow fairly minimal contact with the patient. objectives: during the conduct of a phase clinical trial on the efficacy of tolevamer or g tid for days compared with vancomycin mg qid for days, we collected serial faecal samples on study entry, days , , , , , and to determine if non-antibiotic therapy can neutralise c. difficile toxin b in faecal filtrates, promote restoration of the normal microbiota and achieve clinical response. methods: patients were randomised into the study at calgary study sites (out of patients/ centres). faecal filtrate concentrations of c. difficile cytotoxin b, quantitative counts of c. difficile vegetative organisms, c. difficile spore counts were determined. quantitative aerobic/anaerobic cultures using serial dilutions of faeces e- , , , , g À wet weight were performed using criteria as outlined in the wadsworth anaerobe laboratory manual. stools from healthy donors served as normal microflora controls. results: thirty of patients provided one or more samples, and / provided serial samples beyond days and up to days. normal flora controls showed an average of four different bacteroides species in counts of À g À faeces wet weight, plus other anaerobic genera in a more inconsistent manner. using bacteroides species as a marker genus for the anaerobic microflora, , , and patients had bacteroides counts below the limit of detection, between À , or > cfu/g faeces, respectively, at study entry. vancomycin treatment eliminated vegetative c. difficile with variable spore persistence, and the bacteroides genera remained suppressed in the majority of patients during and after the course of therapy. on the other hand, response to tolemaver therapy appears to be accounted for by the inter-relationship between toxin neutralisation, c. difficile growth/persistence, and the pattern of recovery of the microflora. in general, patients who responded to toxin binding therapy exhibited non-emergence of toxin combined with increase in the numbers of anaerobic organisms. recovery of the anaerobic microflora appeared not to be complete at days in the majority of patients. objectives: the treatment of choice for a. baumannii bacteraemia has not been established. there are few data to guide the selection of agents for treating these infections. carbapenems are generally considered the drugs of choice, but an increasing of the resistant strains has been described. several alternatives guide lines have been proposed: ampicillin-sulbactam (sam) alone or associated with an aminoglycoside, piperacillin-tazobactam (tzp) or tetracyclines. the aim of this study is to know the best alternative in the empirical treatment of these infections according to the temporal evolution of the nosocomial outbreaks or endemic infections in our hospital. methods: from june to december we collected all a. baumannii strains from bacteraemia infections and their related focus. all the isolates were characterised by molecular methods in order to obtain different clones using pfge and rep-pcr. susceptibility study was performed by disk diffusion to antibiotics and mic-e-test in the mainly treatment alternatives (imipenem, meropenem, sam, tzp, tobramicine (tm), amikacine (an), and ceftazidime) and interpreted according to nccls criteria. results: in - the empirical antimicrobial treatment (eat) of choice was imipenem because all isolates were carbapenem sensible (s), with two mainly molecular clones ( isolates c -aminoglycosides resistant (r), and c -gentamicin-r, but an, netilmicin and tm-s). according to detection of an outbreak carbapenem-r in ( of the isolates) clone c multiresistant (only some strains sam or an-tn-sensible) the eat changed to sam and an or tm. this clone was persistent until and replaced with another multiresistant outbreak (c b - % sam-r and - % aminoglycosides-r). then the eat was chosen as monotherapy with an or tn (the only ones sensible of the antimicrobial tested). in the last period ( - ) emerges a new clone (c -carbapenems, sam, aminoglycosides and doxycycline-s) and imipenem returns like the actual eat in our hospital to control bloodstream a. baumannii infections. conclusions: empirical antimicrobial treatment on patients with bloodstream a. baumannii infections in a hospital, with changes in the temporal evolution of the clones associated to outbreaks or endemic infections must be established according to the susceptibility test and molecular characterisation of the strains in different clones. gentamicin-resistant enterococci in paediatric blood-stream infections in a tertiary hospital in tanzania b. blomberg, s.c. mohn, k.p. manji, n. langeland on behalf of the joint study group on antimicrobial resistance at muhimbili national hospital, dar es salaam, tanzania and the university of bergen, norway objectives: enterococci have emerged as major pathogens causing urinary tract, wound and blood stream infections (bsi). nosocomial spread of enterococci resistant to multiple antimicrobials is a great therapeutic challenge. little is known about the role of these pathogens in bsi in east africa. the objective of the study was to assess the prevalence and resistance patterns of enterococcal isolates causing bsi in children at muhimbili national hospital, dar es salaam, tanzania. methods: blood cultures were obtained from children (age - years) with fever or signs of serious infection admitted to the hospital during the period august to august . isolates were identified by standard methods. the identities of enterococcus fecalis and e. faecium isolates were confirmed by polymerase chain reaction (pcr), the isolates were susceptibility tested by e-test and assessed for genetic relatedness by pulsed field gel electrophoresis (pfge). twelve e. faecium isolates were also investigated by mlst. results: thirty-two of children ( . %) had growth of enterococcal isolates in blood culture. nine of e. faecium isolates showed combined resistance to ampicillin (are), ciprofloxacin and high-level gentamicin resistance (hlgre). six of e. fecalis isolates were hlgre, but none of these were resistant to ampicillin or ciprofloxacin. all except one of the hlgre were also resistant to chloramphenicol. the resistant strains were recovered from several geographically separated wards, including the neonatal ward. the majority of the e. faecium and e. fecalis were closely related when investigated by pfge. mlst conducted on e. faecium strains also confirmed this result. conclusion: this is the first study to identify outbreaks of bloodstream infections caused by combined are/hlgre e. faecium and hlgre e. fecalis in tanzania. e. faecium was more frequent than e. fecalis. the commonly used treatment regimens at the hospital (ampicillin and gentamicin or penicillin and chloramphenicol) are insufficient for infections caused by are/hlgre enterococci. nonrepetitive (one per patient) resistant to fox k. pneumoniae strains were isolated from clinical specimens ( from blood, two from bronchial secretions, four from urine, two from wound and five from catheter tips). patients were cared in different wards including intensive care unit (icu) and neonatal intensive care unit (nicu). species identification was done by using the vitek system (biomerieux, france). mics were determined with vitek automated microdilution system and by disk diffusion method. the criteria of the nccls were used to define susceptibility or resistance to antimicrobial agents. expanded spectrum a-lactamase (esbl) production was assessed by the double disk synergy test. the isolates were typed by enterobacterial repetitive intergenic consensus (eric) pcr with the eric- primer. isoelectric focusing (ief) of blactamases was performed to representative group isolates. results: antimicrobial profiles demonstrated that all isolates were resistant to third-generation cephalosporins, to aztreonam, cefoxitin, amoxicillin/clavulanate, ticarcillin/clavulanate and piperacillin/tazobactam. four isolates were also resistant to cefepime and cefpirome. all isolates were susceptible to imipenem. ief showed that all isolates expressed two b-lactamases, one with pi of . which correlated with the shv- and one with pi of . which corresponded to lat- . eric-pcr analysis demonstrated three strain types. type i, consisting of two subtypes, was common to strains, indicating that the clonal spread was mainly responsible for the outbreak. type ii comprised two isolates and type iii was unique. five isolates were not identified with eric-pcr. conclusions: k. pneumoniae strains, harbouring plasmid-coding for ampc-type b-lactamase, have been established in our hospital. nosocomial infection surveillance, such as restriction of particular antibiotics and adjustment of the infection control measures, has been recommended. acinetobacter baumannii producing the per- extendedspectrum b-lactamase objectives: recently per- extended-spectrum b-lactamase (esbl) was discovered in a peudomonas aeruginosa strain in france and was subsequently detected in acinetobacter spp. and pseudomonas aeruginosa in other countries including turkey. the purpose of this study was to clarify the molecular epidemiology of infection caused by a strain of cefepime-resistant a. baumannii and also to determine the mechanism of drug resistance. methods: cefepime-resistant a. baumannii strains were isolated from clinical specimens of nine patients hospitalised in an intensive care unit in busan, korea. antimicrobial susceptibilities were determined by the disk diffusion and agar dilution methods. the double disk synergy (dds) test was performed for screening of esbl production. isoelectric focusing and conjugation experiments were performed. blaper- and blaper- alleles were detected by pcr, and sequences of amplified products were determined by using the dideoxy-chain termination method. pulsed-field gel electrophoresis (pfge) was performed for molecular typing of isolates. results: the isolates showed same antimicrobial susceptibility pattern, positive dds results and pfge patterns. the isolates contained three b-lactamase bands: pi . , . , and . . pcr-based experiments detected blaper- genes. mics of ampicillin, piperacillin, cephalothin, cefoxitin, cefoperazone, ceftazidime, cefotaxime, cefepime, and aztreonam to these isolates were ! mg/l, respectively, and those of imipenem were - mg/l. despite repeated attempts, the resistance to cefepime of a. baumannii isolates was not transferred to the recipient. conclusions: a. baumannii isolates from clinical specimens of nine patients hospitalised in a same intensive care unit were shown to be of the same clone. all these isolates contained blaper- gene which caused resistance to cefepime. to the best of our knowledge, outbreaks caused by per- esbl-producing a. baumannii have not previously been described. objectives: hospital outbreaks of salmonella spp. infections are not uncommon not only in europe but also in the united states, but in neonatal units it is rarer. the maternity unit has approximately deliveries each year. we described the results as well as infection control that stopped the outbreak. methods: from october to january six neonates were infected in the neonatal unit of our hospital. the index case corresponding to a newborn delivered in our hospital, she was born by normal vaginal delivery. the -day-old patient was admitted again by neurological deficits. seven days at the hospital she developed diarrhoea. the group included five prematures, only case index was not premature. all stool specimens from family case index were negative. stool samples were request for culture from asymtomatic staff and all babies from the neonatal unit (n ¼ ). the isolates were identified by standard methods and serotyped by agglutination with monospecific antisera. the antibiotics (ab) taken in the study were: ampicillin (a), ticarcillin (t), amoxicillin/clavulanate (a/c), cefalothin (ce), ciprofloxacin (cp), co-trimoxazole (co), nalidixic acid (na), gentamicin (g), thirdgeneration cephalosporins ( gc). its was evaluated by a microdilution method and confirmation by e-test. results: eight strains of salmonella enteritidis serotype o , :h g,m were identified. the phage type (pt) involved was same in all cases, pt a. seven were isolated from faeces and one from blood culture. all isolates demonstrated same antibiotic susceptibility pattern with resistance to ampicillin and ticarcillin. fortunately no babies died. no salmonella from stools of nurses, staff personnel and mothers was isolated. conclusions: a hand washing was not sufficiently frequent the infection was probably transmitted by hand contact to prepared milk, infusion and other equipment from the index case. this hypothesis was subsequently confirmed as the outbreak was terminated after eradication of the presumed contamination sources by changing the mattresses, disinfecting the unit and ensuring strict observance of hand washing before and after every manipulation. salmonella enteritidis pt is third commonest phage type in spain. objectives: to investigate the cause of an outbreak of pseudomonas aeruginosa isolations following bronchoscopic procedures. methods: from to january , we detected a cluster of p. aeruginosa isolates associated with bronchoscopy (nine samples from eight patients). laboratory culture, bronchoscope and medical records of all cases were reviewed. all of them were related with one bronchoscope and environmental samples were obtained from it. microbiological identification and susceptibility testing were performed with the microscan walkaway system (dade behring). random amplified polymorphic dna analysis (rapd) and pulsed field gel electrophoresis (pfge) were performed on all available isolates of p. aeruginosa (eight clinical isolates from seven patients and six bronchoscope related isolates). results: two of the eight patients showed clinical evidence of infection and required specific antimicrobial therapy (the index case and other patient with two isolates separated by days). all isolates were ceftazidime, aminoglycosides and ciprofloxacin susceptible and imipenem resistant. rapd and pfge patterns revealed that all the clinical and bronchoscope isolates (eight and six, respectively) were indistinguishable. the bronchoscope was replaced and no further cases appeared. conclusions: we documented contamination of a bronchoscope with p. aeruginosa and possible secondary infection of at least one patient. microbiologists have an essential role in the detection of medical devices contamination, especially by surveillance of the emergence of infrequent bacterial recovery. in all three cases klebsiella pneumoniae was isolated from the blood cultures. the aim of this study was to investigate the epidemiological relation among the isolates and to try to find a common source for the infection. methods: environmental samples, including different i.v. fluids and drugs, and skin samples were obtained in order to detect the source of the infection. microbial identification and in vitro susceptibility tests were carried out automatically with the microscan system (dade). clinical isolates were molecular typed by random amplification of polymorphic dna (rapd) using one mer oligonucleotide, pcr-ribotyping with oligonucleotide of the intergenic s/ s region and pfge using xbai as a restriction enzyme. an unrelated strain was also included in all the experiments, as a control, in order to check the discrimination power of the techniques. results: all three clinical isolates of k. pneumoniae obtained from blood cultures shared the same biotype and antibiotype, and were all resistant to ampicillin, gentamycin and tobramycin. molecular typing methods proved clonal identities among the clinical strains. patterns generated were different from those of the control strain. the source of the infection could not be demonstrated in any of the environmental or newborn skin samples. conclusions: a single k. pneumoniae strain was the cause of the fulminant sepsis in the three newborns. all three molecular typing methods, rapd, pcr-ribotyping and pfge accurately demonstrated clonal identities of the isolates. the common source of the infection could not be detected due, probably, to the logical delay in culture growth and identification. the objectives of this presentation are to describe the outbreak and the infection control measures implemented, and to constitute an example for handling possible future outbreaks with limited resources. this is the first ekc outbreak reported from turkey. methods: eye clinic of kou hospital is equipped with modern devices; however it has limited physical conditions (e.g. insufficient hand-washing facilities) because of temporary settlement of the hospital after the earthquake of .on december , the infection control team (ict) was alerted to ekc cases. an investigation began and infection control measures (icm) were implemented. conjunctival swabs of patients with ekc and environmental swabs were obtained and studied in gata and hacettepe university microbiology laboratories. infection control protocol (icp) was implemented as recommended by apic guidelines with some modifications. in addition, terminal disinfection (td) was applied two times and after tds clinic was closed for first and than days (figure ) . results: a total of ekc cases were diagnosed among the patients who visited the eye clinic during the outbreak (general attack rate: . %). seventy-five of the ekc cases were male and average age was . ae . (range: months to years). primary and secondary attack rates were found to be and %, respectively. adenovirus type d was isolated from patient samples, biomicroscope and device solution. with the implementation of icp and td, ekc cases decreased by time and outbreak disappeared about days after the second td and closing the clinic for four days (figure ) . conclusion: this is the first outbreak reported from turkey. isolation of virus from biomicroscope and device solutions which are used for more than one patient is an evidence of transmission from environment. although several reports have described icm that terminated outbreaks of nosocomial ekc, this study demonstrates that implementing td and/or closing the clinic for four days in addition to icm, may control nosocomial ekc outbreaks. background: because of severity of underlying disease, multiple venous accesses, parenteral nutrition and often increased length of stay, intensive care unit (icu) patients are at increased risk for catheter-related candidemia (crc). we investigated health and economic outcomes in icu patients with crc. methods: in a retrospective matched cohort study ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) attributable mortality and excess length of stay for crc was investigated. matching was ( : ratio) based on severity of underlying disease and acute illness (apache ii score and admission diagnosis) and length of icu stay prior to the onset of the candidemia. as expected mortality can be derived from apache ii; this matching procedure results in an equal prognosis for cases and control subjects. attributable mortality is determined by subtracting the hospital mortality rate of the controls from this of the candidemic cases. excesses in length of icu stay and hospitalisation were determined by subtracting the median length of stay of the controls from this of the cases. results: during the study period icu patients developed a microbiologically documented crc (out of a total of candidemic patients). nineteen catheters were removed within h. cases (n ¼ ) and controls (n ¼ ) had an equal age (resp. ae vs. ae year; p ¼ . ), apache ii score (resp. ae vs. ae ; p ¼ . ) and incidence of respiratory failure ( vs. %; p ¼ . ), acute renal failure ( vs. %; p ¼ . ) and haemodynamic instability ( vs. %; p ¼ . ). the excess length of icu stay was days (median vs. days; p ¼ . ). although patients with crc had a longer length of hospital stay this difference was not significant ( vs. days; p ¼ . ). the attributable mortality of crc was . % ( % ci: À to %) as hospital mortality rates in cases and controls were . and . %, respectively (p ¼ . ). conclusion: our data revealed that, after careful adjustment for severity of underlying disease and acute illness, crc is not associated with a significantly higher mortality in icu patients. it is, however, associated with a significant excess in length of icu stay, thereby representing an important economic burden. patients were hospitalised in two internal medicine departments, two surgical departments, the nephrology department and the intensive care unit, during the period july to november . the catheter tips were cultured using the following methods: (a) semi-quantitative maki's and co. and (b) quantitative cleri's and co. samples for culture were taken also from the site of catheter insertion into the skin and from the hub. blood culture samples were taken from a peripheral vein in cases of clinical suspicion of bacteraemia or sepsis and they were incubated using the bactalert (organon teknika) automated system for days. results: cases of ccbri were recorded. the incidence of ccbri was . per catheter days. in of the cases of ccbri the origin of colonisation was determined. in cases which all had positive catheter tip and hub cultures with the same strain, the gram-negative bacteria prevailed ( / , analytically e. aerogenes three, k. pneumoniae two and p. aeruginosa one) while in four cases candida spp. (three cases) and coagulase negative staphylococcus (cons) (one case) were isolated. in contrast, in cases of ccbri with positive catheter tip and skin point entry cultures with the same strain, the gram-positive bacteria prevailed ( / , analytically s. aureus eight, cons six and corynobacterium spp. one). conclusions: ( ) the incidence of ccbri was . per catheter days. ( ) ccbri caused from gram-positive bacteria was mainly derived from the catheter site entry, whilst colonisation of hub caused mainly gram-negative ccbri. ( ) the preventive measures should be focused on better aseptic techniques and hand hygiene, care of the catheter's entry site and better training of the medical staff. we studied patients, suspect for catheter-related infections (cri) - from icu and from hdu with central venous catheters used for parenteral nutrition, drug administration or haemodialysis. the preferred vein in icu was v.subclavia, , and in hdu, v. femoralis, catheters. mean duration of catheterisation - days in icu and . days in hdu. signs for colonisation of the catheters were found in cases - in icu and in hdu. the most common microorganisms in icu were gram-negative rods (kesgroup, b. cepacia, pseudomonas spp.) - ( . %) followed by coagulase-negative staphylococci (cns) - ( . %). in hdu in most of cases were isolated cns - ( . %) and s. aureus - ( . %) (p < . ). as catheter-related bacteraemias (crb) were considered in cases, of them in icu and in hdu. causative microorganisms of crb in icu were most gram-negative rods - ( . %) and syaphylococcus spp. ( . %) in hdu (p < . ). conclusions: the frequency of crb in icu is significantly higher, . - . %, in hdu (p < . ).they developed earlier and were caused by gram-negative rods. more probable way to development of crb in icu is the catheter hub and hdu is the skin of the patients. catheter-related infection (cri) is considered as a cause of increased hospital morbidity but its influence on hospital mortality remains a matter of debate. in critically ill patients, baseline severity, underlying conditions and various confounding factors may explain the observed increased mortality rather than cri itself. in order to determine the influence of cri on hospital mortality in icu, all episodes of nosocomial septicaemia were reviewed. material and methods: retrospective analysis of all nosocomial septicaemia occurring over a -year period in a teaching hospital. septicaemia episodes were separated in secondary, primary and proven catheter-related bloodstream infections. baseline severity (saps score), delay between admission and infection, and hospital mortality were determined. results: over this -year period, patients were admitted to the hospital and episodes of cri were recorded ( . %, . / catheter-day (ktd)). hospital mortality for all septicaemia was . % while mortality related to secondary septicaemia was . % (p < . ). during the same period, patients were admitted to the icu, corresponding to ktd. four hundred twenty-four episodes of septicaemia occurred in these patients ( / ktd), of which were primary septicaemia and were proven cri ( . / ktd). mean saps score for all icu patients was and hospital mortality . %. icu patients developing infection had a mean baseline saps score > . cri occurred more than weeks after icu admission (median : days, mean . days). pathogen-associated cri were scn %, s. aureus %, e. faecalis %, candida spp. %, other %. hospital mortality in patients developing cri was / ( . %). conclusions: in this study, hospital mortality in critically ill patients developing cri was high but seemed to be primarily determined by baseline severity and underlying conditions as reflected by saps score and prolonged delay between icu admission and septicaemia. staphylococcus epidermidis is the most important pathogen of these systemic infections. objectives: to study the genomic dna profiles of s. epidermidis isolated from catheter-related infections and bloodstream infections comparing with the strains isolated from skin and nasal swab in patients hospitalised in a tertiary care university hospital. methods: catheter-related infections were defined according to the cdc definitions. patients with a culture for s. epidermidis from blood and catheter tip (> cfu) were selected to have swabs from skin and nasal for s. epidermidis. the s. epidermidis were typed using pfge, antibiotic susceptibility testing and biofilm detection, by congo red method, were performed. results: twelve patients with episodes of catheter-related infections were included in this study and strains were analysed. in episodes, the same dna profile was detected in cvc/blood and in the skin/nasal and in seven episodes the clone causing cvc/blood infections were not found in skin/nasal. the mean time of isolation of s. epidermidis with clonal relation between cvc/blood and skin/ nasal colonisation from the first day of hospitalisation until the detection in cvc/blood was . days. in episodes without s. epidermidis clonal relation, the mean time was . days. pfge identified three hospital endemic profiles that were present in . % ( / ) of all strains from episodes, including the strains from cvc/blood infections and in skin/nasal colonisation. in the strains from skin/nasal colonisation, the endemic profiles were present in . % ( / ) of the strains. the endemic dna profiles were biofilm producers and were resistant to penicillin g, oxacillin and ciprofloxacin, variable susceptibility to aminoglycosides and were susceptible to vancomycin. conclusion: patients with long term hospitalisation were previously colonised by hospital endemic s. epidermidis strains that were responsible for catheter-related infections. , and requiring a cvc were included in this retrospective cohort. the following data were analysed: patient and cvc characteristics, risk factors and microbiological results. the diagnosis of cvc-ri was based on brun-buisson methodology ( ) . the comparisons were done using the chi-square and student's t-tests. a multiple logistic-regression model was used to identify risk factors of cvc-ri according to their adjusted odds ratio (aor; % ci) and completed by a survival analysis adjusted on duration of hospitalisation and cvc, the unit and the timing of cvc implementation (before or after admission in the unit). results: a total of patients were included who required cvc ( cvc were implanted as per the hospitalisation in this study units [gr# ] and before the patient admission in this same units [gr# ]). the total of catheter days was (respectively for gr# and for gr# ). the number of cvc-ri was that is . % of cvc and the incidence rate per patient was %. the part number of cvc-ri were respectively ( %) for gr# and ( . %) for gr# . the incidence density of cvc-ri was . / catheter days for the totally cohort, . / for gr# and . / for gr# . in the totally cohort cvc-ri was monomicrobial in cases ( . %). in that case the most prevalent bacteria were: coagulase-negative staphylococcus spp. ( . %) and staphylococcus aureus ( . %) and in case of plurimicrobial infection the most prevalent agents were: staphylococcus aureus ( %), coagulase-negative staphylococcus spp. and enterococcus faecalis ( % for each). intestinum somatoplasty was not was a risk factor for cvc-ri in this study. the crude mortality rate was . % ( / ) and % ( / ) for cvc-ri and non-cvc-ri, respectively (p ¼ . ). conclusion: in these surgical units, the incidence of cvc-ri is high and was related to the frequency of manipulations of the line such as infusion, parenteral nutrition, injections and dressing even after adjustment on the duration of cvc and timing of cvc implantation. an intervention focused on these risk factors is planned to reduced cvc-ri and improve the quality of care. case : a schizophrenic -year-old man was admitted to the hospital because of fever of weeks duration; he was affected by diabetes type ii and nh lymphoma diagnosed months earlier and treated with chemotherapy through a groshong cvc and, subsequently, with chronic steroid. multiple blood cultures, performed from cvc and peripheric veins, were positive for e. faecalis and e. coli; the patient was treated with ceftriaxone g ev qid  w + lock-in therapy with teicoplanin mg (in ml) and ciprofloxacin mg (in ml) for h a day for days. it was obtained a clinical and microbiological resolution without removal of cvc. case : a -year-old man was admitted to the hospital for septic fever; months earlier a groshong cvc had been placed to treat with chemotherapy a rhinopharyngeal carcinoma. multiple blood cultures (from cvc and peripheric veins) were positive for a multi-drug-resistant stenotrophomonas maltophilia (s only to chloramphenicol, trimethoprim-sulfamethoxazole and levofloxacin). the patient was successfully treated, without removal cvc, with systemic trimethoprim-sulfamethoxazole + levofloxacin combined to antibiotic lock (ciprofloxacin mg in ml for h a day for days). conclusion: the cases reported by the aa confirm that many catheter infections can be maintained in place and sterilised with lock-in therapy avoiding to replace expensive intravascular lines with unnecessary and risky insertions. one of the questions to resolve will be whether or not concomitant systemic antibiotic therapy is necessary. background: nosocomial infections influence upon the mortality, quality of patients' life, costs and length of hospitalisation. the source of those infections might be staff members, contaminated water system, air-conditioning or pests. disinfectants are helpful in reducing or eradicating harmful pathogens existing in hospital environment. some bacteria are able to grow on a surface as a biofilm. this form is more resistant to external harmful conditions such as antibiotics, disinfectants or host defence. bacterial adhesion was recognised as the important virulence factor for colonisation of patient or biofilm formation. in our study the susceptibility of bacterial strains isolated in hospital environment (colonising or infecting patients or carried by german cockroaches) to antibiotics and chemical disinfectants was determined. moreover the efficacy of the disinfectant working solution (active ingredients: sodium dichloroisocyanorate . mg/l; glucoprotamine mg/l; potassium persulphate mg/l) on selected bacterial strains adherent to catheter (after growing for days on it) by treating then for min was determined. results: susceptibility profile to antibiotics varied; among grampositive bacteria the mlsb, mrcns strains were found; among gram-negative bacteria the esbl, ampc phenotype were described. determined mic values or disinfectants were in range: sodium dichloroisocyanorate . - mg/l; glucoprotamine . - mg/l; potassium persulphate . - mg/l. the results indicate that the working solution of the disinfectant might be ineffective to some strains of well-known pathogens: serratia marcescens, citrobacter freundii, enterobacter cloacae and staphylococcus epidermidis. the examination of disinfectants efficacy on selected strains showed that some bacterial strains were more resistant when they were grown on catheter for days. the mic value was lower than working solution of that chemical even more than times. moreover it was found that all tested disinfectants were ineffective to some strains adherent to catheter ex. s. marcescens and e. cloacae strains isolated from the body surface of german cockroaches. conclusions: the possibility of biofilm formation could explain the increase of resistance to disinfectants of some strains. german cockroaches carrying them in hospital should be considered not only as nuisance insects, but also as a real source of resistant to antibiotics and disinfectants bacteria. background: indwelling catheters are commonly colonised by skin flora. propionibacterium spp. are among the commonest bacteria of normal human skin but currently recommended catheter-culture procedures would not detect its presence. furthermore, propionibacterium is nearly always regarded as a blood culture contaminant and automated blood culture methods may not detect a proportion of them. our objective was to determine the rate of catheter colonisation by propionibacterium spp. in unselected intravascular catheters submitted for culture. methods: intravascular catheters were processed by the rollplate technique and incubated in air at c for at least days. organisms that were present in significant counts were subcultured for identification and susceptibility testing. when the conventional aerobic processing was finished, all primary culture plates were reincubated in an anaerobic jar. after days of anaerobic incubation the plates were read looking for bacterial colonies that were not initially present. control plates were inoculated with a suspension of p. acnes to assess the influence of aerobic preincubation on the final number of colony forming units (cfu). conventional processing detected significant growth of bacteria in . % of all catheters and no significant number of colonies (< ) in an additional . % samples. anaerobic reincubation yielded p. acnes in significant counts in . % of all catheters ( % of all positive catheters) and no significant number of colonies in an additional . % of samples. three samples yielded significant growth of both aerobic and anaerobic bacteria. of all the organisms recovered in significant counts, coagulase-negative staphylococci represented . %, p. acnes . %, s. aureus . % and corynebacterium spp. %, enterococcus spp. . % and other bacteria and yeast . %. anaerobic bacteria other than p. acnes were rarely recovered in non-significant counts. aerobic preincubation for days did not substantially affect the final number of cfu. conclusion: p. acnes is the second most frequent coloniser of intravascular catheters. anaerobic reincubation of plates used in standard routine is a simple method that could be useful for catheterrelated research projects. the potential of p. acnes as a cause of catheter-related bacteraemia merits further studies. results: nineteen patients included males and nine females, whose ages ranged from to years (mean years). all of the patients were hospitalised in the neurosurgical department. the most common underlying conditions were intracranial haemorrhage ( / cases), followed by hydrocephalus ( / cases) and cranial injury secondary to trauma ( / ). all patients underwent surgical procedures prior to infection, which included craniotomies and four ventriculostomies. all patients were receiving antibiotic therapy at the onset of infection. mean time between surgical procedure and diagnosis of meningitis was days ( - days) . fever and neck stiffness was found in eight and seven patients, respectively. in patients serum leukocyte count was higher than  /cu mm. mean leukocyte count in serum and cerebrospinal fluid was  /cu mm (min  /cu mm, max;  /cu mm) and  / cu mm (min /cu mm, max; /cu mm) respectively. mean csf protein concentration was mg/dl and mean csf glucose concentration was mg/dl. only in of cases the microorganism was isolated from cerebrospinal fluid. acinetobacter spp. ( cases), k. pneumoniae (two cases) and e. cloacae were the isolated microorganisms. most of the acinetobacter isolates were susceptible to carbapenems but all of them were resistant to thirdgeneration cephalosporins. a combination of carbapenem plus an aminoglycoside and/or vancomycin therapy was applied most of the patients. an additional intrathecal aminoglycoside dosage was needed for seven patients who responded poorly. the overall mortality rate in these patients was %. conclusion: there has been an increase of post neurosurgery meningitis cases. in addition, the emergence of strains resistant to third-generation cephalosporins in this group has also been noted in recent years, and has become a great therapeutic challenge. early diagnosis and initiation of appropriate antibiotic therapy is needed in this potentially fatal disease. objectives: pulmonary resection is associated with considerable risk of infection, so antimicrobial prophylaxis has become routine practice in thoracic surgery. the aim of this study was to assess changes in microflora of upper respiratory tract in hospitalised patients with non-small cell lung cancer (nsclc) before and after preoperative antimicrobial prophylaxis. methods: patients with nsclc aged - years were subdivided into two groups: (a) control group ( patients without antimicrobial prophylaxis and surgery), (b) 'prophylaxis' group ( patients undergoing pulmonary operation with preoperative antimicrobial prophylaxis, including piperacillin, cefuroxime or ceftriaxone alone or in combination with amikacin). throat and nasal specimens were taken up two times: examination i -on the day of hospital admission and examination ii -on the third or fourth day of hospitalisation in group a and on the third or fourth day after the surgery in group b. the routine microbiological methods were used for isolation and identification of bacteria and fungi. statistical analyses were performed by nonparametric tests. results: the colonisation of nasal mucous membranes by pathogenic microflora did not differ significantly during hospitalisation between group a and b. similar situation was observed in the case of pathogenic microflora on throat mucous membranes in group a. different results were obtained in group b. the increased prevalence of pathogenic microflora on throat mucous membranes was observed -from . % in examination i to % in examination ii. this difference was statistically significant (p ¼ . ). in group b colonisation of throat mucous membranes by enterobacteriaceae family and candida spp. was increased significantly during hospitalisation (from to . % and from . to %, respectively). conclusion: our results indicate that antimicrobial prophylaxis can be regarded as an important predisposing factor for changes of upper respiratory tract microflora and for colonisation of mucous membrane of throat with enteric gram-negative rods and yeast-like fungi -candida spp. these microorganisms are potential causative agents of endogenous infections in immunocompromised patients with lung cancer. objectives: the purpose of this study was to determine aerobic and anaerobic bacteria colonising pleural drains in patients with non-small cell lung cancer (nsclc) undergoing thoracic surgery and to define antimicrobial agents susceptibility of isolated strains. routine antimicrobial prophylaxis included piperacillin or cefuroxime. in some cases beta-lactam was used in combination with amikacin. methods: material for research was fluid from pleural drains collected from patients aged - years two times -on the day of pulmonary resection and on the fourth day after operation. samples were routinely cultured under aerobic and anaerobic conditions and determined using api system (biomerieux). antimicrobial resistance was estimated by the disc diffusion method according nccls recommendations. results: aerobic ( strains) and anaerobic ( strains) bacteria were found in ( %) and ( %) samples, respectively. among aerobic bacteria, gram-negative rods ( strains; -belonging to non-fermenting rods) and coagulase negative staphylococci (cns; strains) were most often cultured. fifteen strains of non-fermenting rods and isolates of cns were classified as multidrug resistant (mdr) organisms. two isolates of s. marcescens were producers of extended spectrum beta-lactamases (esbls) and inducible beta-lactamases (ibls). all staphylococci were susceptible to vancomycin and teicoplanin. cns strains resistant to penicillin and oxacillin but sensitive to amoxicillin/clavulanate were most frequently isolated. only two methicillin-resistant strains, belonging to s. haemolyticus were found. the most common anaerobic bacteria were from the genera eubacterium (nine strains) and actinomyces (six strains). all of them were highly susceptible to antimicrobial agents except metronidazol ( . % resistant strains) and chloramphenicol ( . % resistant isolates). conclusion: colonisation of pleural drains does not mean infection, however knowledge about bacterial species found in drain fluid in a local population and antimicrobial resistance (especially mdr strains) has a major impact on the success of prophylaxis and therapy of potential postoperative infections. a. artero, j.j. camarena, r. zaragoza, s. sancho, j. tamarit, r. gonzález, j. nogueira valencia, e objectives: to know the clinical and microbiological characteristics of diabetic patients with severe bacteraemia. to identify the differential features of severe bacteraemia between patients with and without diabetes mellitus (dm). materials and methods: during a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) we have evaluated all bacteraemias with severe sepsis or septic shock in an intensive care unit of a teaching hospital. clinical and microbiological features were recorded from clinical charts. the spss package ( . ) was used to identify significant differences between dm and no-dm cases, and to determine if the presence of dm was associated with mortality by a multivariate analysis. results: the prevalence of dm in patients with severe bacteremic infections was . % (n ¼ ). in the group of dm the mean age of patients was . ae . years, the relation between men/women was . , the origin of the bacteraemias was nosocomial in . %, severe sepsis was present in . % and septic shock in . %. the focus of infection in diabetic patients was: unknown (n ¼ ), catheter (n ¼ ), respiratory (n ¼ ), urinary (n ¼ ), abdominal (n ¼ ), vascular (n ¼ ) and cutaneous (n ¼ ). the main microorganisms causing of bacteraemias in patients with dm were: cns ( . %), acinetobacter baumannii ( %), staphylococcus aureus ( . %), escherichia coli ( . %) and enterococcus spp. ( . %). a higher proportion of nosocomial cases in dm was the only differential feature between patients with and without dm (p ¼ . ). the global mortality in patients with and without dm were . and . % (p ¼ . ), respectively, and the related mortality were . and . % (p ¼ . ), respectively. dm was related neither to global (or ¼ . , % ic . - . ) nor related mortality (or ¼ . , % ic . - . ) by multivariate analysis. conclusion: dm is prevalent between critically ill patients with severe bacteraemic sepsis and bacteraemic septic shock. diabetic patients had a higher proportion of nosocomial origin of bacteraemia. we did not find that dm was related to mortality in severe bacteraemic infections. a. poulou, f. markou, x. efthimiou, f. mountaki objectives: brucellosis is a zoonotic disease whose prevalence in northern greece is high and constitutes a significant problem for the local health authorities. the aim of this study is to report a rare case of transmission of brucella melitensis. patients: a female infant showed signs of respiratory distress during delivery. the obstetrician in charge tried to clear the respiratory tract of saliva and amniotic fluid. in his attempt he swallowed some secretions. a blood culture from the infant was incubated in the bactec . after days b. melitansis was isolated. the case was proved to be a rare case of congenital brucellosis. the family of the infant was checked and the mother was found to be positive at / titre by brucella agglutination test though her blood culture was negative. neither her husband nor her other two children were positive on the wright agglutination test. both parents were involved in animal husbandry. two months after the delivery of the infected infant the obstetrician reported pains in the back of his neck and low fever. a blood test revealed leucopenia and neutropenia (white cell count /mm ). the wright agglutination test was positive at titre / . a blood culture was taken and b. melitensis was isolated. transaminases were normal. the obstetrician reported that he had not consumed unpasteurised milk or dairy products. he was treated with vibramycin and rifadin for days. two months later the wright agglutination test was found negative and the white cell count was normal. conclusions: b. melitansis is usually transmitted through consumption of unpasteurised diary products. in these cases we had transplacental transmission and transmission through infectious secretions via the gastro-intestinal tract. therefore it is essential that detailed medical case histories should be taken from pregnant women in order to avoid congenital infections and that medical personnel should be aware of the possibility of such transmission. objectives: coryneforme bacteria have gradually acquired greater importance in infectious pathologies, especially as opportunistic nosocomial pathogens, some of them displaying resistance to various antibiotics. the aim of this report is to describe some of these bacteria with significant implication in different clinical pictures. methods: over a -year period we characterised the coryneforme isolates with presumable clinical significance. clinical significance of the isolates was evaluated according to clinical information received (fever, intravascular devices, underlying disease, prolonged antibiotic therapy, etc.) as well as microbiological criteria (more than one isolation from habitually sterile anatomical areas and/or repeated isolations as predominant flora in sites contaminated with comensal flora). results: in patients the isolations were clinically significant. the most frequent isolations ( ) were found in blood culture: seven corynebacterium amycolatum, five corynebacterium jeikeium, two corynebacterium minutissimum, two dermabacter hominis, one corynebacterium group g, one brevibacterium sp. in another eight cases bacteraemia was accompanied by isolation of the same species in intravenous catheters (two c. amycolatum, one c. striatum, one c. jeikeium, one c. group g), a pace-maker cable (c. minutissimum) or soft tissue wound (one c. urealyticum, one brevibacterium sp.). in addition, four c. striatum were isolated (three in respiratory secretions and one in a lower limb abcess), two c. amycolatum in mammary abscesses, one c. jeikeium in articular fluid and two c. urealyticum in urine. all the isolates were sensitive to vancomycin (mics < . mg/l), while sensitivity to beta-lactamics, macrolides and fluorquinolones was variable. conclusions: ( ) c. amycolatum and c. jeikeium were the most frequently found corynebacteria with presumable clinical significance. dermabacter and brevibacterium were the genera identified among the non-corynebacterias. ( ) outbreaks of nosocomial ssss and impetigo bullosa in infants have been well-described to be associated with the well baby nursery. the source of infection has been traced to health care workers in the delivery room or the newborn nursery. the initial site of s.a. colonisation/infection may be the anterior nares, nasopharynx, conjuctiva, umbilicus and/or the blood rather than the skin. often the personnel are asymptomatic carriers of the epidemic strain of s.a. objectives: the aim was determine the genetic relatedness of s.a. isolated from patients and staff and investigation of the potential source of the infection. material and methods: in november strains of s.a. were isolated from various materials from newborns hospitalised at neonatological and obstetrician departments as well as from the staff. biochemical test api staph was used for the species identification. to molecular typing of isolates was used pulsed field gel electrophoresis (pfge). interpretation criteria for the gels followed manufacturer's guidelines: isolates with identical restriction profiles were assigned the same type, isolates that differed by one genetic event (one to three bands) were considered closely related, isolates with a four-to five-band difference were considered possibly related, and isolates that differed by more than six bands were different strains. results: comparative analysis of the banding pattern for the isolates can be divided into several categories: genetic: type asix strains from newborns with impetigo bullosa and one from staff (baby nursery); type b -two strains from the staff; type c -seven strains from the staff; type d -two strains from the staff; nine other types -each one from one person from the staff. conclusions: all the cases of impetigo bullosa were caused by one genetic type of s. aureus which allows to characterise the infection as a hospital infection. strains isolated from the staff (except one person) belonged to different genetic types (unrelated strains). isolation of the same genetic type from infected newborns and a person from the stuff may suggest that this person was the source of the infection, but we can not exclude that she was accidentally colonised during the hospital outbreaks. to define whether bacterial translocation is a process involved in the series of events following multiple trauma. methods: crushing fracture of the middle of the right femor was performed in new zealand rabbits. blood sampling was performed before and h after fracture for the determination of tumour necrosis factor-alpha (tnf-alpha) and of nitric oxide (no). tnf-alpha was estimated by a bioassay on l fibrosarcoma cell line and no by a colorimetric assay. survival was recorded and after death segments of liver, spleen and lower lobe of the right lung were cut for quantitative culture. < . . conclusion: in this in vivo model of pp ( ) gat was strongly effective on the fully susceptible strain and its efflux derivative, despite the emergence of rm for this later one. ( ) gat was ineffective, as expected on resistant gyra strain, but more surprisingly on parc mutated strains, mainly due to the presence of rm. ( ) these mutants were selected in vivo in a msw more precisely defined by pkpd parameters using mpc. ( ) low levels of resistance to fq should be detected by simple tests to guide the therapeutic options. objective: boost of systemic neutrophil count by g-csf prior to infection leads to diminished growth of pneumococci in experimental meninigitis and improves survival. whether this protective effect also includes attenuation of hearing loss is reported here. materials and methods: rats -infected intracisternally with $  s. pneumoniae serotype -were randomly allocated to receive g-csf ( lg/kg s.c. td) h prior to infection (n ¼ ), late treatment ( h postinfection, n ¼ ) or no g-csf (n ¼ ). all animals also received ceftriaxone started h postinfection. infection was documented by blood and csf tap h post infection. just before, h and days after infection, assessments of hearing was made by measurements of distortion product otoacoustic emissions (dpoae) at f ¼ - khz and by assessment of hearing thresholds by auditory brain stem responses (abr) at khz in levels from to db spl. results: -h postinfection hearing loss was significantly increased in g-csf treated animals compared with untreated (hearing loss in . vs. . % of animals from f ¼ - hz and vs. . % f > hz, respectively, mann-whitney, p ¼ . ). on day postinfection among surviving animals, severity of hearing loss in g-csf pretreated animals was furthermore increased compared with the control group (severe hearing loss in . vs. % from f ¼ - hz, respectively, mann-whitney, p ¼ . ). late g-csf treatment did not affect hearing loss significantly compared with the control group. objective: bacterial meningitis is characterised by an intense inflammatory host response that contributes to the high mortality and morbidity of the disease. doxycycline is a clinically used antibiotic which has anti-inflammatory effects that are separate and distinct from its antimicrobial action, including the reduction of cytokine release and the inhibition of matrix metalloproteases. the present study assessed the effect of doxycycline, when given as adjuvant therapy in experimental pneumococcal meningitis. methods: eleven-day-old rats were infected intracisternally with ll of saline containing . - .  cfu/ml streptococcus pneumoniae. at h after infection all animals received ceftriaxone ( mg/kg i.p., q h) and were randomised for administration of a single dose of doxycycline ( mg/kg s.c.; n ¼ ) or an equal volume of saline ( ll; n ¼ ). at h after infection, surviving animals were sacrificed. albumin concentration in the brain was assessed as an index for blood-brain barrier (bbb) leakage. brain damage was quantified by histomorphometry. results: a single dose of doxycycline ( mg/kg) vs. saline improved survival (survival rate: vs. %, p < . ), protected the bbb (cortical albumin/total protein: . vs. . lg/mg, p < . ) and reduced injury in the cerebral cortex (damage in percent of cortex; median [range] [ - . ] vs. [ - . ], p < . ). conclusion: adjuvant treatment with doxycycline may be a promising approach to prevent death or neuronal injury as a consequence bacterial meningitis. establishing conditions resulting in null survival by antibodies protection or antibiotic treatment. methods: a fully amoxicillin-resistant (mic of mg/l) serotype b streptococcus pneumoniae was used as infecting strain. amoxicillin was administered at a dose ( . mg/kg) producing serum concentration lower than the mic of the infecting strain all over the treatment period (c max : . mg/l). passive immunisation was performed with hyperimmune serum (hs; obtained from mice weekly inoculated with whole cell heat-inactivated inoculum for weeks) diluted in pbs up to dilution / that had shown null protection ( % survival) in preliminary experiments. groups of balb/c mice weighing - g were passively immunised with one-single intraperitoneal (ip) injection of the / dilution of hs, h prior to infection with the b pneumococcus. amoxicillin treatment was started h after inoculation and continued t.i.d for h. groups of animals receiving placebo (pbs), non-immune serum, non-diluted hs, / dilution of hs or amoxicillin . mg/kg alone were included as control groups. mortality was recorded over the -day follow-up period. results: survival rates in all control groups were lower than % except in the non-diluted hs that was %. antibiotic treatment in passively immunised animals produced survival rates of %, with significant differences vs. controls (except the non-diluted hs). conclusion: since amoxicillin concentrations were below the mic ( mg/l) of the infecting organisms all over the treatment period (c max of . mg/l), the presence of specific antibodies produced in vivo efficacy of sub-inhibitory concentrations. the in vivo combined effect antibodies/amoxicillin is synergistic and not only additive considering the survival rates obtained by the antibodies ( % survival) and amoxicillin sub-inhibitory concentrations ( % survival) alone and those obtained when acting together ( % survival). ( mg/kg) and cro ( mg/kg) were injected at hour and v ( mg/kg) were injected at hours and . cro and v were standard doses. d corresponded to high doses in humans. csf samples were repeatedly collected during therapy in order to determine antibiotic levels and killing rates. d serum levels peaked at mg/l decreasing slowly to mg/l h later. d csf levels ranges between and mg/l. d penetration into inflamed meninges was %. results of bactericidal activity of the different regimens are expressed in delta log cfu/ml h and delta log cfu/ml over h. results are presented in table . conclusions: ( ) d is highly efficacious against penr and penr+ qurr pneumococci in experimental meningitis, sterilising the csf of rabbits within h ( out of in both d treatment groups). ( ) d as monotherapy is significantly superior to the standard regimen based on a combination of cro with v against both strains. ( ) the efficacy of d was also confirmed in time-killing assays over h. objectives: skin-temperature is an effective measure of the severity of pneumococcal pneumonia in mice and can be used to predict lung bacterial counts and imminent death. skin-temperatures vary considerably in groups of infected mice and thus, drug intervention at a particular skin-temperature more closely resembles that which is used in humans. in this study, we compared the efficacy of moxifloxacin (mfx) with levofloxacin (lvx) in the treatment of pneumococcal pneumonia using our novel skin-temperature model. methods: swiss webster mice were inoculated endotracheally with -log cfu of the streptococcus pneumoniae a strain (mics: mfx, . lg/ml; lvx, . lg/ml). skin temperature at h was used to assess disease severity prior to drug treatment. a skin temperature of ! c is indicative of a moderate infection with a pulmonary bacterial count of -log cfu whereas temperatures < c but > c are suggestive of a severe infection with a count of -log cfu. all mice with a temperature of c were excluded from the study, as death is imminent within h. a mg/kg subcutaneous dose of mfx or lvx was given twice daily for days. skin temperature was measured daily to monitor clinical improvement or failure ( c for at least h). all mice deemed to have failed therapy were euthanised immediately. viable counts in the lungs were determined for all mice. results: of the mice classed as moderate, / ( %) mice treated with mfx and / ( %) mice treated with lvx survived. complete eradication was obtained in and % of mice treated with mfx and lvx, respectively, in this group. of the mice classed as severe, / ( %) and / ( %) mice treated with mfx and lvx, respectively, survived. complete eradication was obtained in and % of mice treated with mfx and lvx in this group. conclusions: mfx showed significantly enhanced activity over lvx at both an early and late stage pneumococcal lung infection. . a partial knee replacement was performed with a silicone implant fitting into the intramedullary canal of the tibia, and cfu of mrsa, were injected into the knees. rx was started days after inoculation and continued for days intramuscularly. results: mics (mg/l) of lzd, van and rif were . , . and . , respectively. in vivo, lzd reduced significantly the mean log cfu/g of bone ( . ae . , n ¼ ) vs. controls and van ( . ae . , n ¼ ; . ae . , n ¼ ), respectively (p < . ). both rx were not sufficient to sterilise animals ( / and / respectively). the combination of rif with lzd ( . ae . , cfu/ g of bone, / sterile animals) or with van ( . ae . cfu/g of bone, / sterile animals), was significantly more effective than monotherapy (p < . ). emergence of resistance to rif was not detected in vivo. conclusion: in this mrsa joint prosthesis infection, lzd combined with rif was highly effective in vivo and prevented the selection of mutant resistant of rifampin. lzd should be of interest for treating mrsa joint prothesis infection. staphylococcus aureus nasal decolonisation model to study the role of the multidrug efflux system acrab-tolc in resistance of salmonella typhimurium dt to detergents and bile salts. to evaluate the importance of the components acrb and tolc of this efflux system in the colonisation of a multidrug-resistant s. typhimurium dt strain in chicks. methods: acrb and tolc mutants of a multidrug-resistant s. typhimurium dt strain were constructed by deletion or insertional inactivation of the genes. mics of detergents and bile salts were determined for the acrb and the tolc mutants, comparatively to the wild type mutidrug-resistant strain. the effect of sodium choleate on the in vitro growth of these three strains was evaluated. the ld s of the strains were measured in a one day old chicken model, inoculated with several doses ( - log cfu) by the oral route, during days post-inoculation. the colonisation levels were assessed at the subletal dose days post-inoculation by determining the number of cfu of salmonella in the faeces, caeca, spleen, and liver. results: the decrease of resistance to detergents and bile salts was much more important for the tolc mutant than for the acrb mutant. for example, mics of sds decreased of and times, mics of sodium deoxycholate decreased of and times, for the tolc and acrb mutants, respectively. addition of choleate in culture medium had no effect on the growth of the wild type strains and of the acrb mutant but inhibited the growth of the tolc mutant. the ld s in the -day old chicken model, were log cfu and log cfu for the wild type strain and the acrb mutant, respectively, and not calculable for the tolc mutant because of a too small number of dead chicks. furthermore, in contrast to the acrb mutant, the tolc mutant was unable to colonise the caeca, spleen, and liver after week of infection. moreover, in most chicks no intestinal excretion was detected for the tolc mutant. the colonisation levels of the acrb mutant were the same as those of the parental strain. conclusion: tolc but not acrb appears to be essential in multidrug-resistant s. typhimurium dt colonisation of chicks, which is in accordance with their respective roles in resistance to detergents and bile salts. therefore, tolc could be a better target than acrb for the development of efflux system inhibitors. (kp ) and its derivative producing the plasmid-mediated ampc-type b-lactamase cmy- (kp ). in vitro studies: mic/mbc: microdilution method (nccls), inoculum: , and cfu/ml. the in vitro postantibiotic effect (pae) was investigated by exposing the bacteria to imp and cep at concentration equal to two and six times the mics for . h. the pae was quantitated calculating the difference between the times required for the numbers of drug-exposed and untreated organism to increase -fold above the numbers present immediately after removal of the antibiotic. pk/pd parameters (c max and time above the mic) were determined after a single dose of antimicrobials. in vivo studies: experimental pneumonia in c bl/ mice, with intratracheal inoculum of cfu/ml. the animals were grouped in: con (no treatment), cfp ( mg/kg/day) and imp ( mg/kg/day), during h. variables: mortality rates and bacterial clearance from lungs. statistical analysis: chi-squared and fisher tests, anova, and posthoc tests. results imp ( . , . , . ) . in vivo: for kp , cfp and imp decreased the mortality respect to con ( vs. %, p < . ) and ( . vs. %, p < . ); for kp , imp was the only therapy that decreased the mortality compared with con and cfp ( vs. % and %, p < . ). bacterial clearance from lungs: for kp , cfp and imp cleared the lungs respect to con ( . and . vs. . log cfu/ml, p < . ), cfp being better than imp (p < . ); for kp , cfp and imp cleared the lung respect to con ( . and . vs. . log cfu/ ml, p < . ). conclusions: the presence of plasmid-mediated ampc-type b-lactamase cmy- in k. pneumoniae diminished the in vivo efficacy of cefepime and not that of imipenem. the inoculum effect for cefepime and the pae of imipenem partially explain these results. m. abscessus is a rapidly growing mycobacterium (rgm) that is emerging as a significant pathogen in humans, both as a respiratory pathogen in patients with or without recognised comorbidities, and as the agent of inoculation infections. the histopathologic features of the human infection suggest that m. abscessus causes a tuberculosis-like infection. we investigated the systemic challenge of c bl/ mice with the type strain of m. abscessus through intravenous and intraperitoneal routes. with both high ( cfu) and low ( cfu) doses, the initial bacterial load remained stable for days in liver and spleen until the establishment of a granulomatous response. the differentiation of the granuloma (central f / + epithelioid cells with a peripheral cd + and cd + lymphocytic crown) was contemporary to a drastic decrease of the bacterial load in the organs studied. however, days following the challenge some mice still harboured bacteria capable of in vitro growth in their livers and spleens despite an overall effective control of the infection, and all mice infected presented granulomas of various differentiation stages in their livers. this response is highly reminiscent of the ifnc dependent response to m. tuberculosis. mice deleted for the gene encoding ifnc were challenged intraperitoneally with m. abscessus and significantly failed to reduce the bacterial load by day . we show for the first time that the rapidly growing m. abscessus can cause a long lasting, tuberculosis-like, ifnc dependent infection in c bl/ mice. these results show promise for the elucidation of m. abscessus disease since data from m. tuberculosis might be relevant. reciprocally, m. abscessus faithfully models key features of mycobacterial infection. campylobacter jejuni infection is the most common antecedent in the axonal variant of guillain-barré syndrome (gbs). antibodies against nerve gangliosides found in gbs patients recognise cross reactive epitopes in the lipopolysaccharide (lps) of c. jejuni. this led to the molecular mimicry hypothesis of gbs. to investigate the connection among c. jejuni, antibodies anti gangliosides and gbs we designed an animal model employing a lps isolated from a gbs patient. methods: we immunised eleven rabbits with a lps extracted from penner serotype : c. jejuni strain isolated from patient with gbs and freund's adjuvant (cfa) (group i). in a second experiment we immunised seven rabbits with lps, cfa and keyhole limpet hemocyanin (klh) (group ii). results: all rabbits of groups i and ii developed a strong humoral response to lps. elevated igm and igg antibodies to lps could be detected as early as weeks after the first immunisation. igg raised during the immunisation period up to in group i and in group ii. anti-gm igm antibodies were detectable at low titres weeks after the first immunisation in both groups and raised up to in group i and to in group ii. igg anti-gm could already be detected at low titres in both groups weeks after the first immunisation and increased up to in group i and up to in group ii. titre of anti-gm igg showed a steep rise during the weeks following the first immunisation. in western immunoblotting of c. jejuni lps, the serum of immunised rabbits reacted strongly with a band that co-migrated at kd at the same level of ct, pna and serum of the patient with anti-gm antibodies. the kinetics of igm and igg anti-gd b was similar to that of antibody anti-gm but the maximal titres were lower as igg raised up to in group i and in group ii. igm anti-gd a were at low titre in both groups throughout the experiment whereas igg anti-gd a raised up to in group i and to in group ii. igm and igg anti-gq b were not detectable in group i and ii sera. conclusion: c. jejuni lps is a potent b-cell stimulator capable to induce a strong antiganglioside response in rabbits. however to induce the neuropathy is crucial to employ klh a glycoprotein known to stimulate both humoral and cellular responses. this is the first animal model reproducing the pathogenetic process hypothesised in axonal gbs with antiganglioside antibodies post-c. jejuni infection. methods: three separate experiments were conducted in order to screen the ability of five clinical c. concisus isolates and the atcc type strain of oral origin to infect balb/ca mice. all mice were pre-treated with vancomycin, and half of the animals received cyclophosphamide to disturb immune functions, prior to c. concisus challenge by direct intragastrical inoculation with . ml cfu, controls received . ml of pbs. measured parameters were bacterial isolation from stool and internal organs, loss of body weight and histological examinations of tissue samples. mice were sacrificed on days , and of the studies. isolation of c. concisus was performed by the selective filter method and pcr. results: isolation and identification: c. concisus was isolated on day from the cyclophosphamide treated group infected with the clinical isolate (study ). liver ( / ), ileum ( / ) and jejunum ( / ) were culture positive. pcr results from tissue samples were only positive in one mouse from the same group (liver, ileum and jejunum). faecal pellets were consistently negative. during the two following studies, no isolation of c. concisus was possible. histological examination: microabscesses ( / ) were found in the liver in two untreated groups. oedema of villi in the ileum was occasionally noted in infected groups, but not in controls (study ). two mice in the untreated group infected with the atcc type strain, presented leukocyte infiltration of colon. loss of body weight: compared with controls, the c. concisus infected mice had a significant weight loss (p < . ) (study ). loose stools: on days and , c. concisus inoculated groups had loose and slimy stools compared with control groups (study ). one mouse inoculated with the clinical isolate died on day (study ). discussion: the present model mimics a relevant intragastrical exposure to c. concisus infection of imunocompetent balb/ca mice upon cyclophosphamide treatment and results indicate a possible transient colonisation of liver and ileum, with clinical signs of illness as loss of bodyweight and loose stools. histological examination was inconclusive. isolation of c. concisus was not reproducible in two subsequent studies, which severely hampers the present model. future studies should concentrate on the first days of infection, as the organism is rapidly cleared from the gi tract. . they were co-adminis-tered with antimicrobials in an experimental model of sepsis by an mdr isolate. methods: sepsis was induced in rabbits after the iv infusion of an log inoculum of a p. aeruginosa isolate resistant to ceftazidime (cz), imipenem, ciprofloxacin and amikacin (am) by a catheter inserted into the right jugular vein. animals were assigned into five groups of treatment of six animals each: a controls; b iv cz and am; c iv cz, am and alcohol %; d iv cz, am and an alcoholic solution of gla; and e iv cz, am and an alcoholic solution of aa. therapy was administered min after bacterial challenge. cz was given at a mg/kg dose, am at mg/kg and both n À pufas at mg/kg. n À pufas were infused within min. all agents were administered by a catheter inserted into the left jugular vein. survival was recorded; after death segments of various organs were cut for quantitative cultures. results this synergy is tested in an experimental model. methods: thirty-five wistar rats became neutropenic by the intraperitoneal injection of mg/kg of cyclophosphamide on day and mg/kg on day . on day an log inoculum of one mdr isolate was intramuscularly injected into the right femor of animals. rats were assigned into four groups of treatment: a (n ¼ ) controls; b (n ¼ ) rf treated; c (n ¼ ) cl treated; and d (n ¼ ) treated with both agents. therapy was given four hours after bacterial challenge. cl was administered im mg/kg into the left femor and rf iv from a catheter inserted into the right jugular vein at mg/kg. survival was recorded. results: mean ae se survival of animals of groups a, b, c and d were . ae . days, . ae . days (p: . compared with a), . ae . (p: . compared with a) and . ae . (p: . compared with a) respectively. conclusions: co-administration of cl and rf is beneficiary accompanied by prolonged survival in an experimental model of sepsis by mdr a. baumannii. infections by acinetobacter baumannii (ab) with high-degree resistance (hdr) to carbapenems have recently increased. only colistin seems to keep its in vitro efficacy, but clinical practice is scarce. to our knowledge, no clinical data are currently available to evaluate the systematic use of the beta-lactam (bl)-aminoglycoside (ag) combination to treat serious ab infections in a way similar to that in other infections by gram-negative bacteria. objective: to analyse the efficacy of the combination of two bl (imipenem [i] methods: we used immunocompetent c bl/ mice and three strains of ab with susceptibility, moderate-degree resistance and hdr to carbapenems (a, d and e respectively). mics (mg/l) were (strains a, d, e): i: , , ; s: , , ; and t: , , . the in vivo activity was examined by quantitative evaluation of the lung homogenate cultures after h of induction of pneumonia. results: in control (con) animals (n ¼ ), the bacterial counts in lungs at h were (mean ae sd): . ae . , . ae . and . ae . log cfu/g of tissue for strains a, d and e, respectively (p ¼ ns between strains). results of antibiotic activity were expressed as differences between treated (n ¼ in each therapy) and con groups (delta log cfu/g) (see table) . conclusions: in this mice pneumonia model, i or s kept his efficacy for ab with moderate resistance to carbapenems. in infections caused by this strain d, t in combination conferred a possible greater efficacy on these bls. in infections by ab with hdr to carbapenems, t alone was also effective. interestingly the combination bl + ag also showed a higher effect on the infection by this hdr strain e, against which monotherapy with i or s were totally ineffective. although the pharmacodynamics of t in this model may have been overestimated, because of the peak levels achieved are not usually found in humans at the recommended doses (c max . ae . mg/l), these results are promising to treat multiresistant ab infections. objectives: to investigate the effect of orally administered cranberry juice and its organic acids on escherichia coli in an experimental mouse model of ascending urinary tract infection. methods: e. coli c - , a clinical isolate from a patient with uti was used. it expresses type fimbriae but not p or s fimbriae. the transurethrally infected mice were at all times were allowed free access to chow and water (control group) or treatments. the control group and the treated groups all consisted of six mice in every trial; after week, the mice were sacrificed and urine, bladders and kidneys collected for determination of bacterial counts. most of the treatments were repeated two or more times in independent trials and these data were pooled. treatments were commercially available cranberry juice cocktail, freshly prepared cranberry juice, the hydrophilic fraction of cranberry juice (contains sugars and organic acids) and organic acids (quinic, malic, shikimic and citric acid in concentrations corresponding to cranberry juice). results: a reduced number of organisms could be recovered from the bladder (p < . ) and urine (p < . ) of mice orally treated with unsweetened cranberry juice. commercially available cran-berry juice cocktail also reduced the cfu in the bladder (p < . ), as did the hydrophilic fraction of cranberry juice (p < . ). quinic, malic, shikimic and citric acid were administered in combination and one by one. the four organic acids decreased the cfu in the bladder when administered together (p < . ), and so did the combination of malic plus citric acid (p < . ) and malic plus quinic acid (p < . ). these data indicate that the beneficial effect of the organic acids from cranberry juice during urinary tract infection is obtained when the acids are administered together. conclusion: for the fist time the effect of cranberry juice and its dominating organic acids has been tested in an experimental mouse model of long-term ascending urinary tract infection under controlled conditions. cranberry juice inhibited e. coli colonisation of the bladder, and the organic acids were the active component involved. the active treatments reduced the bacterial load in the bladder to sub therapeutic concentrations, which indicates that cranberry juice is no final treatment but a remedy that could help the patient to clear the infection, before it eventually becomes a final cystitis. (mellado et al., mol microbiol ; : - ) . we describe a kinetic microbroth method of measuring the growth rates of aspergillus fumigatus spectrophotometrically. using this method, growth rates (as defined by v max values) were determined for nine aspergillus fumigatus isolates for which an ld value in temporarily neutropenic cd- mice, infected intravenously, had previously been obtained. methods: an inoculum of spores in ll sab medium gave us uniformly shaped growth curves and allowed the measurement of v max values with greater sensitivity. soft max pro software was used to determine the v max value for each growth curve by performing linear regression on as many five data point line segments as possible, calculating the slope for each line segment and reporting the steepest slope as the v max (mod/min). growth rate was determined in quadruplicate in three separate experiments and the average v max measurement across these experiments calculated. results: mean growth rate varied from . (af ) to . (af ). ld varied from  to  . comparison of the growth rates and ld values of these isolates suggests a correlation exists between the two parameters, omitting the one significant outlier (af , which is amphotericin b resistant), r ¼ . . conclusion: these data are important in describing a simple method for measuring the growth rate of the common filamentous fungus a. fumigatus, and proving a direct link between pathogenicity in vivo and growth rate in vitro. objective: to compare the histological changes, viral persistence and localisation of the virus in the pancreas and the small intestines of mice, experimentally infected by oral or intraperitoneal route. method: mice were infected with cvb (nancy) by the oral or intraperitoneal route. doses ranged from  to  tcid . selected organs from each mouse were embedded into paraffin and sections were attached on silanised slides. for histological observation the sections were stained by mayer's haematoxylin eosin method. for localisation of the antigen by immunohistochemical staining, the vp protein served as an indicator for the presence of the virus. the method was standardised. the tissue sections were processed and stained by the avidinbiotin method, using the monoclonal mouse anti-enterovirus antibody against vp protein. results: the histological observations reveal that the tissue of exocrine pancreas showed inflammatory changes on the rd, th, th, th and st day post-infection in exocrine pancreas of the intraperitoneally infected mice. after oral infection no destruction of the exocrine pancreas was observed, but on day th post-peroral infection liposis was seen. vp was detected mainly on the third and seventh days after infection in the small intestine. we found differences in vp localisation between oral and intraperitoneal infection. in small intestine of orally infected mice positive staining was localised in smooth intestinal muscles whereas after intraperitoneal infection. vp was detected within the villi. there was no correlation between the virus concentration and tissue damage. conclusions: the pathogenesis of cvb infection is influenced by the route of virus administration, which has direct implications for the use of mouse models to study the pathogenesis of coxsackieviruses. objectives: the portal of entry of coxsackieviruses may influence the pathogenesis of infections caused by these viruses. in this study an outbred murine model (swiss albino mice) was used for experimental infection with coxsackie b virus (cvb ), strain nancy to follow-up the virus shedding in the stool and the presence of replicating virus in the small intestine of mice after oral and intraperitoneal route of infection. methods: for infection of mice different concentrations of the virus ( , , and ) were used. the stool and small intestine specimens of dissected mice were collected on days , , post-infection (p.i.) and from day in weekly intervals up to a day p.i. the suspensions made from the collected specimens were studied for presence of replicating virus in hep- cell cultures. the virus titre was determined in hep- monolayers on microtitre plates and calculated by reed and muench method. results: the replicating virus in the stool pellets was detectable from day p.i. to day p.i. in both orally and intraperitoneally infected mice with a virus titre reaching the level : tcid / ml. in the small intestine of orally infected mice the presence of replicating virus was detected up to day p.i. in the small intestine of intraperitoneally infected mice the replicating virus was present for a shorter time, up to day p.i., irrespective of the dose of infection. conclusion: there was no difference in the length of virus shedding in stool specimens of mice infected by oral or intraperitoneal route. however a longer presence of replicating virus in the small intestine of orally infected mice in contrast to intraperitoneally infected mice was observed. this was confirmed by the immunohistopathological studies, these observations support the suggestion that the pathogenesis of coxsackieviral infections is influenced by the route of virus administration. object: in xenotransplantation with porcine neonatal pancreatic cell clusters (npccs), the risk of cross-species porcine endogenous retrovirus (perv) infection remained as problem. we used the severe combined immunodeficient (scid) mouse and the lewis rat model to identify the perv transmission with the time course and the differences between the models. methods: npccs were transplanted to scid mice and lewis rats and left for - days before being sacrificed. dna and rna were extracted from the liver, spleen, pancreas, lung, kidney and testis. to examine the perv transmission, nested-pcr and rt-pcr were used upon pol/env/gag regions of perv. the pig mitochondrial cytochrome oxidase ii subunit gene (coii) was amplified simultaneously to monitor the microchimerism. results: total samples from seven mice and five rats were tested. ten weeks after xenotransplantation, two mice and four rats were identified to have permissive perv infection. in the scid mice, . % of tested organs were positive for perv-pol gene and . % were positive for coii gene with dna examination. in the lewis rats, . % of organs were positive for perv-pol gene and . % for coii gene with dna examination. examinations of organs of mice showed that ( . %) organs were positive for the perv-pol gene and coii gene simultaneously that presumed as microchimerism, but ( %) organs of rats were presumed as microchimerism. results of perv-pol positive and coii negative that presumed as permissive perv infection were observed in . % of organs in the scid mice and . % in the lewis rats. organs presumed as permissive perv infection were the spleen (day ), liver (day ), lung (day ), and testis (day ) in the scid mice by dna examinations. in the lewis rats, the spleen and testis of day ; the liver, spleen, and kidney of day ; the testis and kidney of day ; the liver, spleen, lung, and testis of day were identified to have permissive perv infection. conclusion: the cross-species perv infection was identified from these animal models. expression of perv depends on the immunity of the recipients, because the xenotransplanted scid mice had more perv microchimerism but less permissive infection than that of the lewis rats. detection rate was increased with the time course, accordingly in the early period after transplantation, perv considered to exist as an inactive form. the therapy of numerous antimicrobial classes including the recently introduced quinupristin/dalfopristin, telithromycin and the oxazolidinones. clearly the need for antimicrobial discovery persists, and this should be a continued priority for the pharmaceutical industry. this report addresses the spectrum of activity for pdf tested against a collection of recent ( ) clinical isolates cultured from patients infected with pathogens within the spectrum for peptide deformylase inhibitors. methods: pdf was acquired from novartis. the compound was dispensed into reference broth microdilution trays in appropriate media over the range of . - mg/l. mueller-hinton broth was supplemented with - % lysed horse blood when testing fastidious streptococci, and the corynebacteria. nccls qc strains were used concurrently and all pdf mic results were within proposed ranges. results: gram-positive strains were tested with a species rank order of s. aureus ( strains) > cons ( ) objective: nvp-pdf is a new peptide deformylase inhibitor active against a wide variety of gram-positive and -negative bacteria. the current study examines the activity of nvp-pdf compared with those of ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, vancomycin, teicoplanin, linezolid, ranbezolid, dap-tomycin, oritavancin and quinupristin/dalfopristin against s. aureus ( methicillin resistant) and coagulase-negative staphylococci ( methicillin resistant). microdilution using frozen trays containing cation-adjusted mueller-hinton broth and inocula of  cfu/ ml with trays incubated in air. results: mic and mic values (lg/ml) were as seen in the following table. nvp-pdf was equally active against all staphylococcal strains (mics < . - lg/ml), irrespective of susceptibility to other agents. quinolone resistance was mainly seen in methicillin r strains. vancomycin, linezolid, ranbezolid, daptomycin, oritavancin and quinupristin/dalfopristin were all active at mics < . lg/ml and teicoplanin was less active against coagulase-negative strains. conclusions: nvp-pdf , a new peptide deformylase inhibitor, was active in vitro against staphylococci. p antipneumococcal activity of nvp-pdf compared with other agents p. appelbaum, l. ednie, m. jacobs hershey, cleveland, usa background: drug resistance in pneumococci is found worldwide. objective: nvp-pdf is a new peptide deformylase inhibitor active against gram-positive and -negative bacteria. this study tests activities of nvp-pdf , amoxicillin ae clavulanate, imipenem, meropenem, ceftriaxone, cefuroxime, cefpodoxime, cefdinir, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, azithromycin, clarithromycin, linezolid, quinupristin/dalfopristin, vancomycin and teicoplanin against pen s, pen i, pen r pneumococci ( macrolide r and quinolone r strains with defined r genotypes). methods: agar dilution using cation-adjusted mueller-hinton agar + % sheep blood and inocula of  cfu/spot; plates incubated in air. results: mic and mic values (lg/ml) are shown in table . nvp-pdf was equally active against all pneumococci, irrespective of activity of other drugs. beta-lactam mics rose with those of pen g. moxi was the most potent quinolone followed by gati, levo cipro. vanco, teico, linez, quin/dalf were all active at mics < . lg/ml. conclusions: nvp-pdf was active in vitro against beta-lactam, macrolide and quinolone s and r pneumococci. objective: nvp pdf- is a new peptide deformylase inhibitor active against gram-positive and gram-negative strains. this study tested activity of nvp pdf- , ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, vancomycin, teicoplanin, linezolid, ranbezolid, daptomycin, tigecycline, oritavancin and quinupristin/ dalfopristin against six s. aureus ( methi r) and six cons ( methi r). methods: nccls macrodilution mic was used. for time-kills,  -  cfu/ml inocula in cation-adjusted mueller-hinton broth were incubated aerobically in a shaking water bath at Â, Â,  mic. viabilities were done after , , , h. ca þ was added for dapto. results: mic ranges (lg/ml) were: nvp pdf- , . - ; cipro, . to > ; levo, . - ; gati, . - ; moxi, . - ; vanco, - ; teico, . - ; linez, - ; ranbez, . - ; dapto, . - ; tige, nvp / / / / / / / / / / / / cipro / / / / / / / / / / / / levo / / / / / / / / / / / / gati / / / / / / / / / / / / moxi / / / / / / / / / / / / vanco / / / / / / / / / / / teico / / / / / / / / / / / linezolid / / / / / / / / / / / ranbez / / / / / / / / / / / depto / / / / / / / / / / / tigecyt / / / / / / / / / / / oritavan / / / / / / / / / / / quin/dalf / / / / / / / / / / / . - ; orita, . - ; quinu/dalfo, . - . no. of strains at mic/  mic with delta- log cfu/ml ( %), delta- log cfu/ ml ( %) and delta- log cfu/ml ( . %) killing at the various time periods are shown in table . nvp pdf- was not cidal at  mic and  mic, but was static against all strains at mic after h. cipro and moxi were cidal against four to seven strains at  mic after h. vanco was cidal at  mic for nine strains after h. oxazolidinones, tigec and quinu/dalfo were mainly bacteriostatic and dapto and orita rapidly cidal. conclusions: nvp pdf- gave low mics and static activity against all strains, irrespective of methicillin susceptibility status. p time-kill study of the antipneumococcal activity of nvp pdf- , a new peptide deformylase inhibitor, compared with other agents p. appelbaum, g. pankuch, m. jacobs hershey, cleveland, usa background: drug-resistant pneumococci are an increasing worldwide problem. objective: nvp pdf- is a new peptide deformylase inhibitor. this study used time-kill analysis to examine the antipneumococcal activity of nvp pdf- compared with imipenem, meropenem, ceftriaxone, moxifloxacin, levofloxacin, gatifloxacin, azithromycin, clarithromycin, vancomycin, teicoplanin, linezolid, daptomycin, and quinupristin/dalfopristin. twelve strains were tested: three penicillin sensitive, two intermediate, and seven resistant pneumococci. of the strains tested; were macrolide resistant [ erm (b), mef, l ], and two quinolone resistant. methodology: nccls macrodilution mic methodology was used. time-kill analyses were in cation-adjusted mueller-hinton broth with % lysed horse blood, and final inocula of  -  cfu/ml. mueller-hinton broth was supplemented to a final concentration of mg ca þ /l for testing daptomycin. viability counts were done after , , , , and h. results: mics (lg/ml) were as follows: nvp pdf- , . - . ; imipen, . - . ; meropen, . - . ; ceftriax, . - . ; moxi, . - . ; levo, . - ; gati, . - . ; azithro, . to > ; clarithro, . to > ; vanco . - . ; teico, . - . ; linez, . - . ; dapto, . - . ; quin/dal, . - . . the number of strains at mic/  mic with log cfu/ml values of À ( % killing), À ( % killing) and À ( . % killing) at the various time periods are shown in table . conclusions: nvp pdf- had kill kinetics similar to those of linezolid. nvp pdf- at  mic was bactericidal ( . % killing) against six strains after h. linezolid at  mic was bactericidal against seven strains at the same period. daptomycin and quinupristin/dalfopristin showed rapid killing. imipenem, meropenem, vancomycin, and quinupristin/dalfopristin were bactericidal against all strains at  mic after h. objectives: nvp-pdf- is a new peptide deformylase inhibitor antimicrobial with excellent activity against gram-positive cocci, including methicillin-resistant staphylococcus aureus (mrsa) and penicillin-resistant streptococcus pneumoniae (prsp). we used the neutropenic murine thigh-infection model to measure in vivo postantibiotic effects (paes) and determine which pk/pd parameter best correlated with in vivo efficacy. methods: mice had . - . cfu/thigh of staphylococcus aureus atcc and streptococcus pneumoniae atcc when treated for h with - mg/kg/day of nvp-pdf- fractionated for -, -, -, and -h dosing. mice were sacrificed at the end of therapy. ten per cent thigh homogenates were prepared, and serial dilutions were plated for cfu determinations. serum levels after both oral and subcutaneous injection of doses of , and mg/kg were measured by microbiologic assay. non-linear regression analysis was used to determine which pk/pd parameter ( -h auc/mic, peak/mic or time above mic) best correlated with cfu/thigh at h. in vivo paes were measured from serial - h cfu/thigh values after doses of and mg/kg. results: pharmacokinetic studies exhibited linear kinetics with doses from to mg/kg, with peak/dose values of . - . , auc/dose values of . - . and half-lives of - min. oral bioavailability was - %. protein binding in mouse serum was low at %. nav-pdf- produced in vivo paes of - h with s. aureus and - . h with s. pneumoniae. the -h auc/mic was highly correlated with efficacy (r ¼ - % for -h auc/mic compared with - % for peak/mic and - % for time above mic for s. pneumoniae and s. aureus, respectively). because of the rapid half-life in mice, oncedaily dosing was slightly less effective than the more frequent dosing regimens. conclusions: the -h auc/mic is the parameter that best correlates with in vivo activity of nvp-pdf- . the prolonged in vivo paes would support at least twice daily dosing. p in vivo pharmacodynamic activity of nvp-pdf- against multiple bacterial pathogens w. craig, d. andes madison, wisconsin, usa objectives: the -h auc/mic is the pk/pd parameter that best correlates with in vivo activity of nvp-pdf- , a new peptide deformylase inhibitor. we used the murine thigh-infection model nvp / / / / / / / / / / / / imipen / / / / / / / / / / / / meropen / / / / / / / / / / / / ceftriax / / / / / / / / / / / / moxi / / / / / / / / / / / / levo / / / / / / / / / / / / gati / / / / / / / / / / / / azithro / / / / / / / / / / / / clarithro / / / / / / / / / / / / vanco / / / / / / / / / / / / teico / / / / / / / / / / / / linez / / / / / / / / / / / / depto / / / / / / / / / / / / quin/dal / / / / / / / / / / / / in normal and neutropenic mice to determine ( ) the magnitude of the -h auc/mic needed for efficacy of nvp-pdf- with various pathogens (including mrsa and penicillin-, macrolideand tetracycline-resistant strains of s. pneumoniae) and ( ) the impact of neutrophils on the drug's in vivo activity. methods: mice had . - . cfu/thigh of five isolates of staphylococcus aureus (two mrsa) and six isolates of streptococcus pneumoniae (five penicillin-resistant, four macrolide-resistant, three tetracycline-resistant strains) when treated for h with - mg/kg of nvp-pdf- subcutantously every h. streptococcus pneumoniae atcc and staphylococcus aureus atcc were studied simultaneously in normal and neutropenic mice. mice were sacrificed at the start and end of therapy. ten per cent thigh homogenates were prepared and serial dilutions were plated for cfu determinations. serum levels were determined by microbiologic assay after subcutaneous doses of , and mg/kg. a sigmoid dose-response model was used to estimate the dose (mg/kg/ h) required to achieve a net bacteriostatic effect over h. results: pk studies exhibited linear kinetics with auc/dose values . - . and half-lives of - min. protein binding was %. mics ranged from . to . mg/l. static doses for the various organisms ranged from to mg/kg/day. mean -h auc(free)/mic values (aesd) were . ae . for s. aureus and . ae . for s. pneumoniae. the differences were not significant. methicillin and penicillin resistance did not alter the magnitude of the auc/mic required for efficacy. the presence of neutrophils reduced the -auc(free)/mic required for efficacy by about fourfold. conclusion: the -h auc/mic of nvp-pdf- required for in vivo efficacy was relatively similar among various pathogens, was not altered by drug resistance, and was reduced fourfold by the presence of neutrophils. p determination of quality control guidelines for mic dilution and disk diffusion methods when testing nvp-pdf , a novel peptide deformylase inhibitor t. fritsche, t. anderegg, r. jones north liberty, usa background: quality control (qc) guidelines remain necessary for accurate determination of antimicrobial susceptibility testing and should be established early in the development of new antimicrobial classes. nvp-pdf is a pdf inhibitor rapidly progressing into phase ii and iii human clinical trials, thus qc guidelines appear necessary for nccls methods. methods: multi-laboratory (seven or eight sites) trials were initiated using the nccls m -a guideline for qc determinations. key technical details were: mic phase -four mueller-hinton (mh) broth lots, eight participant sites and replicates of four appropriate qc strains; and disk diffusion phase -three mh agar lots, seven sites and replicates of three qc strains. results were analysed by statistical methods found in m -a . control drugs included vancomycin, clarithromycin, linezolid and levofloxacin; . - . % of control results were within published nccls ranges ( and results for mic and zone tests, respectively). inoculum concentration controls averaged .  (mic trial only). results: seven or eight participants provided qualifying results in the two separate qc studies, and the calculated (proposed) ranges were (range; % results in range): e. faecalis atcc ( - mg/ l; . ), s. aureus atcc ( . - mg/l; . ), s. pneumoniae atcc ( . - mg/l; . and - mm; . ), h. influenzae atcc ( - mg/l; . and - mm; . ), and s. aureus atcc ( - mm; . ). all qc ranges were maximised to contain % % of reported results and zone sise variation was elevated due to the bacteriostatic character of this pdf inhibitor, creating non-discreet zone edges. conclusions: qc ranges for nccls methods when testing nvp-pdf have been established. results from these nccls m -a -conforming trials can be utilised to control the accuracy of the susceptibility testing of this pdf inhibitor projected to be among the 'first' to reach human clinical studies. p determination of dry-form commercial reagent reproducibility and mic validations for nvp-pdf , a novel peptide deformylase inhibitor g. moet, r. jones, p. rhomberg, t. fritsche north liberty, usa background: nvp-pdf is a new pdf inhibitor rapidly being advanced to human clinical trials. commercial reagent broth microdilution mic panels will be required for investigator laboratory use, especially those products with extended shelf-lives (dry-form). this study reports the results of reagent qualifying tests. methods: the experiment was performed by nccls m -a guidelines to assess dry-form mic reproducibility ( organisms  tests/day  days ¼ tests) and comparative mic accuracy to the reference mic (ref; m -a , ) using % strains representing the following organism groups: staphylococci, enterococci, s. pneumoniae, other streptococci, h. influenzae, and selected species refractory to pdf inhibitor action. all trays were manufactured by sensititre (trek diagnostics, cleveland, oh). results: reproducibility results showed % of mics were identical and . % of mics were within one log dilution step. validation test results comparing dry-form to ref mics were (% identical/twofold/fourfold): for staphylococci ( / / %), for enterococci ( / / %), for s. pneumoniae ( / / %), for other streptococci ( / / %) and for h. influenzae ( / / %). consistent variations were detected with spn ( % of dry-form panel results being one dilution higher than ref) and hi ( % of results being one dilution lower than ref). nvp-pdf mics were off-scale (mic values, > mg/l) for enterobacteriaceae and non-fermentative gram-negative bacilli ( strains). overall, % of sensititre mic results for were within one log dilution of ref mic values. conclusions: nvp-pdf dry-form diagnostic mic panels have been validated for accuracy and reproducibility using recent clinical isolates from five major pathogen groups. the spectrum of activity for this pdf inhibitor compound appears focused toward gram-positive cocci and specific fastidious respiratory tract pathogens. objectives: the emergence of antibiotic resistance among grampositive pathogens has impacted the clinical management of these infections. paratek pharmaceuticals initiated a programme to apply medicinal chemistry to the core structure of tetracycline (tet) with the goal of creating novel classes of proprietary antibiotics that would (a) be unaffected by the known tet resistance mechanisms and (b) retain the safety and tolerability profile of the tet family. since there is no cross-resistance between the tets and other antibiotics, such new agents would be expected to be active against isolates resistant to all other currently available classes. the aim of the programme was to synthesise new agents active against gram-positive, common gram-negative, atypical and anaerobic bacteria. methods: a series of -position and , -position derivatives of sancycline were synthesised and tested for activity in vitro against mrsa, vre, enterococcus faecalis and streptococcus pneumoniae by microdilution. the presence of tet-resistance determinants was assessed by pcr and confirmed by resistance to currently available tets. results: a number of -dimethylamino- -aminomethylcyclines (amc) and -aryl or heteroaryl sancyclines with potent activity in vitro (mic range less than or equal to . - . mg/l) were identified. both novel series were more potent against one or more of the resistant strains than currently available antibiotics tested (mic range - mg/l). the amc derivatives were active against bacteria resistant to tet by both efflux and ribosome-protection mechanisms. conclusions: this study identified the amcs as a novel class of antibiotics evolved from tet that exhibit potent activity in vitro against tet-resistant bacteria, including gram-positive bacteria resistant to currently available antibiotics. one agent of this class, bay - (discovered by paratek pharmaceuticals, inc., boston, ma, and designated ptk ) has been chosen for development. bay - is a novel antibiotic compound being developed for the treatment of severe bacterial infections. it is the first compound selected from the novel class of aminomethylcyclines and was designed to meet an increasingly significant need for additional therapies for treatment of infections, including those resistant to currently available antibiotics. the efficacy of bay - in different mouse models of skin and soft tissue infection (ssti) was compared with that of vancomycin (van) and linezolid (lin). methods: two mouse models were employed to determine the efficacy of bay - : ( ) infected abscess model (induced by implantation and subsequent infection of gelfoam (tm)) and ( ) infected thigh muscle model in neutropenic mice. staphylococcus aureus strain dsm (mssa) was used to infect the respective structures in the skin and soft tissues. infected abscess bearing mice were treated i.v. bid for days, while thigh muscle infection model mice were treated s.c. min post-infection. cfu reduction of infected tissues and bacterial load in different organs (spread from the infection site) were used as read-out for therapeutic efficacy. results: as measured by reduction of bacterial load, therapy of infected abscesses with bay - (cfu reduction > log units at mg/kg) was superior to van and lin (no reduction in bacterial load) . furthermore, bay - reduced the overall bacterial load in spleen, liver, lung and heart. in the reduction of organ load, bay - was as efficacious as van conclusions: this dal activity survey indicates that this new glycopeptide has significant gram-positive activity ( . - . % inhibited at mg/l), superior to available agents in the class, and the potency was similar for european isolates when compared with prior experience in other geographic areas. background: tigecycline (tig) is a novel glycylcycline with broad spectrum activity. increasing reports of resistance (r) among commonly occurring gram-positive cocci (gpc) that produce respiratory tract and skin and soft tissue infections has created a need for development of new antimicrobial agents. in this study the activity and potency of tig, tetracycline (tc) and other comparator agents was evaluated using contemporary isolates of commonly occurring species of gpc, including the presence of r organism subsets. table. organism ( methods: the activity of tig and nine comparators was challenged with a collection of gpc including oxacillin (oxa)-susceptible (s; strains) and -r ( strains) s. aureus (sa); oxa-s ( strains) and -r ( strains) coagulase-negative staphylococci (cons); penicillin (pen)-s ( strains) and non-susceptible (ns; strains) s. pneumoniae (spn); penicillin-s ( strains) and -ns ( strains) viridans-group streptococci (vgs); beta-haemolytic streptococci (bhs; strains); and vancomycin-s ( strains) and -r ( strains) enterococci (ent). broth microdilution susceptibility tests were performed and analysed using nccls reference methods and interpretive criteria. results: whereas oxa-r subsets of both sa and cons displayed cross-resistance to tc, macrolides, clindamycin and quinolones, no differences were seen with tig (mic / being . and . mg/l, respectively). among streptococci, all spn and vgs (regardless of pen-s), and bhs demonstrated tig mic / s of . mg/l (one exception being pen-intermediate vgs with the mic at . mg/l). tig was also uniformly active against enterococcal isolates, with mic / s of vancomycin-s and -r subsets being . and . mg/l, and . and . mg/l, respectively. when using the nccls tc s breakpoint of mg/l, all staphylococci, streptococci and enterococci tested would be classified as s to tig. conclusions: tig displays a remarkable spectrum of activity and potency against s and r subsets of gpc with the highest mic being . mg/l. in addition to for use in treating communityacquired respiratory tract infections, tig may also be a candidate for treatment of complicated skin and soft tissue infections and, possibly, urinary tract infections caused by gpc. p endemic, highly resistant acinetobacter in the intensive care unit -is tigecycline the answer? objective: to find satisfactory antibiotic treatment against an organism, acinetobacter baumanii, that became endemic on the intensive care unit of a busy district general hospital. this organism is resistant to many antibiotics and in one case was ultimately resistant to all currently marketed antibiotics. methods: ( ) surveillance of patients in the intensive care unit for the presence of acinetobacter baumanii. ( ) clinical assessment of patients with the organism to establish those needing antibiotic therapy. ( ) patients requiring treatment were given an antibiotic combination using colistin (usually combined with oral minocycline) or tigecycline monotherapy, a first-in-class glycylcycline agent. ( ) treatment and outcome were monitored. the study was observational. allocation to treatment categories was not randomised or blinded. the tigecycline was used on a compassionate basis. results: the intensive care unit was free of acinetobacter until the beginning of . by the end of , - new isolates of acinetobacter baumanii were isolated per quarter. initially these pathogens were sensitive to imipenem, meropenem, tobramycin, amikacin, colistin, and minocycline. this sensitivity began to wane and, by the end of , one patient had died with acinetobacter baumanii in his bloodstream that was resistant to everything available. after this death, we tested further isolates of acinetobacter against tigecycline, a new broad spectrum agent currently in phase development, and found it to be active against the endemic strain. two patients with ventilator-associated pneumonia caused by this organism were treated with tigecycline and made a full recovery. there were no adverse effects related to tigecycline treatment. conversely, five patients with ventilator-associated pneumonias caused by the same organism were treated with colistin and failed to respond. acinetobacter finds the respiratory system a favourable environment, and this, combined with the fact that the vast majority of the patients were ventilated resulted in ventilator-associated pneumonia being the commonest infection. conclusion: tigecycline is likely to be a useful agent in clinical practice on intensive care units when dealing with this difficult organism. further evaluation is warranted. it may well be the antibiotic of choice. p antimicrobial activity of tigecycline (gar- ) tested against enterobacteriaceae, and selected non-fermentative gram-negative bacilli, a worldwide sample r. jones, t. fritsche, h. sader, m. beach north liberty, usa background: as resistances (r) among gram-negative bacilli (gnbs) expand, few antimicrobial agents have been developed to address this clinical problem. tigecycline (tig), a novel glycylcycline, has an expanded spectrum of activity and potency, tigecycline covers many routine gram-negative resistant strains and additionally possesses activity versus some uncommonly isolated non-fermentative gnbs. this study compares tig with contemporary broad-spectrum agents using recent clinical isolates from europe and other continents. methods: all strains ( ) were centrally processed by reference, broth microdilution methods against more than antimicrobials. all concurrent qc results were within nccls published ranges, with identifications performed by traditional methods and/or the vitek system. over isolates were tested from the enterobacteriaceae (ent) and non-fermentative gnbs categories. susceptibility (s) for tig was defined as mg/l, that breakpoint used for all tetracyclines by the nccls. results: the ent were divided into three groups for analysis: esbl-producing isolates ( strains), proteae group ( strains; includes p. mirabilis and indole-positive species) and all enteric bacilli. tig was very active against all esbl-producing isolates (mic , . - mg/l; highest among tc-r subsets), and all ent (mic / , . / mg/l). proteae had a mic at mg/l and all but one of tig-r or intermediate strains (mics, and mg/l) were m. morganii or p. mirabilis. p. aeruginosa was marginally inhibited by tig (mic , mg/l). in contrast, acinetobacter spp. (mic , mg/l; . % s) and s. maltophilia (mic , mg/l; . % s) were readily inhibited by tig. among all ent studied, . % were tc-r, but only one strain (p. mirabilis) was tig-r (mic at mg/l). conclusions: remarkable potency and breadth of spectrum was observed for tig against ent ( . % at mg/l vs. . % for tc), s. maltophilia and acinetobacter spp. limited activity was noted versus p. aeruginosa ( . % at mg/l) and some proteae (mic , mg/l). tig should be of value for the treatment of infections caused by several commonly r gnb groups. background: tigecycline (tig, formerly gar- ), is a novel glycylcycline which is currently in phase clinical trials. the in vitro activity of the tig was evaluated in comparison with tetracycline (tet) and other antimicrobial agents against recent ( ) ( ) ( ) clinical isolates collected worldwide from patients with respiratory infections and meningitis. methods: a total of isolates were tested against tig and more than comparator agents by broth microdilution according to the nccls reference methods and interpretative criteria. the collection included, h. influenzae (hi; strains, % betalactamase-producing), m. catarrhalis (mcat; strains, % beta-lactamase-producing), and n. meningitidis (nm; strains). results: tig demonstrated excellent activity against these organisms with all isolates being inhibited at mg/l (tet susceptibility breakpoint). tig was highly active against hi (mic , mg/l) and mcat (mic , . mg/l), and its potency against these pathogens was not affected by beta-lactamase production. tig was fourfold more potent than tet against hi and tetresistant isolates showed low ( mg/l) tig mics. nm isolates were highly susceptible to tig (mic , . mg/l) and to the vast majority of antimicrobial agents evaluated. conclusions: these results indicate that tigecycline has potent in vitro activity against clinically important gram-negative bacteria that cause community-acquired respiratory infections and meningitis, including tet-r isolates. further evaluations of tig activity, as well as, clinical studies are necessary to assess the role of this compound in the treatment of both community-and hospitalacquired infections. background and objectives: beta-lactamase production is the major mechanism of bacterial resistance to beta-lactam antibiotics in gram-negative pathogens, and surveillance of beta-lactamase determinants is an important issue of microbial drug resistance. given the great diversity of beta-lactamases and their overlapping substrate specificities, molecular analysis is necessary to identify the nature of beta-lactamase genes in clinical isolates. in this work we investigated the potential of the dna microarray technology for a rapid and comprehensive detection of beta-lactamase genes in drug-resistant bacteria. methods: a total of oligonucleotide probes were designed for specific recognition of beta-lactamase genes of different lineages ( of molecular class a, of class b, of class c and of class d). a dna chip was designed including a triplicate set of probes, as well as positive hybridisation controls. the microarray was printed on epoxy-modified glass slides using an affymetrix gms robotic spotter. genomic dna was labelled with cy or cy by random priming. hybridisation signals were then detected using an affymetrix laser scanner and images were analysed by the genepix pro (version . ) software. results: the dna chip was tested with gram-negative strains (including both reference strains and clinical isolates) in which the repertoire of beta-lactamase genes was partially known or unknown. all the predicted beta-lactamase genes (among which there were members of the blatem, blashv, blactx-m, blaper, blavim, blaimp, blacmy-lat groups of acquired genes) were correctly detected by microarray hybridisation. in clinical isolates of unknown beta-lactamase content, the microarray detected genes whose presence was subsequently confirmed by conventional pcr assays. false-positives were observed with a subset of probes, which had to be redesigned to overcome the problem. conclusions: successful detection of several different beta-lactamase genes of clinical importance was achieved by using a dna microchip. the dna microarray technology appears to be a sensitive and specific tool for rapid detection and characterisation of beta-lactamase genes in clinical isolates. objectives: some members of the genus citrobacter are potential pathogens of debilitated hospital patients. they can become resistant to beta-lactamases, including third generation cephalosporins due to over-expression of a chromosomal beta-lactamase. eleven species are currently known, but speciation is often difficult using biochemical tests. isolates previously typed as citrobacter diversus are now known as citrobacter koseri. here we measured sequence variation at the beta-lactamase structural gene amongst a group of clinical isolates, originally identified as c. diversus by api e profiling. methods: nine c. diversus isolates were collected from faecal samples of children being treated in the oncology department of bristol children's hospital in the early s. beta-lactamase and s rrna genes were amplified by pcr and sequenced by standard methods. beta-lactamase induction was attempted in liquid-grown cultures using cefoxitin ( mg/l for h). nitrocefin hydrolysis assays were performed using a spectrophotometer. results: analysis of s rrna gene sequences confirm that, of the nine clinical isolates, five, which all have an inducible betalactamase gene whose sequence is closely related to c. diversus nf and ula , are actually citrobacter amalonaticus. given that c. diversus isolates have all been renamed c. koseri, this error in nomenclature must be addressed. the reason for the error is that c. diversus was known to have variability in its ability to utilise malonate, the only differentiation between c. koseri and c. diversus. four of the test isolates do type as c. koseri using s rrna sequencing. these true c. koseri isolates produce a novel, acidic, class a beta-lactamase, named ckoa, constitutively. the sequence of this beta lactamase gene was determined, and is only % identical to the c. diversus (now c. amalonaticus cdia). conclusions: we present a new beta-lactamase sequence, from c. koseri and shows that c. koseri nf and ula should be retyped as c. amalonaticus. beta-lactamase-specific pcr may provide a valuable tool for typing citrobacter spp. isolates, and is very suitable for separating c. amalonaticus and c. koseri, which are very closely related biochemically. the knowledge that clinical c. koseri isolates produce a beta-lactamase constitutively at low levels may be useful clinically. p a single-tube pcr with mgb eclipse probes for detection of shv-type extended-spectrum beta-lactamases (esbls) a. ekimov, m. edelstein, e. belousov smolensk, rus; bothell, usa objectives: esbls of the shv-type are one of the most common and clinically significant beta-lactamases. the number of shv variants is continuously growing; however esbl activity of shv enzymes has been associated with mutations at relatively few amino acid positions (aa-s) as compared with the tem enzymes. here we propose a simple and rapid method that allows detection of all the known shv esbls in a single real-time pcr reaction. methods: the proposed method is based on amplification of blashv genes in the presence of short ( - nt) fluorogenic probes capable of hybridisation-triggered fluorescence. these probes commercially known as mgb eclipse probes contain a dark quencher with a conjugated minor groove binder at the ¢-end and a fluorescent dye at the -end. this structure allows detection and differentiation of nucleotide polymorphisms at targeted sites by post-pcr melting curve analysis. four probes were designed to perfectly match the wild-type (wt) sequences at mutation sites corresponding to aa-s , , , and . thus, mutations conferring esbl activity were expected to specifically lower the melting temperatures (tm-s) of the probe-template duplexes. each probe was labelled with a unique dye permitting analysis of mutations at multiple sites in a single reaction. results: the method was validated using laboratory strains producing the shv- (wt, non-esbl control), shv- , , , (g s), shv- (g a), shv- (d a), shv- (d n) and strains carrying cloned blashv fragments to which the naturally occurring mutations d g, g d, t s and a v were introduced by site-directed mutagenesis. following careful design of the probes and optimisation of pcr conditions, all the above mutations were successfully detected and discriminated from the wt sequence and each other according to specific tm-s. the detection was precise and highly reproducible in repeated experiments. furthermore, when applied to the analysis of clinical isolates of klebsiella pneumoniae expressing esbl phenotype, the method was able to detect multiple shv alleles (wt and g s or d a) in the same isolates. this observation is particularly important considering the high frequency of co-production of the shv- and esbls in klebsiellae. conclusions: a pcr with mgb eclipse probes has a great potential for studying the epidemiology of shv esbls and possibly for analysis of other antimicrobial resistance mechanisms associated with mutations at defined loci. methods: a total of non-repeat enterococcal blood isolates ( e. faecalis and e. faecium) were collected during to from hospitals located in south east of sweden. the bacterial isolates were identified by standard microbiological methods and susceptibility testing was performed with a -lg gentamicin disk on pdm-agar (ab biodisk) to detect hlgr isolates. all isolates were tested for the presence of the aac( ¢)ie-aph( ¢¢)ia gene using the polymerase chain reaction (pcr) technique. results: there was complete correlation between the gentamicin disk diffusion test and the pcr results. all hlgr isolates, as defined by disk diffusion, and the positive control (e. faecalis atcc ) carried the aac( ¢)ie-aph( ¢¢)ia gene as judged from the pcr results. the resistant gene was not found in the negative control atcc or any of the non-hlgr enterococci. conclusion: this study shows that in our setting the sensitivity and specificity of the disk diffusion method for the detection of hlgr enterococci is very high and there is a total agreement with the results obtained by using a pcr technique for detection of the aac( ¢)ie-aph( ¢¢)ia aminoglycoside modifying gene. objectives: the main objective was to develop a pyrosequencing method for identification of enterococcus spp. species with pyrosequencing method. also, development of antibiotic resistance with special reference to macrolide resistance will be studied by susceptibility testing in samples isolated serially from subject exposed to clindamycin. methods: biochemical identification of the enterococcal strains from faecal samples was done by growth at c, catalase and hydrolyse of -pyrridonyl-beta-naphtylamide (pyr). species identification was done with pyrosequencing method. psq ma pyrosequencing technique enabled identification of different enterococcus species based on their s rrna v -regions signature-sequences. antibiotic susceptibility testing was done by agar dilution method on mü ller-hinton ii medium, according to nccls. mic values were tested against erythromycin, clindamycin, ciprofloxacin, ampicillin, gentamicin, vancomycin and tetracycline. macrolide resistance genes; erm(b), erm(tr) and mef(a) was studied by multiplex-pcr. results: with pyrosequencing method, we identified enterococcus faecium, e. faecalis, e. avium and e. casseliflavus species, and non-enterococci species. the antibiotic susceptibility testing showed that . % of the enterococcus strains were resistant to erythromycin, . % to ciprofloxacin and . % to tetracycline. about . % of the enterococcae had erm(b)-gene. conclusion: pyrosequencing was rapid and easy method for identification of bacterial strains even to the species level. antibiotic resistance varied a lot between different bacterial strains, as e. faecium and e. casseliflavus species being the most resistant ones. pyrosequencing results correlated well with species phenotype and antibiotic resistance. objectives: to determine the species distribution of vancomycin resistant enterococci (vre) isolated from hospitalised patients and detect genes encoding resistance to vancomycin and teicoplanin, by sandwich hybridisation method. and cpha genes by pcr, but not actual enzyme production, may be attributed to so-called 'silent' genes. susceptible strains are known to be able to convert to high-level beta-lactam/carbapenem resistance by increasing the expression of 'nearly' silent metallobeta-lactamase genes. metallo-beta-lactamases have been found to be carried on a small plasmid ( . kb) that appears to be selftransmissible, posing a potential threat of rapid spread of resistance. therefore early recognition of metallo-beta-lactamase producing strains is imperative. to describe the distribution of species in our nocardia isolates and to evaluate the usefulness of an easy and rapid method based on a short battery of susceptibility tests to identify clinical nocardia isolates compared with pcr and restriction analysis of hsp routinely used in our laboratory. methods: nocardia sp. isolated from to were selected to study. molecular identification was performed by hsp pcr-rflp. identification by susceptibility testing was by disk diffusion with gentamicin (cn), tobramycin (tob), amikacin (ak) and erythromycin (e) and by broth microdilution and e-test with ampicillin (amp), ciprofloxacin (c), cefotaxime (ctx) and amoxicillinclavulanate (aug). results: isolates of nocardia sp. were studied. distribution of species according to results from pcr-rflp was: n. asteroides i ( ), n. asteroides vi ( ), n. farcinica ( ), n. nova ( ), n. otitidis-caviarum ( ). n. asteroides i isolates had two different susceptibility patterns, two isolates were cn-s, tob-s, ak-s, e-r and the other two were cn-r, tob-s, ak-s, e-r. all n. asteroides i isolates were amp > lg/ml and c > lg/ml and ctx < lg/ml. eightyseven per cent of n. asteroides vi were cn-s, tob-s, ak-s, e-r, amp > lg/ml, ctx < lg/ml whereas c was variable. hundred per cent of isolates of n. farcinica were cn-r, tob-r, ak-s, e-r, amp > lg/ml, c < lg/ml and ctx > lg/ml. n. nova isolates were cn-s, tob-s, ak-s, e-s, amp < lg/ml, ctx < lg/ml and c < lg/ml. n. otitidis-caviarum isolates were cn-s, tob-s, ak-s, e-r, amp > lg/ml, ctx > lg/ml and c < lg/ml. medium time to obtain results by both methods was h. conclusions: % of isolates belonged to the former n. asteroides complex. n. farcinica and n. nova were easily distinguished from other nocardia species by its susceptibility patterns. the main group n. asteroides vi was more difficult to distinguish from n. asteroides i and n. otitidis-caviarum. a short battery of susceptibility tests permits rapid differentiation of our most frequent nocardia isolates, although genotypic tests are more discriminatory. the ixodes ricinus tick, common ectoparasite of animals and humans, is the main vector of lyme disease in the czech republic. detection of borrelia under microscope, isolation in bsk-h medium and pcr identification was the aim of this work. methods: a tick was crushed in drop of sterile phosphate buffer saline and admired under microscope in dark-field. samples, in which spirochetes had been detected, were incubated in liquid bsk-h medium (sigma) at c and admired weekly for weeks. each strais was passaged twice and was frozen in . -ml aliquots at À c. direct fluorescence assay (dfa) with fluorescein labelled polyclonal antibody to borrelia burgdorferi was used for screening. deoxyribonucleic acid of borrelial strains was isolated with invisorb genomic dna kit iii (invitec). three sets of primers (for b. burgdorferi sensu lato, b. garinii and b. afzelii) derived from srrna gene (rosa and schwan) were used for elementary identification of strains. detailed analysis of strains was made by light cycler real-time pcr (rt-pcr). primers and probe derived from reca gene were used in this method. results: there was a collection of ticks in urban and suburban localities of the czech republic from to years. incidence of spirochetes in tick population differed from to . % in different localities. spirochetes were cultured from at least one of six ticks ( out of ) that were tested positive by dark-field microscopy. all strains reacted positively by dfa and gave positive response with primers specific for b. burgdorferi sensu lato complex. nineteen strains belonged to b. garinii, four to b. afzelii and two to b. burgdorferi sensu stricto genospecies. one strain did not react with srrna primers for b. garinii but had melting temperature of reca gene product identical with b. garinii type strain. we identified the genotype of two strains determined as b. burgdorferi sensu lato neither by pcr, nor by rt-pcr. uors, blood and tissue were subjected to sequencing with the dideoxy chain termination technique using ceq cx sequencer. cultivation, immunocytochemistry and western blots were used for confirmation. results: we cultured four blood, six skin, six csf isolates, numerous tick and two animal isolates. real-time pcr targeted reca, s and ospa genes showed that involvement of the nervous system, joints and skin in czech patients was predominantly caused by b. garinii, serotypes , , ( %), then b. burgdorferi ss ( %) and b. afzelii ( - %). the remaining - % comprise coinfection with anaplasma phagocytophila or mixed borrelial infections. similar results were found in animals. among game animals % tested positive with b. garinii and b. burgdorferi. wild boars and murids hosted borrelia sp. in and % with prevalence of b. afzelii. no significant differences were noticed between the infection of adult and nymphal ticks, both reaching and % in june and september, respectively. diferences were also between regions, in east bohemia with b. garinii prevailing and in moravia with prevalence of b. afzelii and human cases of erythema migrans and acrodermatitis atrophicans. infection prevalence data for patients were in agreement with data for the tick and animals. objectives: the aim of our study was to identify the strains of borrelia isolated from ticks and lyme disease patients in the russian far east and to analyse their taxonomic positions based on ospa gene phylogeny. methods: we have analysed strains of borrelia burgdorferi sensu lato isolated from ixodes persulcatus ticks ( ) and skin biopsies of erythema migrans from lyme disease patients ( ) isolated by standard methods during last years in the russian far east. after amplification with newly designed primers, we obtained full-length ospa gene sequence of each of the strains. results: we identified four strains as b. afzelii completely identical to the strain xj , isolated in japan. all of them were isolated from ticks. the other strains were found to be genetically variable, but the closest homology found was with b. garinii. after phylogenetic analysis of ospa gene we found that these strains form three distinct and well-defined clades at the phylogenetic tree. genogroups and represent only species isolated in the far easter regions of the russian federation and in japan only, whereas genogroup represents mostly european isolates, including seroand genogroups defined in the works of b. wilsske et al. and g. will et al. and four isolates from the russian far east. european serogroups and form the clade localised between genogroups and . human strains were found within genogroups and . conclusion: b. garinii was found to dominate among other b. burgdorferi sensu lato strains isolated from ticks and lyme disease patients form the russian far east. phylogenetic analysis showed that the species identified as b. garinii have significant variability in the ospa gene and form three major groups. two groups consisting only of strains isolated in the far east are significantly remote from all other b. burgdorferi sensu lato species. bootstrap values and distances among these groups suggest their solidity, especially genogroup . this, probably, indicates the distinct origin of defined genogroups and of b. garinii and may suggest another taxonomic status. objectives: for diagnosis of lyme borreliosis (lb) a two-step approach is recommended by cdc and dghm (screening elisa followed by immunoblot (ib) in case of reactive elisa). though borrelia ibs are widely used, they are still poorly defined regarding sensitivity, specificity and standardisation. a recently described recombinant western immunoblot (wib) complemented with borrelia antigens produced in vivo but not in culture (i.e. vlse) could improve previous tests ( ). here a recombinant borrelia line ib (lib) was developed where each recombinant antigen is separately detectable, even those antigens with identical molecular weight. methods: the following recombinant igg and igm ibs were compared: (a) the wib described in ( ) with p /p (strain pko, b. afzelii), p (strain pbi, b. garinii ospa-type ), bmpa (strains pka , b. burgdorferi sensu stricto, pko, and pbi), vlse (strain pka ), ospc (strains pka , pko, pbi, and b. garinii strain ) , and dbpa (strains pko and pbr, b. garinii ospa-type ). (b) the lib with all antigens of the wib and in addition vlse (strains pko and pbi), ospc (strain ple, b. afzelii) and dbpa (strains b and pbi). to verify sensitivity and specificity, sera of patients with early lb ( early neuroborreliosis, erythema migrans) and control sera ( blood donors, rheumatoid factor positive, syphilis patients and patients with fever of unknown origin) were studied. results: ib interpretation criteria defining a serum as positive with at least two reactive bands or in case of igm at least one strong ospc band were used ( ). sensitivity significantly increased from % (wib) to % (lib) for igg and from % (wib) to % (lib) for igm while specificity remained unchanged ( % for igg tests and % for igm tests). the increase of sensitivity was mainly due to the line blot technique, which allows detection and identification of antibodies differently reactive with homologues of the same protein. conclusion: the lib is more sensitive than the wib for both igg and igm antibody detection in acute lb while specificity remains unchanged. the lib is better to standardise and results are easier to interpret. background: tick of the ixodes ricinus group are well known as major vectors of the causative agents of lyme borreliosis, granulocytic anaplasmosis, ehrlichiosis and babesiosis in european countries. the humans infected with these agents can experience a wide range of clinical manifestations. i. ricinus is a widely distributed tick in lithuania and may transmit pathogens to mammalian hosts, including human beings. a single tick may contain several different pathogens so double-infection with borreliosis and ehrlichiosis may be seen. objectives: the aim of this study was to determine whether i. ricinus ticks collected in different regions of lithuania were infected with the causative agents of lyme borreliosis, anaplasmosis, ehrlichiosis and babesiosis agents and to estimate the prevalence of mixed infections in them by pcr. no investigations have been carried out to assess the prevalence of borrelia, anaplasma, ehrlichia and babesia infection in i. ricinus in lithuania using the pcr method before. methods: altogether, i. ricinus ticks collected from different regions of lithuania, were included in this study. all ticks were analysed individually. the presence ehrlichia/anaplasma group pathogen was determined by using pcr with ehrlichia/anaplasma-specific primers hr /ehr , multiplex pcrs using species-specific borrelia primers gi-r/gi-l (borrelia burgdorferi s.s.), gii-r/gii-l (b. garinii), giii-r/giii-l (b. afzelii). real-time pcr method with the abi prism system was used to detect babesia divergens. ehrlichia/anaplasma species were determined using the reverse line blot hybridisation. results: of the individually processed ticks, ( %) were positive for ehrlichia/anaplasma (hge - , hge variant - , e schotii - and were not identified), ( %) for borrelia (b. burgdorferi s.s -one ( . %), b. garinii - ( %), b. afzelii - ( %) and ( %) were positive for babesia divergens. one tick contained both ehrlichia/anaplasma and babesia, two contained both babesia and b. afzelii and one ehrlichia/anaplasma and b. garinii. conclusions: our results represent the first study in lithuania in which borrelia, ehrlichia, anaplasma and babesia parasites were directly identified in i. ricinus ticks by pcr, multiplex pcr, reverse line blot hybridisation and real-time pcr. it was detected that b. afzelii was the dominant genospecies in lithuanian ticks ( %) and ehrlichia/anaplasma and babesia were found in ticks too and might cause human diseases. molecular bacteriology: characterisation of agents p improved automated ribotyping using hindiii to discriminate previously uniform listeria monocytogenes serotype b strains i. heller, k. grif, m. dierich, r. wü rzner innsbruck, a objectives: to develop improved automated subtyping approaches for listeria monocytogenes, we characterised the discriminatory power of different restriction enzymes for ribotyping. pvuii and hindiii were evaluated for their ability to differentiate among isolates representing one of the two major serotype b epidemic clones, having ribotype reference pattern dup- (which differs from the other clone dup- in the ecori pattern only). this is of utmost importance, as the presence of only two major patterns within the serotype b does not allow sufficient epidemiology of listeria infections. methods and results: the eight selected l. monocytogenes isolates (serotype b) with the ribotype reference pattern dup- were responsible for human listeriosis outbreaks in france, canada, switzerland and turkey from to , and for sporadic foodborne cases in austria ( ), england ( and ) and the usa. ribotyping was performed using the riboprinter microbial characterisation system according to the manufacturer's instructions using ecori, pvuii and hindiii as restriction enzymes. we found that the eight isolates belonging to dup- (i.e. indistinguishable by ecori) were also indistinguishable by pvuii but yielded two clearly different patterns when using hindiii. conclusions: we conclude that automated ribotyping using hin-diii allows discriminating previously uniform l. monocytogenes b isolates. this discrimination may facilitate the tracing of outbreaks and may also improve epidemiological surveys. p detection of bft, the isoforms of the enterotoxin gene and cfia gene in bacteroides fragilis isolates of different origins g. terhes, j. soki, k. ago, e. urban, e. nagy szeged, hun objectives: bacteroides fragilis is an obligate anaerobic, gram-negative rod constituting % of the normal intestinal flora of humans, and is the gram-negative anaerobic rod most frequently isolated from human clinical samples. some of the b. fragilis isolates produce a zinc-dependent metallo-protease, enterotoxin coded by the bft gene. this protein has enterotoxic activity; it causes fluid accumulation in a lamb ligated ileal loop model. to date, three different isoforms, designated bft- , bft- and bft- , have been identified. the literature regards the enterotoxin-producing property of b. fragilis as a virulence factor since these strains can be isolated more often from severe infections such as sepsis, or abdominal and deep soft-tissue abscesses. it is also thought to be involved in diarrhoea in - -year-old children. aims and methods: the aim of the present study was to examine the prevalence of enterotoxin production among b. fragilis strains isolated between and from specimens originating in clinical wards of our university or in other hospitals by ht- cytotoxicity testing or pcr detection of the bft gene. the results obtained with the two methods were compared. the frequencies of three alleles of bft genes in enterotoxigenic strains from different sources were determined by using pcr-restriction fragment length polymorphism analysis. the b. fragilis strains can be divided into two major groups by molecular typing methods and most importantly according to the carriage of the cfia gene. we therefore also examined the occurrence of the cfia gene by pcr and the co-incidence of bft and cfia among the above collection of strains. results: the average occurrence of toxigenic b. fragilis strains in the different groups of clinical samples was % and in deep-tissue infections was % by both the pcr method and the cytotoxicity assay. bft genes were found only in the cfia-negative group. the prevalence of the cfia gene corresponded to our earlier findings and data from the literature and we did not observe co-incidence of the bft and cfia genes in this study. introduction: in addition to the two large clostridial cytotoxins (lct -toxins a and b) some strains of clostridium difficile also produce an actin-specific adp-ribosyltransferase (binary toxin cdt). cdt may serve as an additional virulence factor. methods: we used pcr and southern blotting methods for detection of genes encoding the enzymatic (cdta) and binding (cdtb) components of binary toxin in strains isolated from patients with suspected c. difficile-associated diarrhoea or colitis. binary toxin production was assessed by western blotting using antisera against the iota toxin of c. perfringens (anti-ia and ib). toxin activity was detected with an adp-ribosyltransferase assay. pcr amplification was performed to detect the gene encoding for toxin b. binary positive strains were subjected to toxinotyping and were characterised by phenotypic (serogrouping) and genotypic markers (pcr-ribotyping, arbitrarily primed pcr (ap-pcr) and pulsed-field gel electrophoresis (pfge)). results: twenty-two strains (prevalence %) harboured both genes cdta and cdtb; out of the strains reacted with antisera against the iota toxin of c. perfringens; the binary toxin activity was positive in only of the strains. all strains also produced toxins b. however, they had significant changes in tcda and tcdb genes and belonged to variant toxinotypes iii, iv, v, vii, ix and xiii. with typing methods used we could differentiate profiles, indicating that most of binary toxin positive strains were unrelated. conclusion: binary toxin-producing isolates of c. difficile are widespread but prevalence varies from one country to another. more studies are needed to define the role of binary toxin in pathogenesis. clostridium difficile in singapore w.y. leong, r. das ramadas, t.h. koh, k.p. song singapore, sgp objective: occurrence of nosocomial clostridium difficile-associated diarrhoea and pseudomembranous colitis is related to the production of toxins a and b (encoded by tcda and tcdb, respectively) from the pathogen. tcda and tcdb, together with their accessory genes, tcdc-e are arranged within a well-defined chromosomal region termed pathogenicity locus (paloc). another virulence factor, adp-ribosyltransferase binary toxin (encoded by cdt genes) was reported to be found in approximately % of pathogenic strains of c. difficile. despite the availability of a number of detection methods, the identification methods commonly used are not designed to detect all the virulence factors known. we present here an alternative characterisation of the toxigenic and the related genes of c. difficile based on genotyping. the correlation between paloc and cdt genes was also examined. methods: all clinical isolates from singapore general hospital (sgh) were screened with pcr and multiplex pcr for the presence of tcda-e and cdta-b in the paloc region and cdt operon, respectively. the production and activity of toxins a and b were analysed by commercial kit and cytotoxicity testing. results: the isolates could be classified into groups based on the genotypic analysis of the paloc and cdt genes. approximately % of them shared a common profile with the reference strain vpi , and about % were completely devoid of the genes tested. variations demonstrated in tcdc-e were complicated and no specific profile could be attributed to a particular genotype. an atypical toxigenic variant was discovered which contains only tcdb. in contrast to data reported elsewhere, none of the pathogenic strains was found to contain complete cdt genes. when tested for tcda and tcdb production, six strains were identified to be toxins a-negative, b-positive. conclusion: the great genetic polymorphisms displayed by the c. difficile isolates here confirm that these strains were highly heterogeneous and could originate from endogenous source. there is no significant correlation between presence of the structural genes (tcda-b), accessory genes (tcdc-e) and cdt genes. pathogenic strains do not necessarily contain all the genes in the paloc. in conclusion, our results using this toxino-genotyping method for the studies of genetic distribution of toxinogenic genes correlates well with the phenotype of the bacteria i.e. toxin expression. p characterisation of clostridium difficile strains isolated in different time periods and belonging to different ribotypes p. spigaglia, v. carucci, p. mastrantonio rome, i objectives: seventy-four clostridium difficile clinical isolates, collected in different time periods, were typed by pcr-ribotyping. strains belonging to the two main pcr-ribotypes were characterised for virulence determinants and for antibiotics resistance. methods: paloc genes analysis, detection of binary toxin gene and antibiotic resistance determinants (ermb, tetm and catd) were performed by pcr assays. erm(b) sequence type was identified by a rflp-pcr. mics for erythromycin, clindamycin, tetracycline and chloramphenicol were determined by e-test. results: two main pcr-ribotypes named a and r, respectively, were identified. pcr-ribotype a collected strains whereas strains belonged to pcr-ribotype r. old strains (from to ) belonged to pcr-ribotype a, whereas recent strains (from to ) belonged to pcr-ribotype r. all strains with pcr-ribotype a had classical paloc genes and did not have the binary toxin gene. ninety percent of these strains were multiresistant and the sequence type of the ermb genes was similar to that of c. difficile . all strains belonging to pcr-ribotype r had the binary toxin gene, four of them showed major variations in the toxin a gene and % had a mutated toxin negative regulator. none of these strains was multi-resistant although one showed all three antibiotic resistance determinants. fifty three percent had a tetm gene, % tetm and ermb genes and % only an ermb gene with a sequence similar to that of c. perfringens cp . interestingly, as far as resistance is concerned, there was no correspondence between phenotype and genotype in % of these strains. in particular, all strains with a tetm or a catd gene were susceptible to tetracycline and chloramphenicol in vitro, whereas five strains, resistant to erythromycin but not to clindamycin, did not have an ermb gene. all these strains showed, after induction with erythromycin, some clindamycin resistant colonies. conclusions: the results seem to indicate a recent spread of c. difficile clones that add together a potential increase of virulence by acquisition of the binary toxin, variations in genes belonging to the paloc and acquisition of different mechanisms of antibiotic resistance. enterococci are natural inhabitants of the gastrointestinal flora of humans and animals and are widely distributed in the environment. members of this genus are recognised as important opportunistic pathogens responsible for serious infections but the molecular mechanisms of enterococcal virulence are not yet completely understood. in this study enterococci from different sources, including clinical isolates (from human and veterinarian origin), non-clinical isolates and reference strains from enterococcal species, were typified and their virulence potential characterised. the relationships among these enterococci were first analysed using smai pulsed-field gel electrophoresis and m pcr-fingerprinting, in order to evaluate the genomic heterogeneity of the isolates. enterococci were also screened for several virulence traits such as cytolysin (cyl genes), adhesins (agg, esp, efaafs and efaafm genes) and gelatinase (gele), revealing distinct virulence potentials. in enterococcus faecalis, it was recently described that some virulence determinants can be clustered on large pathogenicity islands and not only in pheromone-responsive plasmids. dot-blot dna-dna hybridisation was used to locate virulence determinants in the bacterial genome of the enterococci under study. no conclusive results were obtained for esp and gele, whereas efaafs and efaafm were found on the chromosome as expected. although cyl genes and agg are plasmidic, in most isolates, they were detected on the chromosome of five strains, suggesting that these enterococci may harbour a pathogenicity island. beyond the widespread nature of virulence traits, chromosomal integration of virulence genes seems to occur in different enterococcal species and isolates from non-clinical sources. p identification of salmonella serotypes in sheep by pcr t. zahraei-salehi tehran, ir introduction: salmonella abortusovis, s. dublin, s. montevideo and s. typhimurium are more common serotypes in sheep. one way of transferring of contamination is from visceral organs specially gallbladder, intestine and liver, which can be transferred from meat to human. because of this, this research was essential to consider about it. objectives: ( ) isolation of salmonella serotypes from visceral organs of sheep and goats. ( ) detection of inva gene in isolated serotypes by pcr. materials and methods: for these goals, samples from livers, gallbladders, mesenteric lymph nodes and faeces (totally samples) were taken, and then cultured in enrichment and selective media. doubtful colonies were selected and transferred to tsi agar, urea agar, sim, mr-vp broth and nitrate broth. pcr reaction was carried out in master cycle (eppendorf). for dna extraction isolated salmonella serotypes was cultured in lb broth for h at c. lb broth ( ll) was boiled for min and centrifuged at Âg for min. a total of . ll of the supernatant was used for amplification by pcr with salmonella-specific ( and ) primers. results: three salmonella serotypes were isolated from mesenteric lymph nodes (two cases) and gallbladder (one case). serotyping test showed that two of them belong to group b and one of them to group d of salmonella. when subjected to salmonellaspecific primer inva, all isolates, including positive control, generated a single -bp amplified dna fragment, on . % agarose gel. conclusion: salmonella-specific pcr with primer set inva is rapid, sensitive, and reliable for detection of salmonella in many clinical samples. the present research supports the ability of this specific primer set to confirm the isolates as salmonella. all isolates, including positive controls (s. typhimurium and s. dublin), screened by pcr resulted in -bp amplified product. no amplified products were obtained from negative controls (water and o k escherichia coli serotype). objectives: the variability of salmonella typhimurium strains was studied by pcr-based methods. methods: strains of s. typhimurium were isolated from food or animal sources in the course of surveillance programmes. strains were phagotyped and their antibiotic resistance was determined by disk diffusion method. fluorescent aflp was done using eco-ri and msei enzymes and aflp products were separated by capillary electrophoresis. results: the presence of integrons was analysed in all strains of s. typhimurium and three different integron profiles (ips) were detected by amplification of variable region of the integrons. the ip- profile, characterised by two pcr products of . and . kb, was present in six strains. all these strains were multiresistant with resistance acssut or acssutna. the ip- profile contained single . kb pcr product and was present in six strains resistant to asutmp or assutmp. the dhfra gene was confirmed to be an integral part of ip- integron. a total of . kb pcr product (ip- ) was amplified in two strains sensitive to all antimicrobials. as lysogenic bacteriopahges could frequently transfer their dna into the bacterial cell and thus change chromosomal composition, phage-related sequences were probed in s. typhimurium strains by pcr with primers complementary to four genes of phage p (g , g , eae, eac). three different types of pcr products were detected in multiplex reaction: the presence of g sequence only, the simultaneous occurrence of g and eac or presence of g and g . nine strains did not contain any from the tested phagerelated genes. the relatedness between strains was further monitored by aflp. we observed high strain-to-strain similarity as dice coefficients fell in the range of - %. according to the presence of several dna fragments, strains were separated into eight aflp clusters. conclusions: by comparison of all methods we obtained corresponding results in strain clustering. all methods can be used for subtyping of s. typhimurium strains. producing klebsiella strains isolated from nosocomial infections k. matusiewicz, b. maczynska, d. olejniczak, a. przondo-mordarska, r. franiczek wroclaw, pl objectives: klebsiella bacilli present many pathogenic properties, which determine their ability to survive and rapid spreading in hospital environment. the adhesive properties of klebsiella bacilli associated with the presence of fimbrial and non-fimbrial adhesins play a very important role in pathogenicity of these bacteria. rapid spread of patogenical factors is often connected with presence of their plasmid-mediated genes. the aim of our study was to detect plasmid and chromosomally born fimh and mrkd genes encoding main adhesins: ms and mr, respectively. methods: a total of klebsiella clinical isolates obtained from patients hospitalised in different hospital wards were studied. the phenotypic activity of fimbriae was characterised by haemagglutination method. the genomic and plasmid dna were isolated using manual method as well as qiagen dna kits. the presence of genes encoding main adhesins were detected using pcr-method with primers detected fimh and mrkd genes results: % of strains displayed phenotypic activity of both type and type fimbriae, . % showed only activity of type fimbriae, . % only of type fimbriae and . % strains showed the lack of hemagglutination activity. the percentage of detected genes using pcr, was higher then showed results of phenotypic activity. the presence of mrkd genes was detected in % investigated strains in chromosomal dna and . % showed both mrkd and fimh genes. a total of . % strains demonstrated only fimh genes in chromosomal dna and . % strains showed no genes. in plasmid dna, the presence of main adhesin genes confirmed in % klebsiella strains (mrkd genes in % strains, both fimh and mrkd in % and only fimh in % strains). conclusions: the presence of fimh and mrkd genes in genomic and plasmid dna not always leads to phenotypic expression of fimbrial adhesins. the activity of type fimbriae is connected with chromosomal variant of mrkd gene. in case of fimh genes, the plasmid variant is enough for haemagglutination activity of type fimbriae. the percentage of detected fimh and mrkd plasmid genes depended on hospital units from which these strains were isolated. this suggests the spread of plasmid-encoded adhesins among klebsiella strains. objectives: bacteria of the genus klebsiella are opportunistic pathogens responsible for an increasing number of multiresistant infections in hospitals. the two clinically and epidemiologically most important species, klebsiella pneumoniae and k. oxytoca, have recently been shown to be subdivided into three and two respective phylogenetic groups. the aim of this study was in-depth evaluation of the amplified fragment length polymorphism (aflp) genetic characterisation method. methods: first, we investigated the variability of aflp patterns for klebsiella strains within and between different outbreaks. second, by use of carefully characterised, phylogenetically representative strains, we examined whether different klebsiella species and phylogenetic groups can be discriminated using aflp. twenty-four strains originating from seven presumed outbreaks and non-associated strains were investigated. results: the aflp fingerprints of all epidemiologically associated strains showed three or fewer fragment differences, whereas unrelated strains differed by at least four fragments. cluster analysis of the aflp data revealed a very high concordance with the phylogenetic assignation of strains based on gyra sequence and ribotyping data. the species k. pneumoniae, k. oxytoca, k. terrigena and the possibly synonymous pair k. planticola/k. ornithinolytica each formed a separate cluster. similarly, strains of the phylogenetic groups of k. pneumoniae and k. oxytoca fell into their corresponding cluster, with only two exceptions. conclusion: this study provides a preliminary cut-off value for distinguishing epidemiologically non-related klebsiella isolates based on aflp data, confirms the sharp delineation of the recently identified phylogenetic groups and demonstrates that aflp is suitable for identification of klebsiella species and phylogenetic groups. objectives: k-serotyping, i.e. determination of the capsular antigen, has been the preferred typing method for klebsiella isolates, as it is highly discriminatory ( -types are known) and as k-types are known to differ in their pathogenic potential. unfortunately, k-serotyping requires a large collection of sera and is restricted to a few reference centres. moreover, k-serotyping suffers from cross-reactions and is not applicable to non-capsulated strains. the objective of this work was to develop a molecular method that would enable to determine the k-serotype without using antiserum. methods: we amplified by pcr the capsular antigen gene cluster (cps) and the pcr product ( - kb long) was digested with hincii, followed by agarose gel electrophoresis (cps pcr-rflp). results: the profiles (called c-patterns) obtained for strains representing the known k-serotypes showed four to bands in the size range . - . kb. a total of distinct c-patterns were obtained. the following important observations were made: (i) the c-patterns obtained for strains of any k-serotype were distinct from the c-pattern of all other k-serotypes, with the only exception of serotypes k and k , which are known to cross-react. (ii) for k-types, c-pattern variation was found among strains with the same k-serotype; in most cases, the strains with variant c-patterns belonged to other klebsiella species than the reference strain. thus, cps pcr-rflp has a higher discriminatory power than classical k-serotyping. (iii) within k. pneumoniae, we observed c-pattern identity among strains of a given k-type, for example k or k , that were collected many years apart and from distinct sources. this stability of the c-pattern indicates that cps pcr-rflp is suitable for long-term epidemiology of capsular types. (iv) only . % (compared with - % for classical k-serotyping) of the strains analysed by cps pcr-rflp were non-typable, because pcr amplification failed. (v) the value of cps pcr-rflp for k-serotype determination was tested on recent k. pneumoniae clinical isolates. the k-serotype of ( %) of them could be deduced from the comparison of their c-pattern with the database. (vi) four of five non-capsulated strains analysed showed a recognisable c-pattern. conclusions: cps pcr-rflp allows determination of the k-serotype, while being easier to perform and more discriminatory than classical serotyping, and allowing the characterisation of non-capsulated strains. ( ) the composition of the vaginal microbial community of eight of these vaginal swabs (three grade i, two grade ii and three grade iii), were studied by culture and by cloning of the s rrna genes obtained after direct amplification. ( ) species-specific pcr for atopobium vaginae and gardnerella vaginalis was carried out for all vaginal swab samples. ( ) forty-six cultured isolates were identified by tdna-pcr and cloned s rrna gene fragments were sequenced, yielding a total of species. results: cloning revealed that a. vaginae was abundant in four out of the five non-grade i specimens and that lactobacillus iners was the only lactobacillus species that was present in non-grade i specimens, while it was absent from grade i samples. respectively . % (grade i), . % (grade ii) and . % (grade iii) of the vaginal swab samples were positive for both a. vaginae and g. vaginalis species-specific pcr (p < Á , chi square). discussion: culture independent, molecular analysis revealed a higher microbial diversity in non-grade i specimens than did culture. together, culture, s rrna gene cloning and species-specific pcr point to the presence of nine presumptively novel bacterial species and to a strong association between a. vaginae, g. vaginalis and bacterial vaginosis and to an ambiguous role for l. iners. it appears as if a. vaginae may be a constituent -in low numbers -of the human vagina, possibly attaining replicative dominance in association with decreasing lactobacillary grading. the presence of a. vaginae in bacterial vaginosis(-like) microflora may shed new light on the aetiology of this condition. using multilocus pcr tests with various primers in the genomes of strains isolated in the territories of russia and turkmenistan we were able to detect three housekeeping genes (hapa, toxr, rtxa) and nine virulence genes located in prophages and 'pathogenicity' and 'persistence' islands: ctxphi (ctxa, zot, ace), rs phi (rstc), vpi (tcpa, alda, toxt), vpi- (nanh), epi (mshq). besides, we used the methods of ribotyping and pcr typing which involved the 'random' primer, , to elucidate genetical relationship between the strains of varying epidemic significance. the genome of clinical isolates obtained from patients during several epidemic outbreaks, was shown to be stable and to contain all the genes tested. c. vibrios isolated during the interepidemic period from natural ecosystems, formed a heterogenous population represented by single virulent clones that had retained the complete set of the genes under study, by non-toxinogenic strains, that had lost only individual genes and (or) pathogenicity blocks of genes, i.e. either ctxphi and rs phi, or ctxphi, vpi and rs phi, or by those carrying deficient prophages ctxphi (ctxaÀ zot+ ace+) and vpi (ctpaÀ alda+ toxt+), as well as by clones containing only housekeeping chromosomal genes and sometimes a gene from the 'persistence island'. as soon as virulent clones get into water environment, they lose their virulence blocks in the following order: ctxphi and rs phi, then vpi, gene vpi- being the last one to be lost. in conformity with the results of the above three genotyping methods, epidemically hazardous strains, represented a homogenous group, suggesting a single clonal origin. close genetical relationship between these strains and non-toxinogenic vibrios, that partly retained their virulence genes, was also established. at the same time, as shown by ribotyping and pcr typing studies, avirulent 'water' vibrios formed an independent group, because their genotypes manifested quite distinct features, in contrast to the first two vibrio groups. thus, the observed genotype heterogeneity of el tor cholera vibrios living in water ecosystems was likely to be a result of the loss of dna fragments varying in their length and functions. the genotyping procedures used in the work made it possible to discover evolutional relationships among the bacterial strains under study. bacteroides fragilis gram-negative anaerobic rods, strains isolated in poland and in france (from intestinal and extraintestinal sources) were compared in this study. the identification of bacterial strains was done on the basis of gram staining, growth on selective bbe (bacteroides bile esculine) medium, and biochemical characteristics determined by the api a test (biomérieux, france). for assessment of the presence of enterotoxin (fragilysin) gene in analysed strains, the pcr method was used. dna for pcr was isolated using genomic dna prep plus (a&a biotechnology, poland) and amplification was performed in a techne thermocycler with primers ( ¢-gag ccg aag acg gtg tat gtg att tgt- ¢-tgc tca gcg ccc agt ata tga cct agt- ¢). the pcr program consisted of the following steps: °c for min, cycles of c ( min), c ( min) and c ( min). among the polish strains, contained the fragilysin gene. of the french strains contained the fragilysin gene. for all these strains, pulsed field gel electrophoresis (pfge) was performed. bacteria were suspended in se buffer ( mm nacl, . edta ph . ), embedded in . % agarose plugs and lysed overnight at c. plugs were washed five times in se at room temperature afterwards. dna in the plugs was digested using not i (boehringer mannheim, germany). electrophoresis was performed in a chef mapper (biorad, venendaal, the netherlands). the voltage was v/cm for h with linear ramping from to s at ae angles. in conclusion, % strains isolated in france and % of those isolated in poland contained the fragilysin gene. the pfge analysis revealed that strains isolated in poland and in france show genetically differentiation (these strains are genetically not homogenous). objectives: different molecular mechanisms of resistance to azole antifungal agents, that can exist simultaneously, have been described in candida albicans strains. one of these mechanisms includes alterations in the gene encoding the target enzyme erg . in the present study we used pyrosequencing method to conduct an epidemiologic survey in ketoconazole-susceptible and -resistant strains of clinical c. albicans strains isolated in our region, to determine differences in the gene encoding lanosteroldemethylase (erg ). methods: the strains of c. albicans were obtained by swabbing the oral mucosa of subjects with oropharyngeal candidiasis. susceptibility to ketoconazole was tested using the broth microdilution method recommended by the nccls document m -a. concentrations of ketoconazole tested were in the range . - mg/ml. the mic endpoint was defined as the lowest concentration at which % of growth was inhibited, compared with the drug-free control. yeasts were grown in sabouraud agar and dna was extracted by using qiaamp dna mini kit (quiagen). pcr primers matched an erg gene region of bp. one of the primers of pcr fragment was biotinylated, a single strand of pcr products was obtained with streptavidin-coated beads method. samples were analysed using a psq system with sqa software and sqa reagent. results: a total of . % of strains exhibited dds or resistance to ketoconazole (mic > . lg/ml). the sequence analysis was designed to cover a region of the erg gene including codons - . previous studies showed that in this region, the mutations g s, g s, r k and i t are associated with azole resistance in c. albicans. in our study the sensitive strains have shown no mutations. among dds and resistant strains, only the mutation g s was found in two strains, while no mutations were demonstrated in the remaining isolates. conclusion: this study is the first to use the pyrosequencing system to characterise changes in nucleotide sequence of the erg gene fragment involved in azole resistance of c. albicans strains. the observation of one point mutation in only two resistant strains tested suggests a limited role of the region of the erg gene analysed in the azole resistance among c. albicans strains present in our region. however, the pyrosequencing system has shown to be a fast and specific technique for detection of point mutations in the region of erg gene of c. albicans strains. escherichia coli verocytotoxin variants. correlations to the clinical manifestations s. persson, f. scheutz, k.e.p. olsen copenhagen, dk background: verocytotoxin (vt ) of verocytotoxin producing escherichia coli (vtec) is a potent toxin, capable of producing serious complication, when excreted from the bacteria colonising the intestinal tracts. the mature toxin is composed of one a-subunit and five identical b-subunits, and is encoded by the approximately bp vtx ab operon. based on the variable nucleic acid sequence of both subunits, several toxin variants have been identified. objectives: the subtype designation, important sequence motifs and clinical significance of the vtx variants, are not consistent throughout the literature. to shed more light on these features, a novel typing method was developed for the investigation of subtype-specific correlations to the clinical outcome. methods: the subtyping method relies on pcr and sequencing. by use of vtx universal primers, a -bp fragment covering the most variable regions of subunit a and b was amplified by pcr, and subsequently sequenced. results and conclusion: the present method was used for the analysis of vtx -positive strains from our strain collection, counting strains, isolated from patients with known clinical manifestations (hus, hc, bloody diarrhoea, diarrhoea, fever, etc.). compared with traditional subtyping, our preliminary results indicate that most strains in our strain collection harbour the vtx or vtx c subtype, in addition to a few strains containing the activatable carboxy-terminus of subunit a, referred to as vtx d. correlations between these subtypes and the clinical complications will be presented. additionally, the novel sequences from our strain collection will be investigated for other sequence motifs connected to the clinical outcome. as sequencing has become more accessible and less expensive, we believe that this method, offers a good and reliable alternative for diagnostic subtyping of vtec strains from these infections. p shiga toxin-producing escherichia coli o in slovenia p. zabukovnik, a. andlovic, a. zore ljubljana, si objectives: in the institute of microbiology and immunology (department for bacterial diagnostics of diarrhoeal infections), medical faculty in ljubljana, we wanted to introduce multiplex pcr test for detection of shiga toxin-producing escherichia coli (stec). until recently we used only enzyme immunoassay (eia) to detect production of shiga toxin (stx) in specimens. institute of microbiology and immunology has extensive collection of e. coli isolates from human faeces (mostly from hospitals in ljubljana). we decided to test isolates in our collection from to , with serogroup o . we used multiplex pcr assay that amplified sequences in four virulence genes (shiga toxin (stx ), shiga toxin (stx ), intimin (eaea), enterohemolysin (ehxa)). methods: all isolates were serotyped with rabbit o antisera. we used multiplex pcr to detect presence of shiga toxin , shiga toxin (and sub variants, but did not discriminate between them), intimin and enterohemolysin genes. we also tested those strains for production of stx with eia. results: we tested e. coli isolates with serogroup o and found stec. stx and ehxa genes were present in almost all stec o isolates. the most common pcr profile (five of ) of o isolates had stx , eaea and ehxa genes. one isolate had stx gene but did not produce shiga toxin (or possibly eia did not detect produced shiga toxin). most of those stec o were isolated in summer months of july and august. two o stec were isolated in the year shortly one after another. they had identical multiplex pcr profile. the same happened in the year . conclusion: we notice increase in the number of stec o isolates per year in years after . this may be because of use of better diagnostic methods. in last years stec o with pcr profile stx , eaea and ehxa is dominant. in years to the dominant pcr profile had stx , stx , eaea and ehxa genes. background: chronic prostatitis is recognised to be caused by infectious and non-infectious prostatic inflammation as well as non-inflammatory diseases, but the separation of various prostatitis syndromes is difficult to perform. bacterial prostatitis is a common diagnosis and a frequent indication for antimicrobial therapy. however, confirmation of aetiology of inflammation is exceedingly uncommon. objectives: the aim of this study was to determine the prevalence and aetiology of chronic bacterial prostatitis among the patients with clinically confirmed diagnosis. methods: between october and october the patients with suspected prostatitis were examined. the clinical diagnosis was confirmed in patients within months or greater duration of the following signs and symptoms: perineal discomfort, pain following ejaculation, urinary frequency, urgency, dysuria, low back pain, suprapubic pain, palpation of a tender prostate on physical examination. the bacteriological diagnosis was determined in patients, who had not been taking antibiotics in the previous month, by meares and stamey technique. prostatitis was categorised according to nih classification. results: a total of patients were examined. chronic bacterial prostatitis (nih category ii) was found in nine patients ( . %), inflammatory chronic pelvic pain syndrome (nih category iiia) -in ( . %), non-inflammatory chronic pelvic pain syndrome (nih category iiib) -in ( . %). the following pathogens were isolated in nih category ii: staphylococcus spp. -in three ( . %), anaerobic bacteria (prevotella spp., prevotella spp. and peptostreptococcus spp.) -in three ( . %), escherichia coli -in two patients ( . %), acinetobacter lwoffii -in one ( . %). conclusions: chronic bacterial prostatitis is an important but rare clinical entity. careful examination using quantitative segmented bacteriologic cultures leads to proper categorisation into the recognised forms of the prostatic syndrome. the most common pathogens of chronic bacterial prostatitis were staphylococcus spp., anaerobic bacteria (prevotella spp. and peptostreptococcus spp.) and e. coli. objectives: a prospective multicenter urology outpatient survey, undertaken to examine prostatitis in italy, is used to compare the prevalence, characterisation, diagnosis and treatment of prostatitis patient with the north american (na) prostatitis patient. methods and materials: seventy urologists, representing a crosssection of urologic centres in italy, counted and recorded the overall total male patients reported in the clinic and the overall total patients diagnosed with prostatitis over a -week period. results were compared with published practice prevalence and cohort data (in particular the nih chronic prostatitis cohort study -cpc and seattle prostatitis cohorts) examining similar data in na. results: a total of patients were identified with prostatitis ( . %). the mean age of the prostatitis patients was . (range - ). the most common urinary diseases were benign prostatic hyperplasia ( . %), recurrent urinary tract infections ( . %) and urinary calculogenesis ( . %), while the most common concurrent diseases were diabetes ( . %) and depression ( . %). the most frequently reported and most severe symptoms at time of evaluation were irritative voiding symptoms, perineal and suprapubic pain and discomfort. over three quarters of the patients were dissatisfied with their quality of life. bacteria were cultured in . , . and . % of eps, vb and semen specimens, respectively. comparison to na data suggests that the european prostatitis patient and the european urologists' approach to the diagnosis and treatment of prostatitis are not that dissimilar to prevalence and management of prostatitis in na. conclusion: prostatitis is a common worldwide outpatient diagnosis, comprising a significant percentage of male outpatient visits to urologists in both europe and na. the similarities in prevalence, characterisation and management of the typical prostatitis suggests that an international collaborative research effort is indicated in this important urological condition. observation unit for < h. before discharge ( . %) or admitted ( . %). the two factors that significantly correlate to hospital admission were the severity of uti ( % of complicated ac, % of aup, % of complicated ap and . % of acute prostatitis) and patients' age ( . ae . years in those admitted with complicated ac vs. . ae . years in the non-admitted, . ) . demographic factors, underlying conditions, symptoms and signs, laboratory, radiological and microbiological data, antimicrobial therapy, outcome and final diagnosis were evaluated. results are expressed by percentages or median as appropriate. results: median age was years, % were female and % were nursing home residents. seventy-four per cent were dependent for activities of daily living, % had a permanent urinary catheter and % had cognitive impairment. the most frequent symptoms were fever ( %), decline in function ( %) and dyspnoea ( %); only % referred dysuria. stupor ( %), crackles ( %) and ronchi ( %) were the commonest signs. leucocytosis ( /ul), elevated urea ( mg/dl), respiratory failure ( %) and high c-reactive protein ( mg/l) were the main laboratory abnormalities. pyuria was observed in %, chest x-ray showed a pulmonary infiltrate in %, and % of cases fulfilled criteria of severe sepsis. blood and urine cultures were positive in and % of patients, respectively; gramnegative bacilli (gnb) were found in % of positive cultures, escherichia coli being the most common agent. no pneumococci were isolated either in blood or sputum. amoxicillin-clavulanate was the antimicrobial therapy most frequently administered ( %). median hospital stay and mortality were days and % respectively. urinary tract infection was the commonest final diagnosis ( %). conclusion: respiratory manifestations predominate in disabled old patients with gnb severe urinary sepsis initially diagnosed as suari. respiratory distress may underlie this presentation. further studies are required to support this contention. enterococcus in patients hospitalised through the emergency department d. raveh, i. rosenzweig, b. rudensky, a.m. yinnon jerusalem, il objectives: to determine the incidence of, and risk factors for, isolation of pseudomonas aeruginosa or enterococcus from urine cultures obtained from patients in the emergency department (ed). methods: one year prospective, non-interventional study of all urine specimens collected in the ed, out of which one organism was isolated at a concentration of > cfu/ml. in this study were included all patients with p. aeruginosa or enterococcus bacteriuria (study patients), and control patients with escherichia coli bacteriuria subsequently hospitalised, at a ratio of two controls for each study case. patients were interviewed with a structured questionnaire and charts were reviewed for demographic, clinical and laboratory indicators of enterococcus or pseudomonas bacteriuria as compared with e. coli bacteriuria. results: over the -year study period, positive urine samples were obtained from ed patients: ( %) enterobacteriaceae (including isolates of e. coli) and ( %) other organisms, of which ( %) were p. aeruginosa and enterococcus ( %). comparison with a randomly chosen control cohort of patients with e. coli bacteriuria revealed several indicators for pseudomonas bacteriuria, including male gender (odds ratio . , %ci . - . , p < . ), presence of a permanent urinary catheter (or . , %ci . - , p < . ), past prostatectomy (or . , % ci . - , p < . ), hospitalisation in the previous months (or . , % ci . - . , p < . ), and pregnancy (or . , %ci . - . , p < . ). in addition, both enterococcus and pseudomonas, as compared with e. coli, significantly more often indicated asymptomatic bacteriuria in patients with other diagnoses, as opposed to clinically manifest bacteriuria, than isolation of e. coli (or . , %ci . - . , p < . ). conclusions: pseudomonas ( %) and enterococcus ( %) are isolated from a significant minority of urine samples obtained from ed patients with clinically suspected bacterial infection. isolation of these organisms, as compared with e. coli, more often indicates asymptomatic bacteriuria in patients with other infectious disease diagnoses. in addition, several independent clinical indicators for pseudomonas bacteriuria were identified. these data may assist in selecting optimal antibiotic treatment for patients admitted with suspected urinary tract infection. objectives: certain virulence factors (vf), particularly pap fimbriae, are able to trigger production of cytokines, especially through activation of toll-like receptor (tlr- ), and therefore produce inflammation. the aim of this study was to assess the influence of certain vf in the degree of inflammation in febrile urinary tract infections (futi). methods: from to adult patients with febrile community acquired futi ( female with acute pyelonephritis (mean age (sd ¼ )) and acute prostatitis (mean age (sd ¼ )) caused by escherichia coli were prospectively included. levels of c reactive proteins (crp), white blood cell count (wbcc) and days until apirexia after beginning antibiotic treatment were recorded in all patients and considered as indirect markers of inflammation. genes encoding haemolysin, type fimbriae, pap g fimbriae, cytotoxic necrotising factor, aerobactin and autotransporter toxin were detected by a pcr. additionally expression of type fimbriae and haemolysin were detected by agglutination and growth on blood agar. results: strains carrying pap g fimbriae were involved in futi with higher crp levels than pap g fimbriae negative strains ( . vs. . ; p ¼ . ). the relation between the rest of vf and crp levels did not reach statistical significance. no differences were found regarding the wbcc and the duration of the fever. conclusions: these data indirectly suggest that the degree of inflammation in futi caused by e. coli is associated with the presence of pap g fimbriae, which is coherent with the fact that pap g fimbriae are coreceptors of tlr . mycology: candida and aspergillosis p initiation of an active surveillance programme on yeast-related bloodstream infections in france (aspyrif) an active surveillance program has been implemented in france to prospectively analyse yeast-related blood stream infections. a pilot study was conducted from october through september in medical centres in paris and suburbs. for each patient, one isolate of each identified species was sent to the nrcm together with clinical data filled on a standard form. identification was confirmed using phenotyping tests and a pcr assay was performed on all candida albicans isolates to identify c. dubliniensis. antifungal susceptibility testing to amphotericin b, flucytosine, fluconazole, itraconazole, voriconazole and caspofungin was performed according to eu-cast recommendations. the median age of the patients was years [ - years], with a male predominance ( %). underlying factors for yeast-related blood stream infections were often multiple for a given patient dominated by recent surgery ( %), central venous catheter ( %), hospitalisation in intensive care unit ( %), malignancy ( %), immunosuppressive therapy ( %), hiv infection ( %), solid organ ( %) or bone marrow ( %) transplantation and prosthetic devices ( %). overall, the mortality rate was high with % of deaths within days after the first positive blood culture. candida spp. was the most frequent genus ( %) with c. albicans ( %), c. glabrata ( %), c. parapsilosis ( %) and c. tropicalis ( %) being the most frequent species isolated. other candida were recovered below % (c. krusei, c. kefyr, c. lusitaniae). non-candida spp. were trichosporon asahii, t. mucoides, geotrichum capitatum and cryptococcus neoformans. our data show that the percentage of nonalbicans species equal that of c. albicans among the yeasts recovered during fungaemia. the proportion of the four major species differed significantly according to the presence of central venous catheter (p ¼ . ). analysis of the antifungal susceptibility testing results revealed that most of the isolates had usual antifungal susceptibility profiles. in conclusion, aspyrif is a powerful tool that should allow us to accurately describe the epidemiology of yeast-related blood stream infec-tions in france without restriction to any underlying disease or species. background: nosocomial candidaemia is associated with significant morbidity and mortality in the critically ill. emergence of fluconazole resistance raises further problems, but the newer antifungal drugs [voriconazole, caspofungin, ambisome and abelcet] offer alternative therapeutic options. they also raise the issue of treatment-associated costs. an -year [ to november clinical audit was conducted across two tertiary care hospitals [western infirmary and gartnavel general hospital, glasgow] . the distribution of candida species and fluconazole/itraconazole resistance, with emphasis on high-risk areas was studied. it also addresses the newer antifungal options, cost implications and patient risk-stratification approach. objectives: to evaluate the outcome and complications in patients with candidaemia treated with antifungals. to identify the most common candida species isolated in the vamc patients with candida and evaluate the risk factors and epidemiological data of the patients. methods: all patients admitted in the vamc from august to august with blood cultures positive for candida were included in this study. epidemiological data, medical history, risk factors, co-morbid diseases and laboratory results were evaluated in record review. candida species were identified to determine the prevalence of candida species in the vamc. the patients were assigned to three different groups according to the therapeutic regime provided to the patient by the primary physician. outcome and complications including nephrotoxicity, electrolytes disturbances and hepatotoxicity were evaluated in each therapeutic group. statistical analysis was performed using the spss (statistical package or social science). a regression model was used for the analysis of risk factors associated with mortality in patients with candidaemia. results: one hundred and seven patients were randomised in the study. c. tropicalis was the most commonly isolated candida species %, followed by c. albicans %. mortality rate is high %, especially in those patients infected with c. tropicalis and c. glabrata % (p ¼ . ). the mortality rate increased to . % if no treatment was given (p < . ) and was worse if c. tropicalis was isolated and not treated %. the patients treated had a similar mortality rate irrespective of the administered agent, amphotericin ( %), abelcet ( %) and diflucan ( %), but was worse in those patients admitted to an icu, amphotericin ( . %), abelcet ( %) and diflucan . % (p < . ). response rate in the patients infected with c. albicans was . vs. % in patients with c. tropicalis. nephrotoxicity developed in % of patients and no difference was found in those patients treated with amphotericin b vs. abelcet. conclusion: candidaemia has been increasing in frequency. c. tropicalis is the most commonly isolated candida species in our institution. candidaemia has a high mortality rate and is worse if c. tropicalis is isolated and the patient is admitted to an icu and no treatment is given. there is no difference in response rate within the different therapeutic options. nephrotoxicity is higher in patients treated with amphotericin irrespective of the formulations administered. background: invasive candidaemia is a life-threatening complication occurring especially in hospitalised cancer patients due to surgical operation and application of aggravating chemotherapy. candida colonisation, dysfunction of humoral and cellular immune system and prolonged periods of hospitalisation are considered to be the risk factors of invasive candidaemia development. early diagnosis and evaluation of the risk factors are still a major challenge. objectives: the aim of our study was to evaluate the relationship between the rate of candida colonisation, disorders in immune responses (associated with adverse changes in concentration of tnf-alpha, il- , and myeloperoxidase) and development of invasive candidaemia in hospitalised cancer patients. methods: study group included patients with lung cancer admitted for surgical operation and women with carcinoma ovariorum after the third course of treatment with taxol and cisplatin. patients were examined for fungal colonisation of mucosal membranes with culture methods. presence of candida antigens and dna of the pathogen in the bloodstream was determined with elisa and pcr assay, respectively. cytokine and myeloperoxidase concentration in serum of the patients was specified with elisa commercial kits. results: the study revealed that ( %) lung cancer patients were colonised with candida in nosepharynx before the operation. pneumonia and wound infections were observed in patients of this group, candida albicans was isolated as the only pathogen from three patients colonised previously with candida. in case of patient group with ovariorum carcinoma, colonisation with candida of two or three sites was demonstrated in five ( %) of women. the candida antigen was present in blood in four of them; positive pcr result was found in blood sample collected from one of them. significant relationships between candida colonisation or infection and myeloperoxidase concentration were found ( . - . vs. ng/ml in healthy persons). conclusions: high rate of candida colonisation and drastic decrease in myeloperoxidase serum concentration in patients with lung and ovariorum cancer are predisposing risk factors for invasive candida infection. detection of candida antigens and dna of the pathogen may improve early diagnosis of the candidosis. p evaluation of bact/alert d system to diagnose bloodstream infections due to yeasts p effect of voriconazole on ergosterol content of s. costa-de-oliveira, c. pina-vaz, e. pinto, a. oliveira, c. tavares, a. gonc¸alves rodrigues porto, p voriconazole (vor) is a new azole antifungal agent with a similar structure to fluconazole (flu). as with other azoles, its primary mechanism of action is through disrupting the normal sterol biosynthetic pathway, leading to a reduction in ergosterol content ( ). nevertheless vor is more potent against most candida spp., and shows a wide spectrum of activity. thus candida krusei, which is intrinsically resistant to fluconazole (by unknown mechanism), shows low mic values to vor. this lack of cross-resistance and the fact of being fungicidal to some fungi suggest a distinct mechanism of action. objective: to study the effect of vor on the amount of ergosterol of c. krusei strains, in comparison with flu. methods: the mic to vor was determined according to the nccls protocol m -a on strains of c. krusei, all resistant to flu (mic ! lg/ml). ergosterol was isolated from c. krusei cells by saponification and the non-saponifiable lipids were extrac-ted with heptane. ergosterol was identified by its spectrophotometric absorbance profile ( - nm) ( ). a quantification of ergosterol was determined after incubation with and without both azoles at mic and sub-inhibitory concentrations. results: in all the strains, mic to vor ranged between . and . ug/ml. all c. krusei have a significant amount of ergosterol, with no significant differences among the strains. after incubation with mic concentrations of vor an - % reduction of the ergosterol content was observed. a similar effect was obtained with fluconazole but only with highest concentrations ( ug/ ml). conclusion: the vor induces a considerable impairment on the biosynthesis of ergosterol by c. krusei strains. it is much more potent inhibitor of ergosterol biosynthesis than flu. background: mycotic infections of hospitalised patients are emerging as a significant public health issue. numerous studies have shown that candidaemia is associated with a significant attributable mortality and prolonged hospital stay, but only a few reports analyse the incidence of candida spp. in wounds. objective: to analyse the species distribution and antifungal susceptibility of candida infection in wounds in our hospital during a -year period ( - ) . methods: the in vitro activities of amphotericin b (ab), fluconazole (fz), itraconazole (iz), ketoconazole (kz) and flucytosine were determined by the broth microdilution method following nccls criteria. mics were visually determined after and h incubation at c results: from to we processed wound samples in our laboratory. of these, ( . %) were positive, ( . %) showed bacterial growth without candida and ( . %) with candida. the rate of isolation of candida in wounds/year was as follows: ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %). globally, candida albicans was the most frequently isolated species per patient ( ; . %), followed by c. parapsilosis ( ; . %), c. glabrata ( ; . %) and c. tropicalis ( ; . %). the trends in species distribution were similar in both the adult and paediatric population. the evolution in the successive years of wounds with more than one species of candida was as follows: / , / , / , / , / and / . overall, the percentages of resistance of candida spp. isolated were: ab ( . %), fz ( . %), iz ( %), kz ( . %) and fc ( . %). conclusion: our study shows an increasing presence of candida spp. among the wound isolates in the microbiology laboratory. a high proportion is due to species other than c. albicans and it can be probably attributed to the increase in antibiotic burden in our hospital. infants were performed to estimate disease burden, short-term outcome and microbiological characteristics of causative organisms. methods: prospective enhanced surveillance of invasive fungal infections in vlbw (< g) infants began in february , with cases defined as meeting of one or more of the following diagnostic criteria: ( ) culture from a sterile site -csf, blood (peripheral sample), urine (supra-pubic aspirate or in-out catheter sample), bone/joint, peritoneal or pleural space; ( ) pathognomonic findings on ophthalmological examination; ( ) pathognomonic findings on renal ultrasound examination; and ( ) autopsy diagnosis of invasive fungal infection. cases were identified through three separate surveillance schemes: monthly notifications from paediatricians to the british paediatric surveillance unit; continuous reports from microbiology laboratories to the communicable disease surveillance centre (england) and scottish centre for infection and environmental health (scotland). reports from the three systems were reconciled and analysed. rates were calculated using office for national statistics total live birth estimates. results: between february and july, confirmed cases of invasive fungal infection in vlbw infants were reported, . / births of vlbw. median age at diagnosis was days (range - ) and birth weight ( - ) g. thirty-four of the infants were of extremely low birth weight (< g). candida albicans was the most common pathogen, found in % of cases, and c. parapsilosis in %. organisms were most commonly isolated from blood ( %), followed by urine ( %), csf ( %) and central line tips ( %). just over a third of cases ( %) had received prophylactic antifungal therapy. one case of drug resistance was identified during this period (fluconazole resistance in a non-albicans candida spp.). of the infants for whom outcome data were available, were alive at weeks post-conceptional age. conclusion: preliminary findings from enhanced surveillance suggest an incidence of invasive mycoses in vlbw infants of one in . as per adult cases, c. albicans was the most common fungal pathogen involved, although c. parapsilosis was relatively more common than in adults. the majority of cases occurred in extremely low birth weight infants, and mortality was found to be high. methods: surveillance swabs of throat and rectum were taken on admission and twice weekly afterwards. diagnostic samples were obtained on clinical indication. all samples were processed using standard mycological techniques. overgrowth was defined as ! + or ! yeast cells/ml of saliva and/or gram of faeces. carriage index is the ratio of the sum of all semi-quantitative growth densities of positive surveillance swabs divided by the total number of swabs; on a particular sampling day. oral polyenes were started following the identification of the carrier state. results: a total of children requiring minimally days of ventilation were enrolled in this -year observational, prospective study [ / / to / / ]. the median paediatric index of mortality was . [iqr . - . ], and the actual mortality was . %. enteral polyenes as part of selective digestive decontamination [sdd] were administered to half of the study population [ %] . the median length of stay was days objective: candida dubliniensis is a newly described pathogenic species, first isolated from hiv-infected patients with oropharyngeal candidiasis. it shares many phenotypic features with c. albicans, including the ability to form germ tubes and chlamydospores. these similarities have caused significant problems in its differentiation from c. albicans in routine clinical microbiology laboratories. this study reports isolation and identification of c. dubliniensis for the first time from kuwait and presents data on antifungal susceptibility profile. methods: over a period of months, germ-tube positive yeasts identified as c. albicans and recovered from different clinical specimens were screened for their ability to grow at c on sabouraud dextrose agar. isolates which failed to grow at c were presumptively identified as c. dubliniensis. the identity of c. dubliniensis isolates was further confirmed by formation of rough colonies and chlamydospores on sunflower seed agar, by vitek system, and by semi-nested pcr using species-specific primers corresponding to unique sequences within the internally transcribed spacer (its ) of c. dubliniensis and by direct sequencing of its . the antifungal susceptibility testing was performed on rpmi medium as recommended in nccls, m a document. results: of the germ tube positive yeast isolates, ( . %) were identified as c. dubliniensis. they were isolated from sputum (n ¼ ), vaginal swabs (n ¼ ), endotracheal secretion (n ¼ ), throat swabs (n ¼ ), urine (n ¼ ) and one each from bronchoalveolar lavage, catheter tip and peritoneal fluid. none of the isolates originated from hiv-positive patients. all the c. dubliniensis isolates were susceptible to amphotericin b, fluconazole, itraconazole and voriconazole. however, % of the isolates were resistant to -flucytosine (> lg/ml) without any known previous exposure. conclusion: identification of c. dubliniensis from . % of the yeast isolates in our study suggests that this species is not uncommon in kuwait. there is a need to carry out a systematic study in high-risk patient groups to know its epidemiologic significance. acknowledgement: the work is supported by kuwait university research grant mpi- . background: fungaemia remains a severe nosocomial complication and the emergence of non-albicans species is posing new challenges both to clinicians and to microbiologists. objective: to assess the incidence and clinical presentation of c. glabrata fungaemia, its susceptibility and its clinical outcome. methods: from to , we had episodes of fungaemias and cases corresponded to c. glabrata ( . %). thirty cases were six cirrhosis patients and miscellaneous). following the eortc/msg criteria, these patients were classified as proven ia (n ¼ ), probable ia (n ¼ ), possible ia (n ¼ ) and 'colonisation' (n ¼ ). mean saps ii score was with a predicted mortality of . %. overall mortality was % (n ¼ ). mortality of the proven and probable group was . and . %, respectively. among the patients who survived, just had 'colonisation' with aspergillus. post-mortem examination in the non-haematooncological group was done in out of the patients who died ( %) and / autopsies ( %) showed hyphael invasion with aspergillus (mainly the lung as target organ). there were five proven cases in patients without compromising host factors according to the eortc/msg definitions (three liver cirrhosis, one pneumonia in a -year-old man, one klebsiella sepsis with mof). conclusion: ia is an emerging infectious disease in non-haematooncological icu patients. there seems to be a broad group of patients at risk of ia. ia was diagnosed in patients without characteristics described in the eortc/msg definitions. it seems worthwhile to investigate the validity of the available diagnostic tools in non-haemato-oncological patients at risk for ia in a prospective manner. p epidemiology of invasive aspergillosis in a teaching hospital, france: a -year survey ( - ) a. cornillet, c. camus, s. nimubona, v. gandemer, p. tattevin, c. belleguic, s. chevrier, c. meunier, c. lebert, m. aupée, b. lelong, c. guiguen, j.-p. gangneux for the aspergillosis study group objectives and methods: the aim of this survey was to characterise a file of patients who developed an invasive aspergillosis (ia) in our institution, their risk factors and management. we analysed retrospectively the cases of ia, which occurred between and , then prospectively all new cases until the end of . the overall survey covered a -year period. cases were classified as suspected, probable or proven ia, using criteria derived from the eortc/msg classification. results and discussion: until / , out of the cases of ia analysed, nine were histologically proven, were probable ia and were suspected ia. the sex ratio was . male:one female with a mean age of years (ranging from to years). fifty percent of cases were diagnosed in the intensive care units, and % in haematology units ( % in adults and % in paediatrics). neutropenia was the major risk factor in % of the patients (during haematological malignancies and solid cancers). however, we also noted an increasing number of ia in patients under corticosteroid therapy for cobp, asthma, rheumatoid arthritis, horton and microvascular diseases, in comparison to available data in the literature. other cases occurred in solid organ transplant recipients and only one out of the patients was infected by hiv. prognosis factors will be discussed. regarding biological diagnosis, good sensitivities of the mycologic examination (microscopy + culture) and the galactomannan antigen detection by enzyme immunoassay (platelia aspergillus, biorad) were noted: and %, respectively. the sensitivity reached % when both tests were combined. pulmonary imagery was less efficient, probably due to the fact that, in our institution, ct scans are performed later than proposed in the literature. during this survey, we observed great modifications in therapeutic approaches. first line treatment progressively switched from deoxycholate amphotericin b (amb) to voriconazole and second line treatments now include lipid formulations of amb and caspofungin acetate. amb deoxycholate and voriconazole were the two drugs used for empirical therapy. the overall mortality was > %. conclusion: ia remains a major life-threatening infection among immunosuppressed patients, although protective measures such as air filtration significantly reduced its incidence in neutropenic patients. however, this -year-survey points out the increasing number of cases in non-neutropenic patients hospitalised in wards without air filtration. this emerging population of patients must be taken into account and imposes to reinforce surveillance for high-risk groups and to rethink our preventive measures. priate chest ct appearance) was the sole basis of the diagnosis in ( %) pts. in the remaining seven ( %) pts, positive elisa accompanied either histopathological or microbiological evidence of ia. five ( %) of these pts were later upgraded to definite ia. nineteen of the pts were assessed for efficacy at the end of cas rx. the favourable response rate was % ( / ). pts whose only evidence of ia at cas onset was elisa (and characteristic chest ct findings) had a % ( / ) success rate. follow-up elisa data was available in pts. four of five pts with a favourable response to cas had negative elisa by the end of rx. the one other pt with a favourable response had quantitative elisa improvement that was temporally associated with clinical and radiographic response. of the pts with unfavourable responses and follow-up elisa data, had no elisa improvement and two had normalisation of elisa while on cas. conclusions: in this study, the use of elisa did not result in an exaggerated favourable response rate. in general, the elisa was associated with clinical/radiographic response. paradoxical elisa increases in pts clinically/radiographically responding to cas were not noted. best rapd patterns with respect to number, spreading and intensity of the bands, but the highest level of discrimination was achieved by a combination of data generated by both of them. therefore, we emphasise the convenience of using at least two primers for rapd typing. objectives: to gain insight into the molecular epidemiology of staphylococcus aureus at a tertiary hospital. methods: all s. aureus isolates recovered from blood samples over a -year period were analysed. demographic, clinical and microbiological data from these patients were collected. antimicrobial susceptibility tests were performed by the wider system and the disk diffusion method; all methicillin-resistant s. aureus (mrsa) isolates underwent confirmatory pcr analysis for the meca gene. molecular characterisation was performed by pulsed-field gel electrophoresis (pfge) following dna extraction and smai digestion. patterns differing by less than seven dna fragments and with a dice coefficient of correlation > % were considered a common bacterial type while subtypes included isolates with indistinguishable pfge patterns. univariate and multivariate analyses were performed with epi-info and spss . softwares. results: one hundred and sixty-two episodes of s. aureus bacteraemia, whether methicillin-resistant or methicillin-susceptible (mssa), were nosocomial in origin ( . %) or were cases associated with the healthcare system ( . %). only a total of cases of bacteraemia ( . %), one mrsa and mssa, were strictly considered to be community-acquired. thirty-five unique s. aureus pfge types were identified among dna macrorestriction patterns. within the isolates of mrsa, four major genotypes were identified, with isolates ( . %) represented by a single pfge type. in contrast, the isolates of mssa comprised different pfge types, of which represented more than one isolate. three pfge types were found to represent % of all mssa isolates. these common strains were found with equal frequency among adults and paediatric patients, and were evenly distributed between nosocomial and community-acquired cases. conclusion: our results provide indirect evidence of ongoing transmission of mrsa and mssa in our hospital. in the case of mrsa, the spread is predominantly due to a single clone, with transmission favoured by increased length of stay in hospital and the administration of beta-lactam antibiotics. in contrast, the spread of mssa bacteraemia in this population is associated with multiple, genetically distinct strains. ( ) to use a real-time pcr to detect the presence of meca gene in s. aureus clinical isolates. methods: seventy-three strains obtained from clinical specimens were identified by microscan (dade behring) and coagulase test ( s. aureus, s. epidermidis and other coagulase negative staphylococci). in vitro susceptibility was determined by microscan and disc diffusion. a total of s. aureus strains were classified according to methicillin susceptibility: resistant and susceptible to methicillin. dna was obtained by incubation at c in lysis buffer. real-time pcr was performed in a lightcycler instrument (roche diagnostics, spain) using two commercially available kits: ( ) lightcycler staphylococcus kit mgrade: pcr was positive in all staphylococci and they were differentiated according to melting temperature ( . ae c for s. aureus, and . - c for cns) and ( ) lightcycler mrsa detection kit: pcr was positive in meca positive s. aureus. an internal control excludes the presence of inhibition. once the dna was extracted the whole process takes h. results: twenty-seven out of s. aureus strains were clearly identified by real-time pcr due to the melting temperature (range from . to . c). one s. aureus showed melting temperature of . c. all s. epidermidis strains showed melting temperature from . to . c. s. lugdunensis showed melting temperature of . , . and . c. other cns showed melting temperature from to . c. twenty-five out of ( . %) strains tested were meca positive by using this lightcycler mrsa kit and realtime pcr. among the meca positive, were fenotipically methicillin resistant ( %) whilst four were methicillin susceptible ( %). all meca negative strains were susceptible to methicillin by phenotypic methods. conclusions: real-time pcr (lightcycler) seems to be an accurate method to identify s. aureus and differentiate it from different cns and to detect resistance to methicillin in s. aureus. both reactions could be done simultaneously and the whole process takes less than h (dna extraction plus real-time pcr). objectives: surveillance of methicillin resistant staphylococcus aureus (mrsa) in canada began in . from this surveillance, six epidemic strains of mrsa have been identified and named cmrsa - . in order to better understand the relatedness of these strains, as well as their genetic content, we have used microarrays to compare their genomes to that of the fully characterised genome of the mrsa strain col. methods: genomic dna from representatives of the six epidemic strains, as well as col was fragmented and labelled using random primers with cy or cy labelled dctp. col and each of the epidemic strains (labelled with different dyes) were hybridised to arrays containing pcr products or bp oligomers representing of the open reading frames (orfs) in the col genome. data were processed with the arraypro software package, positive/ negative cut-off values were determined using genomotyping analysis by charles kim and then analysed using the genemaths program. macrorestriction digest patters were generated using smai. results: results indicate that all canadian epidemic strains have six common regions of deletion, a portion of the type i sccmec region, bacteriophage l a, and four smaller areas composed of two to four orfs. the only gene of known function in these smaller areas was the staphylococcal enterotoxin b. apart from these major deletions, many sporadic, single deletions are seen throughout the strains. larger regions of deletion that are not present in all strains also occur. the only obvious orf duplication is of is in cmrsa , and , which is found in multiple copies in the typeiii sccmec region in these strains. macrorestriction digest data were used to approximate the sizes of the cmrsa genomes. cmrsa shows the smallest genome ($ kb), and the least genetic content in common with col ( %). though cmrsa and appear to have larger genomes ($ and $ kb, respectively), they show fewer orfs in common with col than other strains ( and %, respectively), suggesting a substantial portion of the genome may be novel. conclusions: this is the first study of epidemic mrsa using the comparative genomic hybridisation approach. while the cmrsa strains show a high degree of relatedness to col, there are considerable differences in genetic content. this study also indicates that there may be genetic content which is unaccounted for in the col genome. other studies are being devised to identify and characterise the novel genetic content. objectives: in finland, the annual number of mrsa isolates notified to the national infectious disease register (nidr) has constantly increased, especially outside helsinki metropolitan area. molecular typing has revealed numerous outbreak strains of mrsa, and some of them have been associated with community acquisition. we analysed strain types identified by pulsed-field gel electrophoresis (pfge) of mrsa isolates sent to the national reference laboratory (nrl) during - . methods: all isolates of mrsa notified by the finnish clinical microbiology laboratories were sent to nrl for further verification and characterisation, including pfge analysis. pfge profiles differing by fewer than six bands were interpreted as identical or closely related. one isolate per person were included in the analysis. strain types were categorised as sporadic (strain type only found from one person), domestic outbreak or international epidemic (strain type found from more than one person) as well as community-acquired (strain type associated with community acquisition in our previous study). the proportions of mrsa isolates included in each category were assessed. results: a total of mrsa isolates were studied. the number of mrsa isolates increased from in to in . pfge identified more than different strain types. of the mrsa isolates, % were sporadic, % domestic outbreak and % international epidemic. one strain type disappeared compared with years before , and new strain types appeared during - . the proportion of sporadic strains varied between and % during the study period. of the international epidemic strains, bel ec- increased from < % in to % in , mainly outside helsinki metropolitan area. uk emrsa- decreased from % in to < % in , and helsinki i (a representative of mlst st- strains) from to %, respectively. uk emrsa- varied between and %. the three main strains with community-acquisition fluctuated during the study period (range, - %). conclusions: intensive national surveillance with molecular typing revealed that the predominant mrsa strains change over time. the internationally spread epidemic strains of mrsa have also been found in finland. however, most of them show a decreasing trend or have disappeared. these results encourage us to continue aggressive interventions with each new mrsa case. introduction: according to recent studies, community-acquired (ca)-methicillin-resistant staphylococcus aureus (mrsa) strains often contain a type iv sccmec cassette and panton-valentine leukocidin (pvl) locus. it has also been shown that certain multilocus sequence types (st) seem to be connected to ca-mrsa strains from different continents. materials and methods: we studied finnish ca-mrsa strains for their genotype by pulsed-field gel electrophoresis (pfge) and multilocus sequence typing (mlst), the methicillin resistance genes by sccmec pcr and the presence of pvl gene locus by pcr. mrsa was defined as community acquired if the mrsa specimen was obtained outside hospital settings or within days of hospital admission from a person who had not been hospitalised within years before the date of mrsa isolation. to confirm the functionality of the pvl-pcr reaction and the quality of the dna, nuc gene was amplified at the same time. results: the majority of ca-mrsa strains studied ( / , %) possessed sccmec cassette type iv but only ( %) were pvl positive. all but two pvl positive strains contained sccmec type iv. one strain had sccmec cassette iii subtype, and for one strain the type was not determined. the pvl positive strains were mostly ( / , %) of multilocus st . the four remaining pvl-positive strains were of st (two strains) and st and . the sequence types correlated well with the pfge results: all strains with st were analysed as pfge profile hkiviii and strains with st as pfge profile nurmes. st (sccmec cassette -iii subtype) and st (sccmec not determined) strains were considered as sporadic. the pvl negative ca-mrsa strains belonged to different shared and four sporadic pfge profile types. the mlst analysis of pvl negative strains is currently underway. conclusions: most of the finnish ca-mrsa strains have sccmec cassette type iv but only a minority contain pvl gene locus, which is in contrast to previous reports. majority of the pvl gene positive strains possessed st . in spite of the strict definition for community-acquisition we used, majority ( %) of finnish ca-mrsa were pvl negative and showed heterogeneous pfge profiles. objectives: methicillin-resistant staphylococcus aureus (mrsa) is among the major pathogens. the most common methods currently used for identifying methicillin (oxacillin) resistance in many clinical laboratories are susceptibility tests. the performance of these tests has been erratic because the expression of resistance is variable and commonly heterogeneous within strains. methods: a retrospective laboratory-based study was carried out with clinical isolates of s. aureus in a tertiary care providing uni-versity hospital in thrace, greece. methicillin (oxacillin) susceptibility of s. aureus isolates, which were recovered from various clinical specimens (blood cultures, tracheal aspirates, wound swabs and central venous catheters) were studied by four different methods: ( ) agar screening test [mh-oxacillin ( lg/ml) agar supplemented with % nacl], ( ) susceptibility determination by the vitek (biomerieux), ( ) mic was determined by e-test (ab biodisk), ( ) mec-a gene detection by pcr, using specific primers. the strains were evaluated by using the presence of meca gene detected by pcr, as definitive criteria for mrsa and non-mrsa. the susceptibility tests were carried out as recommended by the nccls. results: among all the isolates, were identified as meca-positive and the remaining as meca-negative. the percentages of correct results (% sensitivity/% specificity) were: oxacillin agar screen, / ; e-test, / ; and vitek- , / . ten isolates, negative for the mec-a gene by pcr, were recognised by at least one phenotyping method as oxacillin resistant. only one strain meca-positive was incorrectly identified as oxacillin-negative by the oxacillin agar screen. conclusions: as shown in this and other studies, no phenotypic method is completely reliable for the detection of oxacillin resistance in s. aureus. the specificity was generally high, especially with the agar screening and e-test methods, while the sensitivity varied between the different methods. in particular, the oxacillin screen test is the most accurate test and approaches the accuracy of pcr. although, the presence of the meca gene, as detected by pcr, still remains the 'gold standard', agar-screening test should be considered in association with other susceptibility methods to maximise the ability to correctly detect oxacillin-susceptibility in s. aureus. p amplification of dna fragments surrounding rare restriction sites (adsrrs-fingerprinting) for typing staphylococcus aureus isolated from patients with recurrent furunculosis w. baranska-rybak, r. nowicki, e. scieburako, j. kur, e. arlukowicz, a. samet gdansk, pl introduction: in the present study we report data on phenotypic and genotypic characteristics of staphylococcus aureus strains. the aim of our research was to identify sa genotypes in the patients suffering from recurrent furunculosis. materials: we obtained isolates from patients with recurrent furunculosis. purulent discharge from furuncle, nasal and throat swabs were taken for culture. methods: the identity strain of sa was confirmed by novel dna-typing technique. amplification of dna fragments surrounding rare restriction sites (adsrrs-fingerprinting ) is an effective and rapid method for molecular typing of isolates of bacteria. this method is based on suppression of pcr (polymerase chain reaction) reaction. sa dna was digested with two restriction enzymes: bamhi ( u/ml) (sigma) and xbai ( u/ml) (sigma). cohesive ends of dna were ligated with adapters (xbai short adapter and bamhi long adapter) and amplified. pcr products were electrophoresed on polyacrylamide gels, stained by ethidium bromide and photographed under uv. results: adsrrs-fingerprinting of sa isolates revealed unique patterns. in most cases the strains isolated from the same patient (nose, throat and furuncle) gave identical pattern. the reverse situation was found in five patients. conclusions: ( ) in most cases we confirmed the identity between nasal/throat and furuncle sa isolates. ( ) we found no specific genotype, which is responsible for recurrent furunculosis. ( ) adsrrs-fingerprinting seems to be a very useful method for epidemiological studies of sa. objectives: rapid and efficient epidemiologic typing systems may be useful to investigate dissemination of the lineages of staphylococcus aureus. we have compared the usefulness of well-established methods to those of newly developed rapid typing methods as epidemiological tools. methods: a total of s. aureus isolates were analysed by pulsedfield gel electrophoresis (pfge), multilocus sequence typing (mlst), repetitive-element pcr technique (rep-pcr) based on the presence of dna sequence that are homologous to mp repeat in mycoplasma pneumoniae, multiple-locus variable-number tandem repeat analysis (mlva), and multiplex pcr-based method with primer mix of the spa gene, the coa gene, and the hypervariable region adjacent to meca gene. results: fifty-nine s. aureus isolates clustered by pfge in different genotypes. mlva, which had the highest compatibility with pfge of all testing methods in this study, clustered into different genotypes, multiplex pcr-based method clustered into , and rep-pcr clustered into different genotypes. rep-pcr differentiated s. aureus isolates in a way similar to mlst that clustered these isolates in groups. conclusion: although pfge is still the gold standard, owing to its high discriminatory power amongst molecular typing methods, genotyping methods based on pcr may be useful in respect of speed and ease of performance. mlva, multiplex pcr-based methodology and rep-pcr are rapid, reproducible, and easy to perform. however, mlva and multiplex pcr-based method generate more unambiguous results than those of rep-pcr. objectives: to determine whether the variable visual outcome in endophthalmitis secondary to coagulase-negative staphylococci spp. are due to different strains causing intraocular infection, with a possible difference in virulence of each strain or resistance to the antibiotics given. methods: twenty-eight intraocular samples infected with coagulase-negative staphylococci spp. were analysed using both biotyping and pulsed-field gel electrophoresis for strain identification. the results were correlated with the visual outcome after months post-treatment. results: four different strains of coagulase-negative staphylococci spp. were found to cause endophthalmitis; s. epidermidis, s. haemolyticus, s. equorum and s. warneri. twenty-one out of the isolates were identified as s. epidermidis and the others were grouped as non-s. epidermidis for correlation with the clinical data. comparing the s. epidermidis with the non-s. epidermidis infected cases, it was found that the mean visual gain was significantly better for the non-s. epidermidis infected cases [(mean visual gain of . vs. . logmar letters, respectively) (p ¼ . )].the visual outcome was significantly worse for patients infected with s. epidermidis and antibiotic resistance was more common among these isolates although all were sensitive to at least one of the three/four antibiotics given. comparing the non-s. epidermidis infected cases to the s. epidermidis infected cases that were sensitive to all four antibiotics used, the visual outcome was still significantly better in the non-s. epidermidis group [mean visual gain . vs. . logmar letters, respectively) (p ¼ . )]. use of arbitrarily primed pcr to study salmonella ecology in turkey production environment detection of salmonella serovars from clinical samples by enrichment broth cultivation -pcr procedure p aetiology and resistance of community urinary tract infections in são paulo, brazil: a three-year survey with positive cultures %) positive cultures were analysed in this survey. chi-square test for trend (altman, ) was performed to evaluate the resistance prevalence ordering in the years surveyed (p < . was considered significant). results: among the positive cultures, . % were from female and . % from male patients. among the positive cultures . % presented growth of enterobacteriaceae followed by . % of gram-positive cocci conclusions: an important difference in the resistance pattern was observed among pathogens and age groups. the difference in age groups suggests the possibility of selective pressure due to previous antimicrobial use in the community setting. ciprofloxacin could be used for empiric therapy in community uti. however, its apparent ascending resistance should raise awareness as to possible usage restriction in this setting. surveillance studies are useful for guiding therapy and helping curbing resistance. p resistance of escherichia coli isolates from pregnant and non-pregnant women with community-acquired urinary tract methods: one hundred and forty-four non-pregnant and pregnant women with signs of upper or lower communityacquired uncomplicated utis were enrolled in two multicentre prospective epidemiological studies (eight medical centres), utiap- and arimb, respectively. the strains isolated from the patients who had significant bacteriuria (> cfu/ml) were included in the microbiological analysis. the mics of antibiotics (ampicillin -amp, amoxicillin-clavulanate -amx-clv, cefuroxime -cfr, cefotaxime -cft, gentamicin -gnt, co-trimoxazole -ctz, nitrofurantoin -ntf, fosfomycin -fsf) were determined by the agar dilution, as described in the nccls ( ) guidelines. quality control was performed using reference strains including e. coli atcc , e. coli atcc . results: resistance rates of e. coli from pregnant and non-pregnant women with ca-uti in russia are shown in figure. there are some statistically significant differences in antimicrobial resistance between studied groups. ampicillin resistance was higher among uti isolates of e. coli in non-pregnant women ( . %) than in pregnant women ( . %), p < . (chi-square statistic) methods: consecutive patients with presumed uti were included during days if they were older than years and had a positive urine dipstick. subsequently, urine culture (uc) was prescribed and patients classified according to nine uti categories. centres were also required to notify all visits motivated by infectious diseases (id) during the study period. results: of potential participants, included uti period, prevalence of id is estimated at . % of nontrauma visits and prevalence of uti at . % of all id. the main uti categories were acute cystitis (ac ¼ . %), acute pyelonephritis (ap ¼ . %), bacterial prostatitis (bp ¼ . %). mean age of patients was . ae . years and sex ratio f in %. however, both differ significantly according to uti category all bc received in the microbiology service were included in our study. all the specimens were performed with bact/alert d (biomerieux) initially during days or days in special cases related to the detection time in aerobic bottles, . % gave a positive result in the first h of incubation (average . h) cumulative percentage of % at h. in the second day, . % were positive. in anaerobic bottles . % gave a positive result in the first h of incubation (average . h) cumulative percentage of . % at h. in the second day . % were positive. candida albicans was isolated in . % cases methods: from / / to / / we studied all yeasts considered pathogens from all body sites, from paediatric pts in all in-hospital locations. isolation and yeasts species identification were carried out by conventional methods. on isolates, flu and vor susceptibilities were assessed by the nccls m -p method, with disks tested in mueller-hinton medium with glucose and methylen blue, . macfarland inoculum. all susceptibility test results were read by biomic plate reader system (giles scientific). c. albicans (ca) atcc was included. nccls flu breakpoints (mcg/ml) were s < , s-dd - , r > with corresponding zone interpretative criteria (mm) s > , s-dd - , r < . breakpoints for vor have not yet been established. results: in the study period we recovered ca, c. parapsilosis (cp), c. tropicalis (ct), two c. krusei, two c. glabrata (cg), two c. lusitaniae (cl) and one tricosporon beigelii. species were isolated: % from urinary tract, %, upper respiratory tract, % miscellaneous fluids, % lower respiratory tract, % blood, % cvc, % various. patients with yeasts infections were hospitalised: % in picu/nicu, % haematology-oncology, % surgery, % infectious diseases, % nephrology, % pneumology, % medicine, % orthopedics, % dermathology. distribution of bloodstream isolates were: four cp, three ct, one ca and one cl. seventy percent of cp strains were recovered from picu/nicu pts. the average zone diameter (mm) -mic /mic (mcg/ml) (agar disk gradients) were: ca flu flu with vor mics > mcg/ml, one ct was flu sdd, one gg was r to flu and inhibited with . mcg/ml of vor. conclusions: our results show that ca is still the predominant species recovered from paediatric pts; cp and ct appear to be recovered with increased frequency in serious infections of critically ill pts p trends in species distribution and antifungal susceptibility in candida wound infections: an overview of a -year period when compared with c. albicans, patients with c. glabrata fungaemia were older ( vs. ), had received more previous antifungals ( vs. %, p ¼ . ) and antimicrobial agents ( vs. %, p ¼ . ), had more indwelling bladder catheters ( vs. %, p < . ) and had more septic metastasis ( vs. %, p ¼ . ). iv catheters were more commonly withdrawn in patients with c. glabrata fungaemia ( vs. %, p ¼ . ), whereas these patients received fewer antifungals ( vs. %, ns). mic of c. glabrata were fluconazole (flu) mg/l, itraconazole mg/l, amphotericin b (amb) mg/l and voriconazole . mg/l. surprisingly, flu was more frequently selected to treat patients with c. glabrata ( vs. %). mortality was similar ( vs. %). six of the patients treated with flu died, as well as four of the seven treated with amb. two patients had persistent fungaemia despite catheter withdrawal and flu therapy cs p invasive aspergillosis in patients with copd of patients with copd and aspergillus spp. in respiratory samples to determine risk factors and outcome. results: we identified patients with copd and aspergillus spp. in respiratory samples. median age was ae . years. eighty-three percent were men. forty-one patients had criteria for 'probable' ifi none of cases had criteria to suspect an ifi, however, nine were treated and all but one died. the remainder were colonisations. conclusions: a progressive increase of copd patients with aspergillus spp. has been observed but frequently, this is a colonisation. however, we observed that patients in 'probable' category have a high rate ( %) of 'proven' ifi, similar to other known risk groups. we think that these categories could help in clinical practice and to identify homogeneous groups for clinical research in diagnostic methods and therapeutic interventions p evaluation of serum galactomannan elisa during caspofungin therapy: results from the caspofungin salvage invasive aspergillosis study a sandwich elisa assay, which detects circulating aspergillus galactomannan antigen using a rat monoclonal antibody has recently been licensed (plateliaâ, biorad). yet, animal models of ia have shown that treatment (rx) with an echinocandin may result in a paradoxical increase in antigenemia despite clinical/radiographic improvement. concern also remains that using elisa as the sole means of ia diagnosis may result in exaggerated favourable outcomes. to address these concerns, we reviewed the elisa experience from the caspofungin (cas) salvage invasive aspergillosis (ia) study. methods: patients (pts) with proven/probable ia were eligible for enrolment. probable ia was limited to pulmonary sites. probable pulmonary ia could be diagnosed serologically provided the pt had an appropriate chest ct appearance (halo sign, air-crescent sign) and positive elisa on more than two consecutive tests. all pts were refractory (> days) or intolerant of prior antifungal rx. cas, with doses ranging from - mg/day, was administered as monorx. efficacy was assessed at the end of cas rx. favourable responses were limited to complete or partial responses. results: of the pts enrolled, ( %) had consecutively positive serum elisa at the onset of cas underlying diseases were: lymphoid: %; myeloid: %; non-malignant: %. clinical efficiency of the test was tested at three different cut-off values . , . and . . results: results are summarised in the table. the overall incidence of invasive aspergillosis (ia) was . % ( / admissions). following eortc definition criteria, the repartition was: two definite ia, probable ia and possible ia. the definition of 'probable' ia was substantiated by positive gm antigen tests (eight cases); both by microbiological (positive cultures) and positive gm antigen tests (four cases) or only by microbiological criteria (four cases). gm antigen was detected at all different cut-off values in cases corresponding to: / definite ia, / probable ia. results were considered as false-positives in patients: four cases without clinical context; cases with a negative chest ct-scan conclusion: detection of circulating gm antigen may be helpful for the diagnosis of ia, particularly in the absence of microbiological data, but a substantiated number of false-positive results do occur among patients undergoing antibiotic therapy with pipera/ tazobactam or amoxi/clavulanate. considering different cut-off values did not improve the sensitivity or the specificity of the assay a results were reported as the number of isolates of each species per plate of the pair. results: a total of pairs of samples were evaluated. of these, showed growth of mucor spp. ( in sd and two in czapeck) and could not be studied for aspergillus. of the remaining pairs, pairs ( . %) were positive for aspergillus spp a. fumigatus was the most frequently isolated species, pairs ( . %) were positive [ ( . %) on both plates, ( . %) only in sd and ( . %) only in czapeck conclusions: our data supports the recommendation that both media (czapeck and sd) should be used for correct air sampling antifungal combination of caspofungin with flucytosine has been shown to be additive to synergistic in vitro against aspergillus fumigatus. the aim of the present study was to evaluate the interaction between these two drugs in vivo in an animal model of disseminated aspergillosis. methods: for in vivo experiments survival rates of mice treated with the combination of caspofungin at . mg/kg/day with flucytosine at and mg/ kg/day were and %, respectively. mice treated with caspofungin at . mg/kg/day combined with flucytosine at and mg/kg/day had a and % survival the study was performed on strains of enterococci all from patients with severe underlying diseases. strains were isolated from urine ( . %), blood cultures ( . %), pus ( . %), peritoneal fluid ( . %), intravenous catheter ( . %), infection of the drainage site ( . %). identification to the species level was performed by vitek (bio-merieux, france). antibiotic susceptibility testing was done by kirby-bauer and mic by e-test and vitek . the sandwich hybridisation method was performed in all strains using the commercially available evigenetm vre detection kit (statens serum institute), for the presence of vana and vanb genes.results: from the stains tested, were vancomycin and teicoplanin resistant (vana phenotype) and susceptible to these antibiotics, as determined by kirby-bauer and mics by vitek and e-test methods. of them, were e. faecalis, e. faecium, three e. casseliflavus and two e. hirae. all the vres strains, which were suggesting the presence of vana phenotype by kirby-bauer and mic, were identified to be vana positive by the sandwich hybridisation method. the susceptible strains were negative for the detection of the genes vana and vanb. conclusions: identification of vre to the species level and knowledge of the type and the profile of resistance is critical for infection control purposes in the hospital environment. the sandwich hybridisation is a rapid ( . h) and easy to use commercially available molecular method to detect the vana and vanb genes, while the phenotypic resistance determination requires incubation for at least h and other molecular methods require specific instruments and experienced technicians. the sensitivity and specificity of the method is %.p evaluation of the evigene tm vre detection kit for detecting of enterococci including vancomycin resistance genes a. kilic, m. baysallar, g. bahar, a. kucukkaraaslan, l. doganci ankara, tr objectives: evaluating the correlation of the evigenetm vre detection kit using pcr, which is the golden standard for gene detection and correlating the minimum inhibitory concentration (mic) for vancomycin and teicoplanin are the aim of this study. methods: the vancomycin-resistant enterococci (vre) detection kit is based on microwell plates where to dna probes specific for the bacterial targets dna are bound. test wells include: a positive ( s rrna) and a negative control, a vana microwell and a vanb microwell. the pcr detects the vana, vanb, and vanc- genes. the mic determination was performed by e-test according to the nccls guidelines. results: we tested a total of diverse vancomycin resistant enterococci: enterococcus casseliflavus (n ¼ ) and enterococcus faecium (n ¼ ). all strains were vana positive (od: all strains > . ). all results obtained with the vre kit were confirmed by the pcr. the mic determination correlated with the pcr and kit results for all vana positive strains with high mic for vancomycin. conclusion: as a result, the evigene vre detection kit can clearly distinguish vre with the vana and vanb genotypes among a large collection of enterococci and with the same specificity as pcr.p development of antibiotic resistance in enterobacteria s.d. nyberg, a. hakanen, m. Ö sterblad, p. huovinen, c. edlund, j. jalava turku, fin; stockholm, s objectives: the main objective is to get a better knowledge of the human microflora in gastro-intestinal organ by following variations among intestinal enterobacteria in four healthy subjects receiving oral clindamycin. the microflora in the chosen subjects will be monitored for a -year period. the presence and stability of specific resistance genes will be studied in samples collected serially from selected antibiotic exposed subjects. blatem and blashv that code for an extended spectrum beta-lactamase in enterobacteriaceae will be studied. the study will be done by using identification, susceptibility testing, pcr and molecular fingerprinting methods. methods: serially collected faecal samples from four healthy subjects who had received clindamycin perorally for days were cultured and screened for enterobacteriaceae. sampling was performed pretreatment, day , weeks, , , , and months after clindamycin administration. between and colonies of suspected enterobacteriaceae were picked from each sample. biochemical identification of the bacterial isolates was done by oxidase, indole production and activity of beta-glucoronidase. mics were determined according to nccls by standard agar dilution method on mü ller-hinton ii medium. the following antimicrobials were tested: ampicillin, cephalothin, cefuroxime, piperacillin/ tazobactam, amoxicillin-clavulanic acid, ceftazidime, cefotaxime, imipenem, aztreonam, gentamicin, streptomycin, chloramphenicol, tetracycline, nalidixic acid, trimethoprim, sulfamethoxazole and ciprofloxacin. results: a total of isolates were identified as oxidase negative, gram-negative rods and thus belonged to the enterobacteriaceae. the isolates were then screened for indole and betaglucoronidase activity. these results showed that % of all strains were e. coli. of all strains, % were resistant to ampicillin, % against sulfamethoxazole, . % against cephalothin and . % against nalidixic acid. the variation of antibiotic resistance between subjects is broad. conclusion: enterobacteriaceae are naturally resistant to clindamycin. however, after clindamycin treatment alterations in the susceptibility to other antimicrobial agents still occur in the microflora. additional research needs to be done to clarify if these alterations in antibiotic resistance are caused by variation of strains/species or exchange of resistant elements.p prevalence and implication of the cfia and cpha genes in imipenem resistance among bacteroides spp.m. theron, m.n. janse van rensburg, c. roussouw bloemfontein, za objectives: bacteroides is a major cause of intra-abdominal and female genital tract infections as well as subcutaneous abscesses. beta-lactam agents and carbapenems are currently used in monotherapy against anaerobic infections. the study was done to: ( ) investigate the susceptibility of bacteroides strains isolated from bloemfontein academic hospitals; ( ) compare results with a previous study; ( ) determine the prevalence of carbapenemases/ metallo-beta-lactamases in bacteroides spp. methods: fifty-one bacteroides spp. strains were isolated from patients in the universitas and pelonomi hospitals in bloemfontein. mics of antimicrobial agents were determined by the nccls agar dilution method. a bioassay was used to screen for carbapenemase or metallo-beta-lactamase production. pcr amplification was performed for the detection of cfia and cpha genes. plasmids were extracted using a high pure plasmid isolation kit. results: susceptibility levels were relatively high for imipenem ( %), meropenem ( %) and metronidazole ( %). comparing the results with a previous study (isolates from / ), showed a reduction in susceptibility to imipenem ( - %), meropenem ( - %) and metronidazole ( - %). the bioassay results gave no indication of the presence of significant concentrations of a carbapenemase or metallo-beta-lactamase. pcr amplification showed the cfia gene ( bp) in / strains (imipenem mic to > lg/ml) and the cpha gene ( bp) in / of the isolates (imipenem mic - lg/ml). no plasmids were detected. conclusions: although > % of the isolates were susceptible to the carbapenems, it is evident that resistance has increased over the last decade. fortunately the production of metallo-beta-lactamases has been found to give rise to mics that only range from to lg/ml. this study supports these findings with the exception of one isolate with a mic > lg/ml. demonstration of the cfia p application of molecular biological techniques to the study of alterations in hamster gut microflora and assessment of treatment with saccharomyces boulardii l. coroler, g. philippe-taine, e. bayart, t. cécile, j.-m. gillardin, h. goïot compie`gne, f objectives: studies of the intestinal microbial ecosystem by classical culture techniques suggest that only % of the microflora can be cultured. pcr procedures based on s rrna gene specific for bacteria were developed to detect bacterial populations in hamster faeces. methods: a total of populations of bacteria were characterised by their genomic dna sequences and targeted by pcr probes: actinomyces group, bacteroides distasonis, bacteroides fragilis, bifidobacterium group, b. adolescentis, b. angulatum, b. catenulatum, b. infantis, b. longum, clostridium group, c. clostridiiforme, c. coccoides, c. difficile, c. leptum, c. perfringens, fusobacterium prausnitzii, lactobacillus group, peptosteptococcus productus, propionibacterium group, pseudomonas aeruginosa, ruminococcus obeum, citrobacter group, c. freundii, escherichia group, enterobacteria group, enterobacter cloacae, morganella morganii, proteus mirabilis, staphylococcus group, salmonella group. results: sensitivity was measured by extraction of total genomic dna and pcr amplification and a significant detection level of bacteria/faecal sample was obtained. qualitative variations of bacteria population were observed during the first weeks of acclimatisation, suggesting a stabilisation period for hamster microflora in new environmental conditions. after oral antibiotherapy, with one dose of mg/kg amoxicillin-clavulanic acid, some groups were eradicated from hamster faeces: propionibacterium, staphylococcus and c. leptum, c. clostridiiforme. as reported in the literature, no antibiotic effect was observed on levels of dominant faecal groups: bifidobacterium, peptostreptococcus. antibioticassociated perturbations are linked with the disruption of the normal intestinal flora leading to a colonisation of pathogen bacteria species. in order to understand the role of saccharomyces boulardii (s.b.) in prevention of antibiotic-associated diarrhoea, Â cfu/kg/day of s.b. were administered to hamsters during oral antibiotic treatment. the results showed that populations that were eradicated by antibiotic administration remained expressed and stabilised with concomitant s.b. treatment, suggesting an effective protection by s.b. on the intestinal flora. conclusions: these pcr results should be used to quantify the intestinal microflora by dna microarray analysis. objectives: the acinetobacter calcoaceticus-acinetobacter baumannii complex (acb complex) includes a. calcoaceticus (genospecies ), a. baumannii (genospecies ), unnamed genospecies and tu. these species are difficult to differentiate by phenotype. in this study, the feasibility of using sequences of the s- s rdna spacer region (its) for identification of the acb complex was evaluated. methods: the bacteria-specific universal primers bf (gtgaa tacgt tcccg ggcct) and r (gggtt ycccc rttcr gaaat) (y ¼ c or t, and r ¼ a or g) were used to amplify a dna fragment that encompassed a small portion of the s rdna region, the its, and a small portion of the s rdna region. the its regions from reference strains ( species) of nonfermenters including strains of acb complex were amplified by pcr and sequenced; the sequence data in combination with those available in genbank were used to construct an its sequence database for the identification of acb complex. for reference strains of each species of the acb complex, the sequence similarities of the its regions were obtained by comparing their its sequences with that of the type strain of the same species. the database was used to test clinical isolates of acb complex, including isolates of a. baumannii and isolates of a. calcoaceticus, as identified by api ne. results: a. baumannii had the shortest its fragment ( - bp) followed by genospecies tu ( - bp), genospecies ( - bp) and a. calcoaceticus ( - bp). the intraspecies its similarity of the acb complex was very high, ranging from . to . , whereas the interspecies its similarity was relatively low (range: . - . ). among the clinical isolates of a. baumannii, two isolates were genospecies and isolates were ungroupable, as revealed by its sequence analysis. therefore, about % of clinical isolates of a. baumannii was misidentified. furthermore, among the clinical isolates of a. calcoaceticus, isolates were genospecies and three isolates were ungroupable. therefore, the designation of a. calcoaceticus to clinical isolates is, under most conditions, not correct. these results were confirmed by amplified rdna restriction analysis (ardra). conclusions: its sequence analysis provides a simple and useful alternative for species delineation of the acb complex. objectives: enteroaggregative escherichia coli (eaec) are increasingly implicated in acute and persistent diarrhoea around the world. phenotypically, eaec have a defining 'stacked brick' pattern of aggregative adherence (aa) to epithelial cell lines in vitro. genotypically, they are diverse, and while a range of eaec pathogenicity factors are known, their distribution amongst strains varies. the most widely used dna probe for eaec is cvd , which has been reported to have limited sensitivity in some studies, but it is presumed to be specific for eaec. the aim of this study was to determine whether the cvd probe is a specific tool for identifying eaec strains imported into the uk. methods: a total of e. coli isolates (four per patient) were obtained from consecutive stool samples of diarrhoeal patients with a recent history of foreign travel ( different countries). all were screened for hybridisation with the cvd probe, as well as eaec plasmid encoded virulence factors aggr (aggregative adherence regulator), aap (dispersin) and the chromosomal pathogenicity-island-encoded mucinase pic. other pathogenic e. coli were identified using standard probes. cvd probe positive strains were then examined for adherence to hep cells after coincubation for h. results: the prevalence of eaec-associated genes amongst the isolates was: cvd . %; aggr . %; aap %; pic . %. adherence assays on the isolates that were cvd positive revealed a mixture of aggregative ( isolates) and non-adherent strains (nine isolates) plus isolates that gave an unusual pattern of loose, highly localised aggregation, present on < % of the hep- cells. of the cvd positive strains, % were aap, aggr, and pic positive as well, but this group also contained strains of all three adherence types. none of the other eaec-associated probes was unequivocally predictive of actual aa among cvd positive strains. unexpectedly, cvd positive isolates that hybridised with the enteropathogenic e. coli probe eae were isolated from one patient (returning from turkey). conclusions: this study suggests that the cvd probe may not be specific for true eaec, even when combined with the other probes used here. the significance of the newly described adherence pattern in relation to diarrhoeal disease remains to be elucidated, as does the finding of e. coli with both eaec and epec properties. objectives: otomycosis represents a significant percentage of clinical external otitis and is usually caused by candida, aspergillus, penicillium and malassezia. clinical symptoms such as otorrea, erythema and stenosis of the external auditory canal are commonly present and create appropriate conditions for fungal growth. the objectives of this study were to determine the prevalence of candida otomycoses and to evaluate the relationship between albicans and non-albicans species. methods: from april to november , a total number of patients were found to be suffering from symptoms indicating otitis externa. the specimens were taken by cotton swab from bony portion of external ear. all specimens were inoculated on sabouraud dextrose agar, incubated at and c for days and examined macroscopically every day. suspected cultures were examined microscopically in order to confirm finding of candida spp. the identification of isolated candida strains was carried out by germ tube test and api c aux assimilation test (biomerieux, france). results: in a base of microbiological findings ( . %) patients considered to be negative, ( . %) confirm bacterial or mould results and in ( . %) patients candida spp. was found. out of patients with diagnosed candida otomycosis, in patients only candida spp. was isolated and in five patients otitis externa was caused by candida associated with bacterial or mould infection. c. albicans was identified in three ( / ) cases, while all other was non-albicans strains as three cases of c. guilliermondii ( / ), four of c. famata ( / ) and six of c. parapsilosis ( / ) . conclusion: in clinical finding of otitis externa mycological examination could be very important in setting the accurate diagnosis and appropriate therapy. these results suggest that c. albicans is not the predominant causative agent of otitis externa. isolation of non-albicans species has particular interest in therapy of otitis externa because of their reduced susceptibility to antifungal agents. the study was focused on the species involved and their in vitro antifungal susceptibility. molecular typing of the isolates involved in subsequent episodes of rvvc allowed establishing if the strains showed the same dna type. methods: isolates were identified by standard morphological and biochemical methods. mics of amphotericin-b, itraconazole, fluconazole, ketoconazole, -fluorocytosine, voriconazole were determined by sensititre yeastone colorimetric antifungal panel plates according to nccls document m -a. the strains were typed using pulsed-field gel electrophoresis (pfge) and repetitive extragenic palindromic-pcr dna fingerprints. results: c. glabrata was isolated in . %, c. albicans in . %, c. krusei in . % of cases. the yeasts involved in each recurrence were characterised by identical biochemical profiles and drug resistance phenotypes. c. albicans strains isolated from one rvvc resulted in in vitro resistant to azoles. the genotyping by pfge revealed that c. albicans and c. glabrata obtained from different patients were clinically unrelated to each other while an identical profile, indicating clonal relatedness, was observed with yeasts recovered from the same patient. conclusion: our data underline the persistence of strains, with the same antifungal susceptibility profile and clinically related genotypes in patient with recurrent infections, suggesting a colonisation with the same strain over different periods of time despite therapy. these results stress the need for molecular tools for strain typing in order to clarify the epidemiology of the rvvc and to control drug-resistant fungal agent spread. objectives: using criteria designed for invasive aspergillosis (ia) in neutropenic patients, the present study aimed to determine the impact of invasive aspergillosis in different groups of non-haematooncological icu patients. methods: this study is a retrospective analysis of all patients that were hospitalised in the -bed medical intensive care unit (micu) between january and january . any admitted patient fulfilling one or more of the following criteria was included in the study: (a) histopathological evidence of aspergillosis (including autopsy) or (b) microbiological evidence of aspergillosis during stay in the micu (positive culture or positive circulating galactomannan). ia was classified as proven, probable or possible, according to the eortc/msg definitions. aspergillus isolation from a non-sterile site in patients without appropriate clinical setting was considered as 'colonisation'. results: between and , of patients ( . %) fulfilled the inclusion criteria. thirty-eight patients ( %) had haematological malignancies and were not further analysed. eightynine ( %) were non-haemato-oncological patients ( copd, nine solid organ transplant recipients, autoimmune diseases, objectives: we evaluated the value of aspergillus pcr as a tool for diagnosing invasive aspergillosis during antifungal therapy from whole blood samples. methods: in a -year study, patients receiving antifungal therapy due to chest radiographic findings highly suggestive for fungal pneumonia were evaluated. the pcr results of whole blood samples were compared with those obtained from bronchoalveolar lavage fluids and/or tissue specimens. results: a total of whole blood samples, fine needle aspirations or tissue biopsy specimens, bronchoalveolar lavage fluids and tracheal secrets were analysed using pcr. fifteen patients had proven, nine probable and possible invasive aspergillus infections according to european organization for research and treatment of cancer/mycosis study group definitions. in patients with proven infections, the sensitivities of pcr of lung and blood samples were and %, respectively. the specificities were %. the negative predictive value of blood monitoring under antifungal treatment was %. in patients with probable infections, the sensitivities of pcr of lung fluids and blood were and %, respectively. the specificities were %. the negative predictive value of blood monitoring under antifungal therapy was %. conclusions: the benefits of pcr diagnosing of whole blood are limited if sampling takes place once treatment has started. the performance of aspergillus pcr should be recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection. objective: air is considered the main vehicle of aspergillus spores causing community or nosocomially-acquired invasive aspergillosis (ia). air surveillance is nowadays performed in protected air environments in many institutions. sabouraud dextrose agar irradiated (sd) is used for the control of air in our institution but czapeck agar is also recommended for this purpose. the aim of our study was to compare the efficiency of both media for aspergillus isolation in air samples. methods: we collected samples using the merck air sampler mas Ò with a volume of air per culture of l. every sample was cultured in both media (pair of samples), and agar plates were incubated at c for days. aspergillus spp. was identified by conventional methods. the pairs were checked daily to observe the growth of fungi and after the incubation period the objectives: the spreading of aspergillus hyphae into the brain of immunocompromised patients is a complication of invasive asper-gillosis that leads to death in nearly % of the cases. the most frequent species for induction of cerebral aspergillosis is aspergillus fumigatus. our aim was to study the interaction of a. fumigatus with the complement system to determine the reason for the failure of the cerebral immune system. furthermore, these experiments might give first approaches for a putative immune therapy to support current antimycotical treatment. methods: different pools of cerebrospinal fluid (csf) were tested for their ability to opsonise fungal hyphae with different complement factors. germinated conidia were fixated, incubated in csf, and the deposition of complement was shown via indirect immunofluorescense (if) by suitable specific antibodies. the extent of surface labelling on aspergillus was compared with pseudallescheria boydii, another neurotropic fungus. immunohistochemical (ihc) staining of paraffin-embedded tissue sections derived from patients with cerebral aspergillosis allowed the comparison with the complement deposition in vivo.results: the levels of the complement factors c q, c , c , c , c and c in the csf of normal persons were sufficient for opsonisation of the fungal hyphae, although the deposition was much weaker than in human serum. however, the recognition of aspergillus surface was not optimal in comparison to p. boydii that showed a clearly stronger deposition. concentrations of different complement proteins and complement activation products were highly elevated in csf derived from a patient with cerebral aspergillosis. this csf showed a significantly stronger complement deposition on the fungal surface than the non-inflammatory csf. however, ihc-analyses in tissue sections of patients with cerebral aspergillosis showed only limited opsonisation on the fungus. conclusion: csf harbours the ability of complement deposition on the surface of neurotropic fungi. frequent pathogens like aspergillus fumigatus have adopted their surface to minimise recognition by the complement cascade. cerebral complement production is upregulated as a consequence of fungal infection, which might contribute to antifungal immune defence but also to inflammation and tissue damage. the amount of deposited factors on the fungal hyphae in vivo is low, indicating the expression of complement inhibitory factor(s) by a. fumigatus. objectives: staphylococcus epidermidis is a major pathogen in nosocomial infections, and infectious isolates display a high prevalence of oxacillin resistance (oxar). tn mutagenesis of rsbu, encoding a positive regulator of the alternative sigma factor sigma b lead to a reduced oxar in s. epidermidis . however, the mechanism of this regulatory pathway is still unknown. the role of sigma b in the regulation of oxar in s. epidermidis was investigated in this study. methods: two mutants with inactivation of the entire sigma b operon ( rsbuvwsigb) or the regulatory cascade rsbuvw ( rsbuvw) were generated by allelic gene replacement in s. epidermidis , which displays a heterogeneous oxacillin resist-ance phenotype. rna was extracted at and h from cultures in mueller hinton + % nacl (mhnacl) and mhnacl supplemented with lg/ml oxacillin (mhoxa). quantitative transcriptional analysis of meca, femabcdf, fmta, mrp (fmtb), and mprf (fmtc) were performed by real-time rt-pcr. at least a . -fold difference compared with the wild type in the average of three independent experiments was defined as cut-off for differentially expressed genes. results: population analysis of the mutants and the wild type strain revealed that mutant rsbuvwsigb displayed a more heterogeneous phenotype with a smaller subpopulation expressing methicillin resistance compared with the wild type. mutant rsbuvw with constitutive expression of sigma b displayed a strong increase of methicillin resistance and a homogeneous resistance phenotype compared with the wild type. transcriptional analysis revealed that the homogeneously resistant mutant rsbuvw displayed no differences compared with the wild type under all conditions investigated, except of the gene fmta, which was downregulated in mhoxa at h. interestingly, in the less resistant mutant rsbuvwsigb the genes meca, femb, femd, fmta, and mprf were upregulated in mhnacl compared with the wild type at both time points, whereas in mhoxa only the genes femd, fmta, and mprf were upregulated at or h. conclusions: none of the investigated genes including meca is responsible for the homogeneous expression of oxar in mutant rsbuvw. mutant rsbuvwsigb displayed a less resistant phenotype compared with the wild type strain, despite the upregulation of several genes required for oxar. therefore, an additional sigma b dependent factor must be required for homogeneous expression of oxar in s. epidermidis. objectives: to develop methods to measure the initial response of s. aureus after exposure to antimicrobial agents. such an approach has the potential to allow both the sensitivity and mechanism of resistance to be rapidly determined from isolated bacterial strains. methods: mrna was extracted from a selection of s. aureus isolates either with or without min exposure to antimicrobial agents (including oxacillin and mupirocin). the mrna extracted was then used to produce labelled nucleic acid suitable for hybridisation to a low-density flow through oligonucleotide array targeting specific genes. these arrays are suitable for high throughput screening and provide very rapid hybridisation kinetics.results: distinctive changes in mrna levels were detected for each agent tested and for isolates with different phenotypic susceptibilities. oxacillin resulted in a significant increase in the levels of penicillin binding protein (pbp ) mrna in both sensitive and resistant isolates and an increase in the levels of pbp prime mrna in resistant isolates only. in contrast mupirocin resulted in very high levels of ile-trna synthetase in both strains with high-or low-level mupirocin resistance but not in sensitive strains. conclusion: future developments in rna extraction and labelling as well as the increased availability of dna array technology will allow this approach to be more widely used. this and similar methods have the potential to provide information on both the resistance phenotype of the isolate and the mechanism of resistance, in contrast to 'classical' molecular tests for drug resistance which generally target known genotypes. key: cord- -m cuuehi authors: nan title: abstracts cont. date: - - journal: clin microbiol infect doi: . /j. - . .clm_ _ .x sha: doc_id: cord_uid: m cuuehi nan addition of a biocatalytic oxygen-reducing agent may be required in the absence of fresh media (< hours old) when testing tigecycline using broth microdilution t. stevens, b. johnson, s. bouchillon, j. johnson, d. hoban, m. dowzicky, p. bradford (schaumburg, pearl river, usa) objectives: tigecycline (tgc), a new glycylcycline antimicrobial in development has demonstrated excellent in vitro activity against gram-positive and -negative pathogens. recent reports suggest that tgc mic values, against some organisms, may be elevated if broth used in microdilution panels is (> hours old (aged). the nccls has tentatively recommended that testing of tgc be performed in broth that is (< hours old (fresh). this study looks at the difference between panels using fresh broth, aged broth and broth with a biocatalytic oxygen-reducing agent (bora) added to compensate for any potential broth differences. materials and methods: testing was performed on approximately organisms including: e. coli; k. pneumoniae; m. catarrhalis; s. epidermidis; and s. pneumoniae. the bora used in this study is oxyrase Ò for broth at % concentration. tig microdilution panels evaluated in this study include: panels and aged broth without oxyrase (aged); panels and aged broth with oxyrase (ao); panels and fresh broth without oxyrase (fresh); and panels and fresh broth with oxyrase (fo). panels and aged broth were prepared by microscan. fresh broth was prepared internally. each organism was tested on all four panel types. quality controls were performed using nccls approved atcc strains. results: combined test results showed an mic correlation (with in log dilution) as follows: . % between fresh/ao; . % between fresh/aged; . % between fo/ao; and . % between fo/aged. quality controls ranges for fo, fresh and ao were all in compliance, but aged panels were out of range . % of the time. conclusion: the addition of a bora to aged broth produced results equivalent to fresh broth without a bora. objectives: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many grampositive pathogens including methicillin-resistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillin-tazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from multinational evaluation centres in the test program. methods: a total of clinical isolates were identified to the species level at each of sites in countries and confirmed by the central laboratory. isolates were collected between january and november . mics were determined by each participating laboratory using supplied broth microdilution panels from dade microscan. all testing was performed according to nccls guidelines and manufacturer's instructions. results: the mics of tigecycline ranged from . to mcg/ml for all isolates of s. aureus. tigecycline's mic /mic of . / . mcg/ml, respectively, against mssa was similar to imipenem and minocycline and / fold lower than the remaining comparative agents. tigecycline's mic /mic of . / . mcg/ml, respectively, against mrsa was / fold lower than vancomycin, / fold lower than minocycline and / fold lower than linezolid. all isolates of s. aureus were inhibited by tigecycline at an mic of mcg/ml regardless of methicillin phenotype. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. background: rapid increasing resistance in nosocomial pathogens has always been a challenge for clinicians and hospital infection control. tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of enterobacteriaceae (mainly e. coli, klebsiella spp., enterobacter spp., and serratia spp.) collected from hospitals in north america, europe and asia. methods: a total of clinical isolates of enterobacteriaceae were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout . minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity was equivalent to imipenem presenting a mic /mic of . / mcg/ml against all strains of enterobacteriaceae. in comparison to other antimicrobials tested, the mic of mcg/ml for tigecycline was also the lowest being fold lower than commonly prescribed broad spectrum antimicrobials such as ceftriaxone, levofloxacin, and minocycline and fold lower than ceftazidime and piperacillin/tazobactam. the frequency of esbl production among k. pneumoniae and e. coli was found to be . % and . %, respectively. tigecycline inhibited > % of all e. coli and k. pneumoniae esbl producers at an mic of mcg/ml. approximately % of enterobacter spp. and serratia spp. presented resistance to third generation cephalosporins (ceftazidime and ceftriaxone) suggestive of ampc-type resistance. tigecycline also inhibited a majority of these isolates with an mic of mcg/ml. conclusion: tigecyclines in vitro activity was comparable to the activity of a broad spectrum antimicrobial, carbapenem (imipenem) , and greater than other commonly prescribed broad spectrum agents tested in this study. the presented data suggest that tigecycline may be an effective therapeutic option against both susceptible strains of enterobacteriaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have a potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram-positive, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against isolates of enterobacteriaceae collected from hospitals across the usa. methods: a total of clinical isolates, collected in , were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: the efficacy of all broad spectrum antimicrobial agents still remain highly active against enterobacteriaceae in the united states. the susceptibility rates for amikacin, cefepime, ceftazidime, ceftriaxone, imipenem, levofloxacin, minocycline and piperacillin/tazabactam are . %, . %, %, . %, . %, . %, . % and . %, respectively. tigecycline's activity was similar to imipenem presenting a mic /mic of . / mcg/ ml against all strains of enterobacteriaceae. the frequency of esbl production among k. pneumoniae and e. coli was found to be . % and . %, respectively. tigecycline successfully inhibited > % of all e. coli and k. pneumoniae esbl producers at a mic of mcg/ml. it was also noticed unusual resistance to imipenem in of these isolates. while still under more detailed analysis, preliminary data have shown that tigecycline presented a mic / mic of / mcg/ml against these multi-resistant isolates. conclusion: most of broad spectrum antimicrobial agents still remain active against enterobacteriaceae from the us. tigecycline's activity was comparable to the activities of broad spectrum antimicrobials and with greater activity against most esbl and ampc producing isolates. tigecycline also showed in vitro activity against isolates that were intermediate or resistant to imipenem, which in many instances is considered as a last therapeutic option. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible strains of enterobacterieaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered gram-positive and gram-negative species, including anaerobic pathogens responsible for community and hospital infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/ tazobactam against gram negative rods in addition to linezolid, penicillin and vancomycin for the gram positive species. isolates were collected from hospitals in the united states throughout . methods: a total of clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with a mic equal or lesser than mcg/ml. tigecycline also showed in vitro activity with a mic mcg/ml against imipenem resistant enterobacteriaceae strains. although similar to other classes of broad spectrum antimicrobial agents against non-fermenters, tigecycline was especially active against acinetobacter spp. with the lowest mic of mcg/ml. tigecycline inhibited s. aureus with mic of . mcg/ml for both mssa and mrsa isolates. against enterococci, tigecycline's mic was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable or greater than most commonly prescribed broad spectrum antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible common gram-positive and gram-negative pathogens, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, and piperacillin/tazobactam against gram negative rods in addition to linezolid, penicillin, and vancomycin for the gram positive species. isolates were collected from hospitals located in germany, italy, spain, and united kingdom throughout . methods: a total of clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with mics equal or lesser than mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against glucose non-fermenters, tigecycline was especially active against acinetobacter spp. presenting the lowest mic of mcg/ml. tigecycline inhibited s. aureus with a mic of . mcg/ml regardless of sensitivity or resistance to methicillin. the same results were noticed against enterococci where tigecycline's mic of . mcg/ml was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered spe-cies responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/ tazobactam against gram negative rods in addition to linezolid, penicillin and vancomycin for the gram positive species. isolates were collected from hospitals located in asia throughout . methods: a total of clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity was similar to imipenem against enterobacteriaceae with mic /mic of . / mcg/ml. resistance to third generation cephalosporin was found in . % of e. coli and . % of k. pneumoniae consistent with esbl phenotype. tigecycline inhibited esbl and ampc producers with mics equal or lesser than mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against glucose non-fermenters, tigecycline was especially active against acinetobacter spp. presenting the lowest mic of mcg/ml. tigecycline successfully inhibited s. aureus with mic of . mcg/ml regardless of sensitivity or resistance to methicillin. same phenomenon was noticed against enterococci where tigecycline's mic of . mcg/ml was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram-positive, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against isolates of enterobacteriaceae collected from hospitals in germany, italy, spain and united kingdom. methods: a total of clinical isolates, collected in , were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory abstracts using supplied broth microdilution panels and interpreted according to nccls guidelines. results: it was observed that the in vitro activity of all broad spectrum antimicrobial agents still remains highly active against enterobacteriaceae in europe. the susceptibility rates for amikacin, cefepime, ceftazidime, ceftriaxone, imipenem, levofloxacin, minocycline,andpiperacillin/tazobactamare . %, . %, . %, . %, %, . %, . %, and . %, respectively. tigecycline's activity was similar to the most effective antimicrobial agent, imipenem ( % susceptibility) presenting a mic /mic of . / mcg/ml against all strains of enterobacteriaceae. the frequency of esbl production among k. pneumoniae and e. coli was found to be . % and . %, respectively. tigecycline inhibited % of all esbl producing e. coli at a mic of . mcg/ml and at a mic of mcg/ml for esbl producing k. pneumoniae. approximately % of enterobacter spp. and % of serratia marcescens presented resistance to third generation cephalosporins (ceftazidime and ceftriaxone) suggestive of ampc-type resistance. conclusion: most of the broad spectrum antimicrobial agents still remain active against european representatives of enterobacteriaceae. tigecycline's in vitro activity was comparable to the activities of all broad spectrum antimicrobials with greater activity against esbl and ampc producing isolates with a mic of mcg/ml. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible strains of enterobacterieaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, and piperacillin/tazobactam against gram negative rods in addition to linezolid, penicillin, and vancomycin for the gram positive species. isolates were collected from hospitals in north america, europe and asia throughout . methods: a total of clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with a mic equal or lesser than mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against non-fermenters, tigecycline was especially active against acinetobacter spp. demonstrating the lowest mic of mcg/ml. tigecycline successfully inhibited s. aureus with mic of . mcg/ml regardless of sensitivity or resistance to methicillin. the same results were noticed against enterococci. tigecycline's mic was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: resistance to glycopeptides in enterococci was first recognized in the late s, and since then has been a major challenge to clinicians and infection control. tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to vancomycin, linezolid, ampicillin, imipenem, ceftriaxone, levofloxacin, minocycline, penicillin and piperacillin/tazobactam against members of enterococcus spp. collected from hospitals in north america, europe and asia. methods: a total of clinical isolates of enterococcus spp. were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout . minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: of e. faecalis evaluated, resistance to vancomycin in ( . %) isolates was observed. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic /mic ( . / . mcg/ml) among all antimicrobial agents evaluated. among e. faecium, ( . %) were resistant to vancomycin, of which three isolates were also resistant to linezolid. tigecycline also presented the lowest mic /mic of . / . mcg/ml. five isolates of vancomycin susceptible e. faecalis and one vancomycin susceptible e. faecium were nonsusceptible to linezolid. no abnormal resistance phenotype was observed in other enterococcus species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multidrug resistant strains regardless of degree or type of resistance. tigecycline evaluation surveillance trial (test) -global in vitro antibacterial activity against selected species of glucose non-fermenting organisms objective: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many gram-positive pathogens including methicillinresistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillintazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from us centres in the test program. methods: a total of clinical isolates were identified to the species level at each of participating sites and confirmed by the central laboratory. isolates were collected throughout . mics were determined by each participating laboratory using broth microdilution panels from dade microscan. all testing was performed and interpreted according to nccls guidelines and manufacturer's instructions. results: among the isolates, ( . %) were found to be resistant to methicillin (mrsa). besides the cross resistance of mrsa isolates to imipenem, ceftriaxone, penicillin, ampicillin, and piperacillin/tazobactam, it was observed a high rate of nonsusceptibility to levofloxacin ( . %). no resistance was observed against vancomycin and linezolid. the mics of tigecycline ranged from . to mcg/ml for all isolates of s. aureus, and tigecycline presented the lowest mic /mic of . / . mcg/ml against mrsa isolates, being several folds lower than all the comparator agents. the mssa isolates showed the expected profile of high resistance to ampicillin and penicillin. the only unusual pattern was the . % nonsusceptibility to levofloxacin. tigecycline's mic /mic of . / . was also the lowest among all mssa isolates. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. tigecycline evaluation surveillance trial (test) -united states in vitro antibacterial activity against selected species of enterococcus spp. results: of e. faecalis evaluated, it was observed resistance to vancomycin in ( . %) isolates. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic /mic ( . / . mcg/ml) among all antimicrobial agents evaluated. as a typical profile of e. faecalis, fluoroquinolone (levofloxacin) and tetracycline (minocycline) had limited activities against this species. among e. faecium, ( . %) were resistant to vancomycin, of which three isolates were also resistant to linezolid. tigecycline also presented the lowest mic /mic of . / . mcg/ml. five isolates of vancomycin susceptible e. faecalis and one vancomycin susceptible e. faecium were non-susceptible to linezolid. no abnormal resistance phenotype was observed in other enterococcus species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multi-drug resistant strains regardless of degree or type of resistance. background: glucose non-fermenting gram negative rods are known to be highly resistant in hospital settings and have always been a challenge for clinicians and hospital infection control. the degree or type of resistance may be due to several sophisticated mechanisms such as production of broad spectrum beta-lactamases, efflux pumps and altered membrane permeability, inactivating most classes of antimicrobials that are available for treatment (cephalosporins, carbapenems, aminoglycosides, fluoruquinolones). tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae and selected species of non-fermenters, as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of acinetobacter spp. and pseudomonas aeruginosa collected from hospitals in the united states. methods: a total of clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout . minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity against p. aeruginosa showed a mic of ( mcg/ml. towards a. baumannii (n = ), which cephalosporins were ineffective, tigecycline showed the lowest mic /mic of . / mcg/ml outperforming amikacin mic /mic / , imipenem mic /mic . / and minocycline mic /mic / . similar findings were found in other species of acinetobacter genus. conclusion: the presented data suggest that tigecycline may be an effective and reliable therapeutic option against strains of acinetobacter spp., including multi-drug resistant strains regardless of degree or type of resistance. objective: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many gram-positive pathogens including methicillinresistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillintazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from european centres in the test program. methods: a total of clinical isolates were identified to the species level at each of participating sites and confirmed by the central laboratory. isolates were collected throughout . mics were determined by each participating laboratory using broth microdilution panels from dade microscan. all testing was performed and interpreted according to nccls guidelines and manufacturer's instructions. results: among the isolates, ( . %) were found to be resistant to methicillin (mrsa). besides the cross resistance of mrsa isolates to imipenem, ceftriaxone, penicillin, ampicillin, and piperacillin/tazobactam, it was observed that all of mrsa isolates were also non-susceptible to levofloxacin. no resistance was observed against vancomycin and linezolid. the mics of tigecycline ranged from . to . mcg/ml for all isolates of s. aureus, and tigecycline presented the lowest mic /mic of . / . mcg/ml against mrsa isolates, being several folds lower than all the comparator agents. the mssa isolates showed the expected profile of high resistance to ampicillin and penicillin. opposite to mrsa isolates, mssa presented very little resistance to levofloxacin ( . %). tigecycline's mic /mic of . / . was also the lowest among all mssa isolates. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. tigecycline evaluation surveillance trial (test) -european in vitro antibacterial activity against selected species of enterococcus spp. been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to vancomycin, linezolid, ampicillin, imipenem, ceftriaxone, levofloxacin, minocycline, penicillin and piperacillin/tazobactam against members of enterococcus spp. collected from hospitals in germany, italy, spain and united kingdom. methods: a total of clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout . minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: of e. faecalis evaluated, resistance to vancomycin in ( . %) isolates was observed. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic /mic ( . / . mcg/ml) among all antimicrobial agents evaluated. among e. faecium, ( . %) was resistant to vancomycin. tigecycline also presented the lowest mic / mic of . / . mcg/ml. no abnormal resistance phenotype was observed in other enterococci species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multi-drug resistant strains regardless of degree or type of resistance. tigecycline evaluation surveillance trial (test) -european in vitro antibacterial activity against selected species of glucose non-fermenting organisms background: glucose non-fermenting gram negative rods are known to be highly resistant in hospital settings and have always been a challenge for clinicians and hospital infection control. the degree or type of resistance may be due to several sophisticated mechanisms such as production of broad spectrum beta-lactamases, efflux pumps and altered membrane permeability, inactivating most classes of antimicrobials that are available for treatment (cephalosporins, carbapenems, aminoglycosides, fluoruquinolones). tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae and selected species of non-fermenters, as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of acinetobacter spp. and pseudomonas aeruginosa collected from hospitals in germany, italy, spain, and united kingdom. methods: a total of clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout . minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity against p. aeruginosa showed a mic of ( mcg/ml. the cephalosporins were ineffective towards a. baumannii (n = ). tigecycline showed the lowest mics against a. baumannii mic /mic of . / mcg/ml, outperforming amikacin mic /mic /> , imipenem mic /mic . / and minocycline mic /mic / . similar findings were found in other species of acinetobacter genus. conclusion: the presented data suggest that tigecycline may be an effective and reliable therapeutic option against strains of acinetobacter spp., including multi-drug resistant strains regardless of degree or type of resistance. p antimicrobial activity of tigecycline tested against bacterial pathogens from intensive care units h. sader, t. fritsche, r. jones (north liberty, usa) objectives: to evaluate the antimicrobial activity of tigecycline (tig) and selected antimicrobials against bacterial pathogens isolated from patients hospitalized in intensive care units (icus) worldwide. methods: a total of were consecutively collected in > medical centres located in north america ( ), south america ( ), europe ( ) and the asia-australia region ( ). the isolates were collected from (no. of isolates/%): bloodstream ( / %), respiratory tract ( / %), skin/soft tissue ( / %), and urinary tract ( / %) infections in the - period, and susceptibility tested by nccls broth microdilution methods. results: the antimicrobial activity of tig and the frequency of occurrence of bacterial pathogens are summarized in the table: all gram-positive pathogens ( ) were inhibited at < mg/l of tig. resistance (r) to oxacillin was detected in % of sa and % of cons, and r to vancomycin was detected in % of enterococci. tig was very active against enterobacteriaceae (ent; ) with a mic < mg/l, except for serratia spp. % of e. coli and % of klebsiella spp. showed an esbl phenotype while % of enterobacter spp. were r to ceftazidime. % of ent showed r to ciprofloxacin. tig and trimethoprim/sulfamethoxazole were the most active compounds against s. maltophilia (mic , and mg/l respectively). tig was also highly active against asp (mic , mg/l), but psa showed decreased s to tig (mic , mg/l). non-s to imipenem (mic, > mg/l) was observed in % of asp and % of psa isolates. conclusions: isolates from icu patients showed high rates of antimicrobial r. the most alarming problems detected were vancomycin r among enterococci, esbl mediated beta-lactam r and fluoroquinolone r among ent, and carbapenem r among psa and asp. tig exhibited potent in vitro activity against the vast majority of clinically important pathogenic bacteria (except psa) isolated from icu patients and may represent an excellent option for the treatment of infections in this clinical environment. potency and spectrum of tigecycline tested against an international collection ( ) ( ) ( ) ( ) ( ) of bacterial pathogens producing skin and soft tissue infections t. fritsche, h. sader, m. stilwell, r. jones (north liberty, usa) objective: tigecycline (tig) is the sentinel representative of the glycylcycline class to be developed as a parenteral agent targeting bacterial pathogens responsible for pneumonia, intraabdominal sepsis and ssti. the aim of this study was to evaluate the activity and potency of tig when tested against a large collection of bacterial pathogens causing ssti. methods: consecutive, non-duplicate bacterial isolates ( , strains) were collected from to from patients with documented community-acquired or nosocomial ssti in > medical centres participating in the tig surveillance program in north america ( . %), europe ( . %), latin america ( . %) and the asia-pacific region ( . %). all isolates were tested using nccls broth microdilution methods against tig and representative comparator agents used for empiric and directed therapy of ssti. results: ssti pathogen rank order (top ten), potency and cumulative inhibition rates for tig are in the table: all sa, streptococci, enterococci, cons, ksp and ec were inhibited by £ mg/l of tig, along with % of asp and % of ent. the broad-spectrum of activity exhibited by tig included tetracycline resistant subsets as well as mrsa, vre, and esbl-producing strains. only pm and psa isolates were less susceptible (mic values at and mg/l, respectively). conclusions: among the top ten-ranked pathogens producing ssti, % of isolates were inhibited by £ mg/l of tig and % were inhibited by £ mg/l (the current nccls breakpoint for tetracyclines). tig may represent a welcome choice among newer parenteral agents for the common gram-positive and negative pathogens producing serious ssti given in vitro testing results, thus warranting continued investigation for this indication. objective: to assess the activity of tigecycline (formerly gar ), a novel glycylcycline, against recent bloodstream infection (bsi) pathogen isolates from six continents. frequency of clinical occurrence of these pathogens was determined and their antibiograms assessed using nccls reference broth microdilution methods. methods: a total of , strains were tested by the m -a ( ) method with interpretations from m -s ( ) . a tigecycline susceptible (s) breakpoint was defined as £ mg/l for comparison purposes only, although £ mg/l has been used for tetracyclines. the rank order of pathogens was: s. aureus (sa; . %), coagulase-negative staphylococci (cons; . %), e. coli (ec; . %), enterococci (ent; . %), klebsiella spp. (ksp; . %), p. aeruginosa (psa; . %), enterobacter spp. (ebs; . ), beta-haemolyticstreptococci (bst; . %); s. pneumoniae (spn; . %), and viridans group streptococci (vgs; . %). more than comparison agents were tested including tetracycline (tc) and ciprofloxacin (cip) . results: bsi pathogens (gram-positive and enterobacteriaceae) tested against tigecycline are shown in the table. tigecycline was consistently active against tc-resistant (r) strains ( - % s versus - %). mic :% s results for other bsi species were: p. mirabilis ( mg/l: ), acinetobacter spp. ( . mg/l: ), serratia spp. ( mg/l: ), s. maltophilia ( mg/l: ) and indole-positive proteae ( mg/l: ). tigecycline exhibited a broader spectrum of activity against bsi isolates when compared to cip, tc, older aminoglycosides and imipenem. tigecycline was not active against psa (mic , mg/l; . % of bsi isolates). conclusions: tigecycline exhibited a wide spectrum of antimicrobial potency versus bsi isolates collected worldwide. serious infections in nosocomial environments should benefit from tigecycline among the investigational phase agents focused on r strains. evaluation of the in vitro activity of tigecycline against gram-negative anaerobic bacteria objectives: the evaluation of the in vitro activity of tigecycline in comparison to tetracycline, penicillin, piperacillin ( tazobactam, cefoxitin, clindamycin, metronidazole and imipenem against recently isolated gram-negative anaerobic bacteria. materials and methods: a total of gram-negative anaerobic clinical strains ( bacteroides fragilis group, other bacteroides spp. non-fragilis, prevotella spp. and miscellaneous) isolated during the period / - / were tested using the e-test and the agar dilution methods on brucella agar plates supplemented with % horse blood, vitamin k and hemin. incubation in a chellab anaerobic chamber was performed for hours. interpretation of the results was according to nccls guidelines. for quality control the strains b. fragilis atcc and b. thetaiotaomicron atcc were used. results: overall mic to tigecycline, tetracycline, penicillin, piperacillin + tazobactam, cefoxitin, clindamycin, metronidazole and imipenem were , , , , , , and . mg/l, respectively, whereas mic were . , , , . , , , . and . mg/l, respectively. bacteroides fragilis group mic were , , , , , , and . mg/l, respectively, whereas mic were . , , , . , , , . and . mg/l, respectively. at the tigecycline mic concentration of mg/ l the compound inhibited a higher percentage of isolates than clindamycin, cefoxitin or tetracycline (which inhibited , and % of the isolates, respectively). only one strain was resistant to imipenem (mic > mg/l), having a tigecycline mic of mg/l. in addition, six of the eight strains with a metronidazole mic > mg/l had a tigecycline mic < mg/l. conclusions: in general, tigecycline showed better in vitro activity than clindamycin, cefoxitin, tetracycline or penicillin against gram-negative anaerobic bacteria and somewhat lower clinical microbiology and infection, volume , supplement , activity than imipenem, metronidazole or piperacillin + tazobactam. nevertheless, among most metronidazole and imipenem resistant isolates tigecycline displayed good activity. the results of this in-vitro evaluation show that tigecycline should be considered as a possible alternative for the treatment of mixed aerobicanaerobic infections. objectives: to ascertain the variation between mics of tigecycline determined in broth and agar, and by nccls and bsac methods. to assess the effect of adding tigecycline to the medium one day before use and of using media that had been aged for one week before addition of tigecycline and inoculation. method: mics for non-fastidious bacteria and streptococci were determined in mueller-hinton broth (mhb), mueller-hinton agar (mha), iso-sensitest broth (isb) or iso-sensitest agar (isa). fresh antibiotic stock solutions (tigecycline, tetracycline and minocycline) were incorporated into bulk media by rolling, to minimise aeration. preparation conditions otherwise were as fol-lows: (a) freshly-prepared media, antibiotic added upon cooling, then inoculated; (b) freshly-prepared media, antibiotic added upon cooling thenstored for day before inoculationand (c) media stored for days before addition of the antibiotic and inoculation. results: mics in fresh mhb (the nccls reference method) were taken as a standard. for all the tetracyclines, these mics were - doubling dilutions higher than on mha or isa (bsac reference method), or in isb. media with tetracyclines added a day before use gave raised mics, though rarely by more than one dilution. tigecycline mics were increased in -day-old mhb or isb. this effect was greatest ( - dilutions) for the most susceptible strains (mic £ . mg/l) and was absent for the resistant organisms (mic ‡ mg/l); it did not occur in agar. minocycline mics were not raised in aged media, and tetracycline mics were only marginally affected. the addition of blood to the mhb largely abrogated the effect on tigecycline mic, as did boiling the broth prior to adding the antibiotic. conclusion: the raised mics of tigecycline in aged broth probably reflect inactivation by dissolved oxygen. this accords with lack of any mic increase in newly boiled (i.e. degassed) mhb or on aged agar (which is boiled to melt before use). the effect of blood may be to add to the reducing capacity, protecting the tigecycline. at a practical level, broth mic methods for tigecycline (e.g. the nccls reference method) require, freshly prepared or steamed medium; this is not a concern if agar dilution methods are used, such as that of the bsac. sexually transmitted diseases p prevalence of chlamydia trachomatis and neisseria gonorrhoeae in native and immigrant population (ambits study) in barcelona (spain) testing of all danish gc strains isolated in - (n = ) . clinical and epidemiological data were obtained when possible. results: the pip-negative gc isolates were all designated as serovar ib- and similar mic values were identified in the antibiograms of these isolates fingerprints were identified using spei and three using bglii among the ib- isolates. four distinguishable pfge . however, all these isolates differed by less than six bands in both fingerprints. the phylogenetic tree analysis of the porb b sequences, suggested that these minor differences represented the ongoing evolution of the same strain. during the period jan. to nov. the proportions of pip-neg gc per year in denmark were / , / and / , respectively, and the pip-neg gc infected mostly men (only women). the antibiograms of all danish isolates - indicate a spread of at least one pip-neg gc strain. the results of the present study indicate a circulation of at least one n. gonorrhoeae prolyliminopeptidasenegative strain in the danish homosexual community. an increased awareness for pip-negative n. gonorrhoeae, which create large problems in diagnosis using e.g. api nh, rapid nh, gonocheck ii or neisseria pet, is essential. a new recombinant antigen haemagglutination test for the serological diagnosis of syphilis n. chepurchenko, t. ulanova, v. puzyrev, l. loginova, a. burkov, a. obriadina (nizhniy novgorod, rus) introduction: passive treponema pallidum hemagglutination (ha) -the routine treponemal test has equal sensitivity with the fluorescent treponemal antibody absorption (fta-abs) test and with the enzyme immunoassay (eia) in later stages of syphilis, but some studies have indicated that it is less sensitive in the primary stage of the disease. the use of recombinant t. pallidum antigens instead of a poorly defined mixture of antigens from wild-type t. pallidum has the potential for improving the sensitivity of hemagglutination test. objectives: the purpose of this study was to evaluate the diagnostic relevance of recombinant proteins that efficiently model the antigenic epitope(s) of the treponema pallidum proteins. methods: four full-length recombinant proteins (tmpa, , , kda) were expressed in e. coli and then used individually to develop the hemagglutination test (ht) for the detection of anti-treponema pallidum (anti-tp) activity in serum specimens. serum samples (n = ) from patients with clinically proven syphilis in various stages of the disease (primary (n = ), secondary (n = ), latent (n = )) and from normal blood donors (n = ) were tested. samples from patients with primary syphilis, samples from patients with secondary and samples from patients with latent stage were tested initially with commercially available hemagglutination test based on mixture of treponema pallidum antigens (http). all specimens were additionally tested for specific antibodies by commercially available enzyme immunoassay (eia). results: the sensitivity of the ht for the detection of anti-tp activity in human serum specimens varied from . % to . % with primary syphilis sera, from . % to % with secondary, and from . % to . % with latent stage sera for each protein. the tmpa was found as the most immunoreactive when serum samples from patients with untreated syphilis were tested. the sensitivity of tmpa ht was identical to those of the http in cases of untreated secondary and latent syphilis and significantly higher with specimens from primary stage of disease. the overall specificity and sensitivity of the tmpa ht were comparable to those of eia % and . %; % and . % respectively. the results of this study indicate that the tmpa ht may be a reasonable alternative to the routine hemagglutination test and the elisa as a confirmatory test for syphilis. use of treponema + vdrl virablot test as a useful Ôall in oneÕ confirmatory assay for treponema pallidum antibodies a. kostoula, g. vrioni, c. bobogianni, c. stergiopoulou, e. papantoniou, s. levidiotou (ioannina, gr) objectives: as detection of treponema pallidum subsp. pallidum by dark-field microscopy or direct immunofluorescence is possible only in the early stages of the disease, serology has become the method of choice for syphilis diagnosis. except for conventional treponemal tests, the t. pallidum western immunoblot assay has also been used as an alternative confirmatory assay for t. pallidum antibodies. the aim of the present study was the use of a multiparameter immunoassay, which simultaneously detects treponemal specific and lipoid antibodies, for the confirmation of syphilis antibodies. methods: a total of serum specimens were tested including, reactive sera from patients with documented treponemal infection; a specificity panel of sera from normal subjects, sera with rapid plasma reagin (rpr) reactivity (fta-abs and tpha negative), leptospira igm serology-positive sera and borrelia burgdorferi igg or igm serology-positive sera. all serum samples were tested with conventional syphilis tests according to the manufacturersÕ instructions: rpr, venereal disease research laboratory (vdrl), t. pallidum haemagglutination (tpha) at a serum dilution of : , fluorescent t. pallidum absorption (fta-abs) and igm capture enzyme immunoassay (eia). subsequently, the results were compared to those of the treponema + vdrl virablot igg, igm test (viramed, labor -diagnostika), a immunoassay for the qualitative detection of specific igg or igm antibodies to t. pallidum using the specific treponemal proteins p , p . , p and p , and the quantitative determination of vdrl reaction on one nitrocellulose strip. results: treponema + vdrl virablot igg and igm tests confirmed the results for serum specimens positive for t. pallidum antibodies: reactive vdrl band and at least two clear bands from p , p . , p or p for igg treponemal antibodies or at least one clear band p , p or p for igm treponemal antibodies. the rpr reactive sera were vdrl virablot reactive, but treponema virablot specific antibodies negative. none of the remaining sera reacted in tests with the treponema + vdrl virablot assay. conclusions: the treponema + vdrl virablot assay combining the necessary serologic markers in one test strip (i.e. specific and non-specific treponemal antigens) is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques. the great imitator: syphilis with predominant acute severe hepatic involvement the ground of a significant increase of serum transaminiases (up to and u/l of gpt, respectively), frank jaundice, elevated bilirubin (up to . and . mg/dl, respectively), and evident rise of serum alkaline phosphatase and gamma-gt. clinical history did not show risk factors for viral hepatitis, and all laboratory examinations for major and minor hepatotropic viruses proved negative, as well as autoimmmune, dysmetabolic, or drug-induced liver damage. repeated ultrasonography detected increased liver and spleen dimensions, in absence of other abnormalities. in the second p, histopathologic study performed after liver biopsy, showed an aspecific cholestatic hepatopathy, a moderate granulocyte and lympho-mononocyte intralobular infiltrate, appreciable necrotic foci, and a mild portal fibrosis (knodell score ). only syphilis serology, performed despite the absence of significant history or clinical clues, allowed to recognize an igm positive serology associated with tpha titres ranging from : and : , respectively. specific chemotherapy (i.v. penicillin g at mu daily for days, followed by mu/day in the second p),led to a complete resolution of hepatic involvement, associated with an improvement of serology after month. discussion: while in the majority of episodes of syphilis liver involvement remains missed or subclinical, an early diagnosis of syphilis with predominant hepatic disease remains an unresolved problem of differential diagnosis, due to the rare occurrence of isolated organ involvement compared with typical syphilis signs and symptoms, and the aspecific clinical, histopathological, and imaging picture (jozsa l,acta hepatogastroenterol ; : - ; young mf, j clin gastroenterol ; : - ) . during the recent recrudescence of syphilis in italy, a diagnostic suspicion should be maintained by clinicians who face an apparent acute icteric hepatitis, and syphilis serology should be included among initial laboratory workout. the great imitator: syphilis with predominant meningoencephalitic features r. manfredi, s. sabbatani, d. pocaterra, f. chiodo (bologna, i) introduction: a significant recrudescence of syphilis was observed in recent years, not contained by prophylactic measures against hiv and other stds. meningeal and meningoencephalitic involvement may occur also in early stages of syphilis, thus posing problems of differential diagnosis. case reports: a young woman and an hiv-infected male developed a syphilis with predominant meningoencephalitis expression. in the first p, based on an isolated positive borrelia burgdorferi serology (later deemed as a cross-reaction), early ceftriaxone was started, due to a suspected neurological lyme disease. also after the diagnosis of neurosyphilis (on the ground of positive csf serology), antimicrobial therapy was left unchanged for weeks at our day-hospital facilities, until complete clinical-microbiological cure. a second, hiv-infected p was hospitalized owing to mild fever lasting weeks, associated with cephalalgia and anxiety.the favorable virological-immunological status (cd + count cells/ll and undetectable hiv viraemia, under an effective haart regimen), did not exclude all searchs for possible hiv-related disorders.although serum examination detected a potential latent syphilis (isolated positive serum treponema pallidum igg, tpha : ,igg-positive rpr, and mute history for syphilis), only csf examination disclosed an increased cell content ( /ll), altered brain-blood barrier indexes with increased intrathecal ig synthesis, and frank vdrl,tpha ( : ), and borderline t. pallidum igm serology.penicillin g at mu/day for days led to a slow resolution of neurological signs and symptoms, and a tendency to improvement of specific csf-serum syphilis serology during subsequent controls. discussion: focusing on differential diagnosis, a luetic etiology should not be underestimated, when facing young p suffering from a meningoencephalitis of unclear origin.our cases were characterized by a young age ( and years, respectively), when compared with usual mean age of tertiary neurosyphilis. in absence of suggestive history and other syphilis signs, the diagnosis was achieved only after the retrieval of elevated syphilis serology positivity on both csf and serum, together with some clinical signs, such as seizures, altered mentation, cognitive abnormalities, and anisochoria in the first p, and persisting headache and anxiety in the second p. our experience with ceftriaxone (started in the first p when neuroborreliosis was suspected), was favorable like that with high-dose i.v. penicillin g. comparison of the genomes of pathogenic treponemes, treponema pallidum ssp. pallidum, ssp. pertenue and treponema paraluiscuniculi objectives: the cervical human papillomavirus (hpv) infection is common among young sexually active women. because the cytology tests have some limitations, including up to % false-negative results, molecular techniques are increasingly used for identification of hpv-induced cell changes in the cervix. the polymerase chain reaction (pcr) for hpv diagnostics was successfully introduced recently in bulgaria. however limited data on specific prevalence and determinants of subclinical hpv infections are available. in this study we designed a multiplex primers pcr system for simultaneous identification of the most common low-and high-risk hpvs in women with cytologically normal cervical smears. materials & methods: dna from cervical cell samples from women aged between and years with normal pap smear tests was extracted by standard procedure and studied for hpv presence. six type-specific primer sets for e hpv gene were used in a single-tube reaction for detection and identification of . respective controls and statistic analysis were included. results: the overall hpv dna prevalence was . % ( / ). ten of the hpv positive women ( . %) were infected with high-risk (hpv- or hpv- ) virus types; eight ( . %) with low-risk types (hpv- or hpv- ), and six ( %) were infected with both high-and low-risk types ( hpv- /- ; hpv- /- or hpv- /- ). hpv prevalence among women younger than years was . % ( / ). for low-risk types the peak prevalence was observed in women between - years. besides age, there was a positive association between the detection rates of hpv infection and number of sexual partners and oral contraceptive use. conclusion: the used technique is simple, economic, rapid and reliable for screening studies of hpv infections. the prevalence of hpv in cytologically normal bulgarian women is similar to the reported in other countries. our findings suggest that molecular techniques for hpv detection might be useful as an adjunct to pap smear screening. were examined. mycoplasmas were determined by use of a commercially available system (liofilchem s.r.l, italy) and c. trachomatis antigen by dif (cellabs pty ltd, australia). results: of specimens, were positive. the isolated species in each age group are summarized in table . in cases of lower genital tract (lgt) infection, only one species was isolated. in all three age groups, mycoplasmas were the most commonly identified. in particular, the species of the isolated pathogens were: ( ) group a (n = ): mycoplasmas %, streptococcus spp %, candida spp %, c. trachomatis % and anaerobes %, ( ) in women of this group a mixed infection with three species was found. from women with positive cultures, ( . %) were symptomatic, while in asymptomatic women ( . %), sexually transmitted pathogens were identified. noticeably . % and . % of the infected women with candida spp and c. trachomatis respectively, presented no symptoms. conclusions: ( ) the rate of mixed lgt infections was . % ( ) asymptomatic infections were . % ( ) all teenagers in group a were symptomatic. ureaplasma mba size variants in woman with spontaneous preterm delivery j. spergser, a. witt, l. petricevic, p. husslein, a.m. hirschl, r. rosengarten (vienna, a) objectives: despite colonization of the lower urogenital tract, ureaplasmas reach the upper genital tract only in a subpopulation of women, and only in some of these individuals symptoms like preterm delivery ensue. while some studies indicated that certain serovars are more frequently implicated with particular diseases, others have suggested that size variability of the multiple banded antigen (mba) may contribute to ureaplasmal pathogenesis. to address these hypotheses, vaginal and placental samples from women with spontaneous preterm delivery were tested for ureaplasma species, serovars and mba size variants. methods: placenta/amnion specimens and corresponding vaginal samples from women that delivered via caesarean section less than weeks of pregnancy were collected. samples were examined for ureaplasma species and serovars by cultivation and pcr assays. to determine mba size variants, pcr with primers amplifying the non-repetitive and the c-terminal repeat region of the mba gene were performed. results: out of cases examined so far, placenta/amnion specimens and corresponding vaginal samples were positive for ureaplasmas by cultivation and pcr. while ureaplasma positive samples from the lower genital tract were carrying u. parvum ( %) and u. urealyticum ( %), colonization of the placenta/amnion was restricted to u. parvum which was only recovered from women with preterm labor or preterm prom. in contrast, ureaplasmas were not detected in samples from women with hellp syndrome, iugr and preeclampsia. as opposed to some previous studies, particular serovars were not implicated in adverse pregnancy outcome. however, significant differences between number and lengths of mba pcr products among placental and vaginal ureaplasma isolates were obvious. ureaplasma isolates from placenta/amnion membranes gave shorter pcr products than did corresponding vaginal isolates and while a majority of vaginal ureaplasma isolates were composed of multiple size variants, placental isolates were predominantly restricted to a singular mba size variant. conclusion: only u. parvum was found to be frequently present in the placenta/amnion of women with preterm delivery and certain serovars were not more frequently implicated with particular diseases. in addition, mba size variation represents an additional level of phenotypic and genetic variability beyond the serovar category which may provide an important contribution to ureaplasmal pathogenesis. use of kanamicin in the topical therapy of aerobic vaginitis g. tempera, g. bonfiglio, e. cammarata, s. corsello, a. cianci (catania, paternò, i) objectives: demonstrate the efficacy of the topical therapy with kanamicin in a new pathology called aerobic vaginitis (av). methods: eighty-one patients with clinical diagnosis of av in accordance to donders's criteria, have been included in the study. the presence at least of four of the following symptoms were taken as diagnosis of av: objective abnormal yellow vaginal discharge, foul smell (but negative to koh test), elevated vaginal ph (> ), abundant vaginal leukocytes and lactobacillary grade iia, iib and iii in accordance to donder's classification. other characteristics such as vaginal dyspareunia, vulva-vaginal itching, cervical erosion as well as the isolament of vaginal microorganisms were also documented. the patients were randomised treated, with kanamycin ( mg vaginal ovules per days, consecutively) whereas, with meclocycline ( mg vaginal ovules per days, consecutively). the patients were visited before starting the study, - days and days after the end of the study. results: at the first follow-up the patients showed different level of symptoms reduction. particularly, reduction of presence of leukocytes, burning of vaginal mucosa and itching were statistically significant in the group treated with kanamycin with respect to the group treated with meclocycline. moreover, there was also a reduction of isolament of enterobacteriaeae ( %) in the group treated with kanamycin versus % treated with meclocycline. at the second follow-up, vaginal homeostasis (normalisation of ph and presence of lactobacilli) was demonstrated only in kanamycin treated group. conclusion: our data suggested that the topic use of kanamycin could be considered a specific antibiotic for the therapy of this new pathology. objectives: establishing the identity of lactobacillus species colonizing the vagina of women is of importance, because clinical studies have demonstrated an association between the presence of h o -producing strains of lactobacillus and a decreased prevalence of bacterial vaginosis (bv). recently, culture based studies using molecular identification methods showed that l. crispatus and l. jensenii are the most common species of vaginal lactobacilli and that colonization by these species was positively associated with a lower frequency of bv. here were report that l. crispatus can be distinguished from other lactobacilli using gram staining of vaginal smears. methods: several approaches were used to characterize vaginal microflora samples obtained from pregnant women at three time points in pregnancy: /gram stained smears from vaginal swabs, scored according to modified ison and hay criteria; /identification of cultured isolates obtained after anaerobic culture, identified using tdna pcr; and /species specific pcr for atopobium vaginae and gardnerella vaginalis. grade i specimens, representing a normal microflora, were further characterized as grade ia when only l. crispatus cell types were present, grade ib when other lactobacillus cell types were present, grade iab when both l. crispatus and other lactobacilli were present and grade ic when either gram-positive rods, small and short, or irregularly shaped gram-positive rods, with clubbing, curved edges and irregular staining (Ôdiphteroid cell typesÕ) were seen. results: out of the samples, . % showed a normal vaginal microflora (grade i), . % were grade ii, . % grade iii and . % grade iv. based on the presence of different lactobacillus species, grade i specimens were further characterized as grade ia ( . %), grade ib ( . %), grade iab ( . %) and grade ic ( . %). this classification was supported by the finding that out of respectively . % and . % of grade ia and iab specimens l. crispatus was cultured while this species was present in only . % and . % of respectively ib and ic specimens. in addition, . % of grade ic specimens contained bifidobacterium spp. conclusion: further refinement of gram stain based grading of vaginal smears is possible by distinguishing additional classes of normal microflora. these categories of gram scores may facilitate and improve future studies regarding the interpretation of clinical data and therapeutic outcome. objectives: the importance of the isolation of gardnerella vaginalis in bacterial vaginosis is well known. the involvement in male urogenital infection is uncertain, probably due to low rate of isolation for inadequated specimens process. the objective of this study is to evaluate the possible pathogenic rol of g. vaginalis in male urogenital pathology. semen is a recommended specimen in diagnosing of prostatitis. at the same time it also reflects normal genital tract microflora. although its composition may be affected by the sexual activity, there are no data regarding the changes in genital tract microflora during the sexual debut. objective: to identify possible associations between sexual experience and the reproductive tract inflammatory parameters and microflora in healthy young men. methods: a total of healthy men aged - years were included and divided into groups based on their sexual experience (virgins n = , monogamous n = and polygamous n = ). a semen sample of each subject was assessed for basic semen parameters (semen volume, sperm concentration, sperm motility), white blood cell (wbc) counts and quantitative cultures for aerobic, microaerobic and anaerobic bacteria, and possible correlations between the type of sexual experience, wbc counts and seminal microflora were calculated. results: basic semen parameters were similar in all groups. . % of men had high (> m/ml) and . % of men had medium ( . - m/ml) semen wbc counts. there were no correlations between the type of sexual experience and semen wbc counts. in comparison to sexually active men in virgin subjects the total microbial counts in semen ( . vs . log cfu p < . ) and the number of different species ( vs , p ¼ . ) were lower, however, no differences between mono-and polygamous men were found. there was a positive correlation between the total microbial concentrations and number of different species (r = . ; p < . ). staphylococci, corynebacterium seminale, other coryneforms, gamma-and alpha-haemolytic streptococci and peptostreptococci were the most prevalent organisms. no significant differences in microflora were observed between the groups yet the gram-negative anaerobic rods were more often seen in sexually experienced men than in virgins. the subjects with high wbc counts (> m/ml) tended to have higher number of different microorganisms than the rest of the men (median vs , p = . ). the sexual debut is associated with the enrichment of seminal microflora but not with the influx of wbc or changes in basic seminal parameters. strong positive correlation can be observed between the total microbial concentration and the number of different species in semen. sexually transmitted infections among registered female sex workers in athens under investigation were sexually active non-pregnant women aged from to years with diagnosed cervicitis by cytological examination and presence of muco-purulent discharge. fifty five women were included in studied group -with confirmed c. trachomatis cervicitis and women -in control group (with non-chlamydial/non-gonococcal cervicitis). bv was diagnosed using amsel and nugent ( - negative; - intermediate; - positive) criteria. by using amsel criteria bv was suggested in % and % correspondingly among women in studied and control group. by using nugent criteria bv was suggested correspondingly in . % and %. these differences were not statistically significant and can be explained by the presence of muco-purulent discharge among studied women. objectives: helicobacter pylori is recognized as an important human pathogen but the differentiation of the species by classical phenotypic methods is really difficult. therefore the objective of this study was the development of an identification method for helicobacter species based on pcr-restriction fragment length polymorphism (pcr-rflp) analysis of the s and s rrna genes. objectives: since the first identification of helicobacter pylori by warren and marshal in , members of the genus helicobacter increased to more than species, which have been detected in human and animals. the construction of species specific pcr assays is difficult due to the close relatedness between different helicobacter species. the pcr-dgge technique was developed for the identification of helicobacter colonization. the aim of this study was to investigate the effect of multiple regions of the rrna gene on the diagnostic efficiency pcr-dgge. methods: dna was extracted from helicobacter strains, which represent helicobacter species. amplification of . kb of helicobacter s rdna, which contains v and v - regions, was done using previously published helicobacter genus specific primers c and c . the pcr product can be used as a template for two helicobacter genus pcr assays, the v - regions s rdna was amplified using the primers fc and rc , the v- region was amplified using the primers bsf and bsr published at (http://rrna.uia.ac.be/ primers). dgge analysis of the v and v - regions was performed on % polyacrylamide gels containing urea and formamide gradient from - % or - %, respectively. electrophoresis was performed in a dcode electrophoresis unit (biorad) at constant voltage ( v) at °c for hours. results: dgge analysis of two regions of helicobacter srrna gene showed mobility patterns that allowed discrimination of most helicobacter species except those which are closely related such as h. pullorum-h. pametensis, h. ganmani-h. rodentium and h. bizozeroni-h. felis-h. salmonis. conclusions: helicobacter species are widely distributed in the gastrointestinal tract of mammals, birds and other animals. the pcr-dgge technique has proven to be an easy, inexpensive and efficient tool for the identification of helicobacter species and for the detection of colonisation by more than one helicobacter species, without the need for species-specific pcr assays. in addition, the diagnostic efficiency of this technique was increased by analysis of multiple regions of the s rrna gene. helicobacter spp. in chronic pancreatitis, pancreatic adenoma and pancreas cancer objectives: helicobacter spp. efficiently colonise various hostile habitats such as the stomach, colon and biliary tract of animals and man. with the exception of h. pylori, many helicobacter spp. are difficult to culture. nucleic acid based detection has thus become a method of choice for analysis of helicobacter spp. in chronic inflammatory gastrointestinal (gi) tract diseases and cancers. pancreatic exocrine cancer, with few established risk factors and very poor prognosis, is a common cause of cancer death. previously, serological studies found an association between h. pylori and pancreas cancer (pc). methods: pancreatic tumour specimens of patients with pc (n = ), tissues of chronic pancreatitis (n = ), pancreatic adenoma (n = ), and benign pancreatic tissue of patients with cancers of the choledochus, colon and duodenum (n = ), were analysed by semi-nested helicobacter-specific s rdna pcr. stomach (n = ) and gallbladder (n = ) specimens of the pc-patients were also analysed. were characterized by dna-sequence analysis. results: the helicobacter spp. pcr was positive in of ( . %) pancreas cancer patients, and in the pancreas in of ( . %) patients with chronic pancreatitis. adenoma and pancreas tissues of other gi-tract cancers, as well as gallbladder specimens, were all negative. four of ( . %) stomach samples of the pc-patients were helicobacter positive with sequences homologues to h. bilis by blastn analysis. dna-sequencing of pcr-products amplified in pcs were closely related to h. pylori (n = ), h. flexispira sp. (n = ) and h. cinaedi (n = ). two pancreatitis pcr-products matched h. pylori. conclusions: helicobacter spp. were common in pc compared with benign pancreatic tissue. helicobacter pylori infection and low igg immunoglobulin s. kokkinou, k. pavlou, a. varouta, j. villiotou, k. papaefstathiou, a. moutsopoulou, e. papafrangas (halandri athens, athens, gr) it is established that helicobacter pylori (hp) may cause haematological disturbances such as iron malabsorption and idiopathic thrombocytopenic purpura (itp). the aim of this study is to show that hp is involved in human disease in a more complicated way, producing an abnormal immunologic profile. patients and methods: this work reports the presence of a significant low value of igg immunoglobulin in pts suffering from unidentified iron deficiency (id)or iron deficiency anemia (ida coexisting with megaloblastic anemia(ma), in leukopenic pts and in more pts having thrombocytosis, itp and polycythemia respectively. the ratio male/female was / , and their age ranged from - years. in the blood examination there was a -fold titre of hp-igg type antibody in all, while in there was also a high titre of hp-iga type using elisa technique. the histopathological examination of an antral biopsy showed diffuse corpus gastritis, atrophic gastritis and achlorydria secondary to hp infection that take place in gastric area. results: pts had unidentified id or ida coexisted with megaloblastic anemia. serum iron, transferrin saturation, serum ferritin and b levels were found to be significantly low. all of them had also hp infection. their anemia existed for more than years, was unresponsive to treatment and recurrent in nature. twelve pts with undefined eukopenia (l: . · /l) lasting for at least years, had also an hp infection. all of our pts had a significantly low value of igg immunoglobulin. mean value ranged from to mg/dl(normal value: - mg/dl). they subjected to eradication therapy. all but one responded well. conclusions: ( ) hp infection was found to coincide with id, ida, ma and leukopenia. ( ) the normalization of haematological parameters after eradication started months later. ( ) the essential mechanism between the hp infection and low igg is unclear and its value remains low and after the eradication therapy. ( ) studies with larger samples should be done for a better evaluation of the hp infection and it's involvement in human disease. ( ) hp infection acts in a catabolic way for the humans. it must be considered to search for it in haematological conditions of undefined origin. helicobacter pylori from the dyspeptic patients in thailand: diagnosis, antimicrobial susceptibility and correlation to clinical outcomes c. chomvarin, p. kulsuntiwong, p. mairiang, c. kulabkhow (khon kaen, th) objectives: to evaluate methods for routine clinical diagnostic use of helicobacter pylori infection of gastric biopsies, to study the correlation of h. pylori infection to the clinical outcomes and to study antimicrobial susceptibility by disk agar diffusion in thailand. methods: gastric biopsies, obtained from patients underwent at the endoscopy unit of srinagarind hospital, faculty of medicine, khon kaen university, were diagnosed by culture, rapid urease test (rut, pronto dry) and histological examination. a true positive criteria was indicated when culture or both rut and histology tests were positive. one hundred and fifteen isolates of h. pylori isolates were tested for six antimicrobial agents by disk agar diffusion method. results: the h. pylori infection rate was . % ( / ). the sensitivity vs. specificity of the culture, rut and histology were . % and %, . % and . %, and . % and . % respectively. the prevalence of h. pylori in gastritis (gt), duodenal ulcer (du) and gastric ulcer (gu); and gastric cancer (gca) patients were . %, . % and . %, respectively. the chi-squared test showed that gca patients were significantly more often infected with h. pylori than gt patients. the resistance of h. pylori isolates to single antimicrobial agent as metronidazole (mtz), clarithromycin (clr),ciprofloxacin (cip), amoxycillin (aml), and tetracycline (te) were detected in . , , . , . and percent, respectively. the combination resistance to mtz & clr, mtz & cip, mtz & aml, mtz & te; and clr & cip were detected in . , . , . , . and . percent, respectively. the . per cent of h. pylori isolates were resistance to three or more antimicrobial agents. conclusion: rut method was highly sensitive and specific and appropriate for routine laboratory use. the correlation of the gca patients to h. pylori infection was significant difference compared to gt patients. the high resistance rate to metronidazole indicated that the effective for eradication of h. pylori by metronidazole should be considered in the clinical management of h. pylori infection. use of an indirect immunofluorescence test for the detection of caga in h. pylori c. scherer (essen, d) objectives: the cytotoxin associated gene (caga) of h. pylori is a frequently discussed virulence factor which codes for a -kda cytotoxin associated antigen (caga). the caga-gene is usually detected by pcr methods. publications about phenotypic methods for the detection of caga are rare. aim of our investigation was to compare a slightly modified, recently published indirect immunofluorescence test (iift) for the detection of caga with commonly used pcr assays. methods: h. pylori atcc (caga+/caga+) was used as positive control strain, atcc (caga+/caga+) as negative control strain. nineteen clinical isolates of h. pylori were examined for their caga-and caga-status. caga was detected by iift using monoclonal anti-caga-antibody from mice, which were visualised for immunofluorescence microscopy with fitc-conjugated anti-mouse-antibody from rabbits. dna was extracted by a standard phenol-chlorophorm procedure after proteinase k treatment of the cells from a -day old culture. primer pairs and their published protocols were used: f /b and d /r as the most described primer pairs and further five primer pairs. all tests were performed in duplicate. results: from ( %) strains were caga positive in the iift, and also caga positive in each pcr. from ( %) strains were negative in the iift. from these were caga-negative in each pcr and showed varying results in the pcr-assays. from ( %) strains could not be interpreted by iift, both showed varying results in the pcr-assays. the iift might be a simple to use tool in the determination of the caga-status, since it showed no false positive result in neither tested pcr. the differing results in iift and pcr suggest that genotypic positive strains may not always express caga in vitro and maybe also not in vivo. therefore the caga-iift may be an interesting parameter for the determination of strain virulence in addition to pcr-assays. evaluation of elisa serology for the primary diagnosis of helicobacter pylori infection in turkish dyspeptic patients: a prospective study from a developing country b. kocazeybek, y. erzin, s. altun, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: hp is recognized as an important human pathogen by virtue of it's association with peptic ulcer disease, gastric cancer and gastric lymphoma and the high prevalence of infection worldwide. both invasive and noninvasive tests are available to diagnose hp infection but there is still no single gold standard. the aim of the present study was to evaluate the diagnostic accuracy of a commercially available anti-hp igg elisa kit for the primary diagnosis of hp infection. materials and methods: a total of patients who were referred to our endoscopy unit were included. according to our gold standard, a patient was classified as being hp(+) if the culture and/or both histology, rapid urease test were positive and as hp()) only if all of these tests remained negative. standard methods were used to calculate sensitivity, specificity, predictive values of positive and negative results and % confidence intervals of these values. results: after excluding patients with discordant results of patients ( %) were hp (+). the sensitivity, specificity and diagnostic accuracy of the igg-elisa were %, %, % respectively. conclusion: due to it's lack for specificity, we conclude that quantitative anti-hp elisa test may not be a suitable alternative for primary diagnosis of hp in our country, where the prevalence of infection is still very high. comparison of two different stool antigen tests for the primary diagnosis of helicobacter pylori infection in turkish dyspeptic patients b. kocazeybek, y. erzin, s. altun, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: to assess the reliability of two different enzyme immunoassays in detecting the helicobacter pylori(hp) status in stool specimens of turkish dyspeptic patients. materials and methods: patients [ with non-ulcer dyspepsia(nud), with duodenal ulcer(du), with gastric cancer]who were admitted to the endoscopy unit of istanbul university, cerrahpasa medical faculty for upper gastrointestinal endoscopy due to dyspepsia were enrolled in the study. hp infection was confirmed in all patients by histology, rapid urease test(rut) and culture. a patient was classified as being hp-positive if the culture alone or both histology, rut were positive in the absence of a positive culture and as negative only if all of these tests remained negative. stool samples of patients were obtained in order to assess the reliability of a monoclonal (femtolab h. pylori, connex, martinsried, germany) and a polyclonal (premier platinum hpsa, meridian diagnostics inc., cincinnati, usa) stool antigen test and to compare their diagnostic accuracies. the v test was used for statistical comparison of the values. results: using a cutoff . for femtolab h. pylori and . for premier platinum hpsa the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy of the former and latter tests were %, %, %, %, % and %, %, %, %, % respectively. the sensitivity, specificity, npv and diagnostic accuracy of the former test were significantly superior to the latter one's (v = . ; p < . for sensitivity and v = . ; p = . for specificity, v = . ; p = . for npv and v = . ; p = . for diagnostic accuracy). the bacterial load did not affect the sensitivity of both tests. conclusions: the monoclonal femtolab h. pylori, using a cutoff . is a very sensitive, specific and easy to perform diagnostic tool for the primary diagnosis of hp infection in turkish, dyspeptic patients. evaluation of two different enzyme immunoassays for detection of helicobacter pylori in stool specimens of turkish dyspeptic patients after eradication therapy b. kocazeybek, y. erzin, s. altun, s. saribas, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: to assess the reliability of two different enzyme immunoassays(eias) in detecting the hp status in stool specimens of turkish, dyspeptic patients in the post-treatment period. materials and methods: patients with non-ulcer dyspepsia(nud) who were positive for hp underwent a one week regimen of triple therapy. stool samples of patients were obtained - weeks and c-urea breath test(ubt) was performed weeks after eradication therapy in order to assess the reliability of a monoclonal (femtolab h. pylori, connex, martinsried, germany) and a polyclonal (premier platinum hpsa, meridian diagnostics inc., cincinnati, usa) stool antigen test and to compare their diagnostic accuracies. the v and fisher's exact tests were used for statistical comparison of the values. results: using a cutoff . for femtolab h. pylori and . for premier platinum hpsa the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy of the former and latter tests were %, %, %, %, % versus %, %, %, %, % weeks and %, %, %, %, % versus %, %, %, %, % weeks after completion of eradication therapy, respectively. both at the nd and th weeks in the post-treatment period the diagnostic accuracy of femtolab h. pylori was significantly superior to premier platinum's ( % versus %, v = . ;p = . , and % versus %, v = . ; p = . respectively). conclusions: the monoclonal femtolab h. pylori, using a cutoff . is sensitive and specific enough to monitor hp infection in turkish, dyspeptic patients weeks after completion of eradication therapy. carriers develop serious gastroduodenal diseases. so, it is important to define whether strains with specific genotype are associated with the clinical outcome. the aim of this study was to determine the distribution of the caga, cage and vaca subtypes of hp in patients with various gastroduodenal diseases in turkey, and to explore the association between genotype and the clinical outcome of infection. methods: strains of hp were isolated from cultures and pathological archives of patients with non-ulcer dyspepsia (nud), with duodenal ulcer(du) and with gastric carcinoma. the caga, cage and vacas a/s b, m /m genotypes were determined by polymerase chain reaction methodology. results: there were no statistically significant difference among three different clinical outcomes according to the positive rates of vaca s b, s and vaca m /m . but the positive rates of vaca s a, caga and cage were . % ( / ), % ( / ) and . % ( / ) in nud; . % ( / ), . % ( / ) and . % ( / ) in du; . % ( / ), . % ( / ) and . % ( / ) in gastric cancer group, respectively, showing statistically significant difference (p = . , p = . , p = . , respectively). the prevalence of vacas and m in nud patients was higher than that in duodenal ulcer group, but this difference was not statistically significant. conclusions: helicobacter pylori caga, cage and vacas a genotypes have a significant relation with duodenal ulcer and gastric carcinoma in turkish population. there were no statistically significant association of the vaca s b, s and vaca m /m strains among disease groups. clinical relevance of icea , icea and baba genotypes of helicobacter pylori in turkish patients with non-ulcer dyspepsia, duodenal ulcer and gastric cancer b. kocazeybek, y. erzin, v. koksal, s. erdamar, s. altun, a. dobrucali, a. oner (istanbul, tr) objectives: the clinical relevance of the helicobacter pylori(hp) adherence factor, baba gene, and the other virulence determinant, icea gene, has not yet been determined in turkish clinical isolates to date. therefore, the aim of this study was to evaluate the prevalence of icea , icea and baba hp genotypes in turkish patients with non-ulcer dyspepsia(nud), duodenal ulcer(du) and gastric cancer. methods: strains were isolated from patients with nud, were isolated from individuals with du and were isolated from patients with gastric cancer. bacterial dna was extracted from cultures and hp positive paraffin-embedded biopsy samples and genotyping of icea and baba was carried out utilizing polymerase chain reaction molecular technique by using specific primers. results: the icea , icea and baba genotypes were detected in . % ( / ), . % ( / ), . % ( / ) respectively, of the hp strains studied. the prevalence of icea in gastric cancer patients was higher than that in nud group, . % ( / ) and % ( / ), respectively (p = . ). the prevalence of icea in nud group was higher than that gastric cancer patients, % ( / ) and . % ( / ), respectively (p = . ). the positive rates of baba were . % ( / ) in nud; . % ( / ) in du and . % ( / ) in gastric cancer group, showing statistically significant difference (p = . ). conclusions: baba genotype distribution was evaluated in different gastric pathologies in turkish population and was a good marker for the presence of du and gastric adenocarcinoma. thus, our current results confirm previous experimental studies indicating a central role of hp's adhesin and lewis antigens in the pathogenesis of ulcer disease and adenocarcinoma. influences of interleukin b and interleukin rn polymorphisms on the development of non-ulcer dyspepsia, duodenal ulcer and gastric cancer in turkish population b. kocazeybek, y. erzin, v. koksal, s. erdamar, s. altun, a. dobrucali (istanbul, tr) objectives: il- b and il- rn gene polymorphisms (which encode interleukin b and interleukin- receptor antagonist, respectively) have been associated with development of gastric atrophy and with increased risk of gastric carcinoma. the aim of this study was to determine the influences of host il- b - tc, il- b - ct and il- b rn gene polymorphisms on the development of various gastroduodenal pathologies in helicobacter pylori (hp) positive turkish patients [( with nonulcer dyspepsia (nud), with duodenal ulcer (du) and with gastric cancer (gc)]. methods: genomic dna was extracted from hp positive paraffin embedded gastric biopsy samples of nud, du and gc patients. il- b and il- b rn gene polymorphisms were analysed by polymerase chain reaction-restriction fragment length polymorphism. these polymorphic sites include promoter regions of il- b at positions- (c-t transition) and - (t-c transition), and il- rn variable tandem repeats (intron vntr). results: there were no statistically significant difference among three clinical outcomes in the frequencies of il- b - ct + tt and il- b - tc + cc genotypes (as called proinflamatory genotypes) (p = . and p = . , respectively). the prevalence of the proinflamatory il- b rn / + / alleles in gc group was higher than that in nud and du groups. the frequencies of il- b rn / + / alleles were . % ( / ) in nud; . % ( / ) in du; and . % ( / ) in gc group, showing statistically significant differences (p = . ). conclusions: there were no statistically significant difference among three clinical outcomes in the frequencies of il- b - ct + tt and il- b - tc + cc genotypes, whereas proinflamatory il- b rn * allele ( / + / ) was associated with gastric cancer in turkey. rapid detection of clarithromycin-resistant helicobacter pylori in patients with dyspeptic by fluorescent in situ hybridisation (fish), in comparison with e-test m. moosavian, s. tajbakhsh, a. samarbafzadeh (ahwaz, ir) objectives: isolation of h. pylori from more than % of the patients with peptic ulcer have demonstrated a strong relationship between these bacteria and created ulcers or dyspeptic. recovery of the patients will be accelerated, if clarithromycin be added to therapeutic protocol. the objectives of this study are: -rapid detection of the susceptible or resistant strains of helicobacter pylori to clarithromycin, in patients with dyspeptic by fish technique. -comparison of the results of fish & epsilometer test ( e-test) techniques. methods: frozen -sections of gastric biopsies of patients with dyspeptic were hybridized in situ by fluorescent oligonucleotide probes (fish). the prepared slides were examined under a fluorescent microscope, after staining with dapi. also, susceptibility and resistance of isolated strains of h. pylori to clarithromycin were determined by e-test. both results of e-test and fish techniques were compared in final. results: out of examined gastric biopsy samples were positive for h. pylori by fish. out of these h. pylori strains, strains ( %) were susceptible, strains ( %) were resistant and strains ( %) were mixture of susceptible and resistance strains to clarithromycin. this study showed no significant different between fish and e-test results, in view of number in susceptible or resistance strains. conclusions: since the patient gastric biopsies are not routinely cultured in the most of clinical laboratories, also, for isolation of h. pylori, it is needed to enriched and selective media and the other way, clarithromycin is an expensive drug, so, it looks like that fish technique is a suitable method for replacing of the culture, antibiotic sensitivity test and or e-test method. diagnosis of atrophic gastritis from a blood sample p. douramani, s. mavrea, a. arkas, a. adamopoulos, e. anastasakou (athens, paros, gr) introduction: the majority of gastritis is related to infectious agents, where h. pylori is the most important and common. h. pylori gastritis could proceed, over time, to atrophic form of gastritis a serious disease that increases the risk of peptic ulcers and gastric cancer. a new test panel was developed for nonendoscopic diagnosis of atrophic antrum and corpus gastritis from a blood sample. aim: to investigate whether atrophic gastritis can be diagnosed and typed non-endoscopically if the serum levels of pepsinogen i (s-pgi) and gastrin- are assayed in connection with h. pylori testing. materials and methods: the study population consists of selected dyspeptic outpatients with (cases) or without (controls) advanced (moderate or severe atrophic gastritis) who underwent a diagnostic gastroscopy for dyspeptic symptoms. of the selected patients, (cases) had an advanced atrophic gastritis or a resected stomach. of these cases, had an advanced antrum-limited atrophic gastritis, had resected antrum, had a corpus limited advanced atrophic gastritis. two patients had an advanced atrophic gastritis in both the antrum and corpus (multofocal atrophic gastritis) and the whole stomach was removed in one patient. the controls comprise patients of whom had a non atrophic h. pylori gastritis; the antrum and corpus were normal and healthy in patients. the sample for ÔpostprandialÕ gastrin - (s-g- prand) was taken min after a protein drink. the s-g- ,s-pgi and igg class antibodies to h. pylori were determined using elisa methods. results: a low s-pgi (< mg/l) was found in of patients ( %) with and in of patients ( %) without corpus atrophy. a low s-g- prand (< pmol/l) was found in all patients with h. pylori associated antral-atrophy and in of patients ( %) with resected antrum but in one of patients ( %) with h. pylori-related non-atrophic gastritis. the mean values of both s-g- prand and s-pgi decreased with increasing grade of antral and corpus atrophy respectively. among all patients with atrophic gastritis, of patients ( %) had a low s-pgi and/or a low s-g- prand with positive igg h. pylori antibodies. such low values were found only in one of the ( . %) control patients. conclusions: low serum levels of g- and s-pgi are diagnostic markers of atrophic antral and corpus gastritis, respectively. a low s-g- prand is a sigh of the multifocal or antrum-limited atrophic gastritis in patients infected with h. pylori. helicobacter pylori in gastric mucosa: an ultrastructural study e. ozbek, a. ozbek, h. dursun, o. vuraler (erzurum, tr) objectives: helicobacter pylori are extraordinary among bacteria in their ability to colonize the human gastric mucosa, an inhospitable acidic environment, and to stay on there for long periods as decades in despite of host immune and inflammatory responses. in this study, we aimed to research where h. pylori are found within the stomach by transmission electron microscopy (tem). methods: gastroscopic biopsy samples from patients suspected with gastritis or peptic ulcer were processed for both of pcr and tem. the presence of h. pylori in biopsy samples was investigated with pcr method by using specific s ribosomal rna primers. the samples for electron microscopic examination were fixed in % glutaraldehyde, postfixed in % osmic acid, dehydrated in acetone, and embedded in araldite. plastic blocks of positive samples for the presence of h. pylori confirmed by pcr were cut with a nova lkb bromma ultratome. thin sections were stained with uranyl acetate and lead citrate, and examined by using a jeol sx transmission electron microscope. results: we observed that cross-and longitudinal-sectioned h. pylori within the mucus layer overlying gastric epithelium, in the intercellular spaces between gastric epithelial cells (figure ), and between two microvilli protruding from the apical surface of an epithelial cell into the gastric lumen. apart from the extracellular h. pylori, we found also intravacuolar h. pylori within the epithelial cells of the gastric mucosa. we observed h. pylori engulfed by pseudopod-like structures at the apical part of the epithelial cells. there were also late endosomal vacuoles containing h. pylori within the deeper cytoplasmic parts of the cell. conclusion: although h. pylori is considered generally a noninvasive pathogen, our electron microscopic observations prove that h. pylori is able to invade gastric epithelial cells, and to enter large cytoplasmic vacuoles. that h. pylori is able to be found intracellularly might explain why the eradication of h. pylori infection is difficult with the antibiotics such as gentamicin that cannot easily cross the eukaryotic cell membrane. abbreviations for figure : h, helicobacter pylori; n, nucleus of a gastric epithelial cell; e, cross-sectioned epithelial cells; nm, nuclear membrane; is, intercellular space. bar = . micrometre helicobacter pylori and association with histological findings in endoscopic biopsies in western greece c. petropoulos, e. jelastopulu, m. kardara, s. spiropoulos (erymanthia, patras, gr) objectives: there is evidence that helicobacter pylori (h. pylori) infection is strongly associated with gastric and duodenal lesions in general population. in this study we reviewed gastroscopic and histological records of patients who underwent gastroscopy for multiple reasons in a tertiary hospital in order to evaluate the frequency of h. pylori colonization in biopsy specimens and to examine the association between h. pylori infection and histological findings. methods: the medical records of patients who presented to the general hospital Ôagios andreasÕ in patras, western greece, during the period - for upper gastrointestinal complaints and other specified symptoms were reviewed for the presence of h. pylori. all patients have been submitted to endoscopy with biopsy mainly of the gastric mucosa. the statistical analysis was performed using the spss. results: gastroscopies and biopsies were undertaken in patients ( men, women; mean age . , range - years). in ( . %) of the patients dyspepsia was the main indication for gastroscopy, followed by ( . %) patients with anaemia, ( . %) patients with black faeces and ( . %) patients with reflux. usual macroscopic findings were oedema ( %), hyperaemia ( %), erythema ( . %), ulcer ( . %) and erosions ( . %). the histological examination revealed chronic gastritis in ( . %) subjects (in % of mild form, in % of moderate and in % of severe form), atrophic chronic gastritis in ( . %) and adenocarcinoma in ( . %) patients. the overall colonization of h. pylori was detected in ( . %) subjects. in case of h. pylori positive patients, chronic gastritis was found in . % of mild form, in . % of moderate and in . % of severe form, whereas in absence of h. pylori infection the histological type of chronic gastritis was . %, . % and . % respectively. in case of chronic gastritis of severe form h. pylori colonization was found in % (p < . ), whereas regarding the mild form h. pylori presence was observed only in % (p < . ). conclusions: the study reveals that h. pylori infection is strongly associated with the severe form of chronic gastritis. thus, for the optimal management and treatment of the patients with gastritis a biopsy of the mucosa appears to be necessary. objective: the ultimate aim of the study is to explore potential natural products as alternative for treatment of infections from staphylococcus aureus including methicillin resistant staphylococcus aureus (mrsa). the specific objective of the paper is to assay for molecular changes in the genes of mrsa isolates upon inhibition by natural products. five genes of mrsa isolates study include meca, meci and mecri adab and sav and the non-mrsa isolates were adab and sav genes. the adab gene encodes for methyltransferase activity for dna repair mechanism while the sav is the cell wall protein gene. methods: mrsa and non-mrsa isolates were obtained from patients receiving treatment in malaysian hospitals. these isolates were subjected to growth in methanol extract of marine seaweed. inhibition assay of extract on isolates including several genera of the gram-negative bacteria, were determined by the disc diffusion and minimum inhibitory (mic) methods. treated and untreated isolates inhibited by extract were subjected to pcr amplification, followed by commercial sequencing, rt-pcr assay of the mrna followed by sequencing of the cdna and blastn analysis with the genbank sequences. results: the inhibition assay of methanol extract showed activity only in mrsa and non-mrsa isolates but not in gram negative isolates. the nucleotide sequences changes were seen in four genes of treated mrsa isolates which were the meca, mecri, meci and the adab. the nucleotide changes in non-mrsa and mrsa were only seen in the adab gene but not the sav . the blastn analysis showed variation in nucleotide changes in all genes involved in mrsa phenomenon. conclusions: these preliminary results utilizing genomics for study on nucleotide sequences of pathogen can aim at utilizing the biomolecules from natural products to target the affected nucleotides so as to inhibit growth of the organism. the research activity has the potential of speeding up drug discovery programme and the nucleotide changes in several genes treated with the extract indicates the potential target sites of the extracts. the inhibitory effect of extract on the nucleotide of some genes remained unchanged in the pcr and rt_pcr assay, indicating selective effect of extracts on the genes and the extracts has the potential to be applied as antibacterial agents. implication of the findings is directed towards discovery of antibacterial drug target sites, which warrants further study. phenotypic and genotypic characteristics of staphylococcus aureus strains defective in species-specific proteins a. luczak, j. krzyszton-russjan, k. nowak, w. hryniewicz (warsaw, pl) objectives: the objective of this study was to characterise phenotypically clumping factor and/or coagulase defective s. aureus. methods: the study was performed on s. aureus isolates identified between and as clumping factor (cf) or free coagulase (fc) defective by clinical microbiology laboratories and submitted to national institute of public health in warsaw for verification. reidentification included detection of clumping factor, coagulase, thermonuclease, and ability to ferment mannitol. antimicrobial susceptibility testing was performed by diskdiffusion method according to the nccls. glycopeptides susceptibility was determined by screening method, mic evaluation and population analysis. pcr reactions were performed to confirm the presence of species specific genes, as cfa, cfb, coa, nuc, spa. to obtain isolates clonality, multiple-locus variable-number tandem repeat analysis (mlva) was applied. results: based on the phenotypic reidentification methods, ( %) isolates were phenotypically defective in respect to clumping-factor or coagulase production. all but one isolate exhibited the presence of the genes encoding cf or fc. seventyfive ( %) isolates were resistant to methicilin (mrsa) and multiresistant. twenty-six ( . %) isolates showed reduced glycopeptides susceptibility in pap. all hvisa and hgisa isolates were mrsa and all were defective mainly in clumpingfactor production. thirty-one different mlva groups were distinguished. the a and l groups were the most common and variable. the a type was the most prevalent in hvisa/hgisa isolates and found in centres. conclusion: the clonal spread of s. aureus defective in species specific proteins was revealed. the correlation between decreased susceptibility to glycopeptides and defective species specific proteins was observed. detection of panton-valentine leucocidin and other staphylococcal toxins using oligonucleotide arrays s. monecke, r. ehricht (dresden, jena, d) objectives: the recent outbreaks of community acquired mrsa (cmrsa) harbouring panton-valentine leucocidin (pvl) caused serious concern worldwide. in order to screen clinical isolates, we designed and tested an oligonucleotide array which can detect both components of this virulence factor, six staphylococcal superantigens and the two exfoliative toxins as well as several resistance genes and species-specific controls. methods: twenty-two clinical isolates (two suspected cmrsa and twenty randomly selected) from swabs obtained from a dermatological clinic in saxony/germany were cultured. dna was isolated and subjected to a linear multiplex amplification incorporating biotin-labeled dutp into the amplicon. hybridization of the amplicons to the array was detected using an enzymatic precipitation reaction. results: in two cases of chronical furunculosis, pvl-positive cmrsa were identified. one case was a soldier returning from a peace mission to kosovo where he apparently contracted the disease, the other one was his spouse. in three other cases of furunculosis/abscesses, pvl-positive but methicillin-susceptible s. aureus were found. staphylococcal enterotoxins were found in four, toxic shock syndrome toxin in three isolates, and exfoliative toxin a in one isolate. in two cases, multiple toxins were found (pvl + tst + entc and entb + entk + entq + eta). conclusions: while epidemic cmrsa strains often can presumably be identified due to their resistance profile (methicillin, tetracycline, fusidic acid), there is no easy method to detect pvlpositive, methicillin-susceptible s. aureus. therefore, strains carrying pvl are more common than previously suspected. the high prevalence of this virulence factor as well as of other toxins found in the present study warrants further investigations. characterisation of methicillin-resistant staphylococcus aureus harbouring the panton-valentine leucocidine a. anders, m. kaase, s. friedrich, s.g. gatermann (bochum, d) objectives: until now methicillin-resistant staphylococcus aureus (mrsa) have been known only as a major problem in hospitals. lately, a new kind of mrsa has been described. this mrsa is responsible for community-acquired infections (c-mrsa) and harbours the determinant for the panton-valentine leucocidine (pvl).as there are descriptions of c-mrsa possessing the pvl throughout the world with different mlst types we wanted to characterize eleven c-mrsa from our area possessing the gene for panton-valentine leucocidine. methods: the isolates were found from until the summer of , the gene encoding pvl was verified by pcr.we determined the pfge pattern, the mlst type, the sccmec type and the agr allele type of the eleven isolates. results: so far all isolates belong to the st which has been also described elsewhere in germany and in france but differs from the types found in the us ( and ) or in australia ( and ). they possess a sccmec element of type ivb and the agr allele type. all isolates were resistant to oxacillin, which was determined by meca pcr, and all were resistant to fusidic acid. only two were additionally resistant to erythromycin. no resistance could be found to fluoroquinolones.the average age of patients was years and they all had severe communityacquired abscesses of the skin or soft tissue that caused hospital admission. conclusion: mrsa cannot be longer considered as affecting only hospitalized and elderly patients but we should also be aware of them in community-acquired infections. accessory gene regulator (agr), and its locus of s. aureus is a quorum-sensing gene cluster of five genes (hld, agrb, agrd, agrc, and agra) that upregulates production of secreted virulence factors, including the alpha-, beta-, delta-hemolysins, and downregulates production of cell-associated virulence factors. s. aureus strains can be divided into major agr groups (agr i-iv) on the basis of a polymorphism in agrd and agrc. the purpose of this study is to know the proportion of agr i, ii, and iii polymorphisms and to compare clinical characteristics between group ii and non-group ii polymorphism of methicillin-resistant staphylococcus aureus (mrsa) strains in a tertiary care teaching hospital in korea. methods: isolates were identified as s. aureus by conventional methods. susceptibility to methicillin was determined by the nccls guideline. agr locus was identified using restriction fragment length polymorphisms (rflps) analysis of agr bdc amplicons with drai digestion. the factors assessed included the patients' demographics, comorbidities (diabetes mellitus, congestive heart failure, peripheral vascular disease, dialysisdependent renal failure, cirrhosis, malignancy, and alcoholism), infection site (central catheter-related bacteraemia, bacteraemia of unknown origin, device, endocarditis, intraabdominal, respiratory, skin, urine, and ear), receipt of mechanical ventilation and operation, the presence of nosocomial infection and treatment failure, creatinine level, and mortality. results: a total of strains from mrsa-infected patients ( males and females) were evaluated. their mean age was . ± . years old. there were ( . %), ( . %), and ( . %) strains in agr group i, ii, and iii polymorphism, respectively. only nosocomial infections were statistically significant clinical parameter according to univariate analysis (p = . ) and multivariate analysis (or, . ( . ) . ); p, . ). conclusion: agr group ii was most prevalent in this study and nosocomial infections were correlated with group ii polymorphism. this result suggests virulence and nosocomial spread of mrsa strains are originated from group ii polymorphism. a recruit of more strains will increase and clarify the clinical difference between agr groups. distribution of toxic shock syndrome toxin- , exfoliative toxins and enterotoxins seg and sei among methicillin-resistant staphylococcus aureus clonal types v. chini, g. dimitracopoulos, i. spiliopoulou (patras, gr) objectives: staphylococcus aureus produces a variety of exotoxins including the toxic shock syndrome toxin- (tsst- ), the exfoliative toxins eta and etb and the enterotoxins seg and sei. the seg and sei genes (seg and sei) coexist in the same operon. the aim of this study was to investigate the presence of genes encoding the tsst- (tst), eta (eta), etb (etb), seg (seg) and sei (sei) among methicillin-resistant s. aureus (mrsa) clinical isolates, in relation to their clonal types. methods: the mic of oxacillin was determined by the agar dilution method in mueller-hinton agar supplemented with % nacl according to the guidelines of nccls. pbp a production was investigated by a latex agglutination test (biomerieux) in all s. aureus isolates with mic > . mg/l. pcr amplification was performed for the detection of meca gene to the aforementioned isolates, from different patients, during - , resulting to mrsa. the presence of tst, eta, etb, seg and sei genes was also investigated by pcr. clonal types were determined by pfge of chromosomal dna smai digests. results: among the mrsa, ( %) carried both seg and sei genes, ( %) isolates carried the tst, ( %) the eta, whereas no strains exhibited the etb gene. precisely, during the tst gene coexisted in out of seg/sei-positive strains. eleven strains belonged to pfge type a, whereas the other two to type b, mainly associated with wound infections. eight mrsa in belonging to pfge type a, carried tst, seg and sei. two more strains carrying seg/sei, belonged to pfge type c. two out of seg/sei-positive additional mrsa which were characterized as a new clone f, carried also eta. in , four mrsa carrying the tst, seg and sei belonged to clone a, whereas more carrying the seg/sei were characterized as pfge type c. most isolates were associated with wound infections and abscesses. conclusions: during the study period ( ) ( ) ( ) the incidence of the tst gene carriage among mrsa was reduced from % to %, while the seg/sei genes were almost constantly present. the distribution of these genes was mainly associated with strains of clonal type a, exhibiting a drift towards clone type c predominant during the last two years. detection of decreased susceptibility to glycopeptides in staphylococcus aureus using tablet (disc) prediffusion s.v. nielsen, j.b. casals (taastrup, dk) objectives: the aim of the study was to develop a screening and confirmatory method to detect staphylococcus aureus with decreased susceptibility to glycopeptides (hvisa/visa and gisa). methods: the glycopeptide resistance of staphylococcus aureus strains from our strain collection including well-known resistant strains (atcc , atcc , atcc , atcc ) was examined. we compared: (i) zone sizes of vancomycin lg and teicoplanin lg neo-sensitabs (rosco diagnostica) when prediffused for h (with disc) + h (without disc) on mueller-hinton and bhi, both media supplemented with % horse blood, (ii) agar dilution mics (iii) zone sizes around cefoxitin lg neo-sensitabs. the inoculum used was mcfarland . and the plates were incubated for h at °c. results: regression lines were prepared and there was a good correlation between mics for vancomycin and teicoplanin and the corresponding zone sizes. all hvisa/visa/gisa strains tested showed zones less than mm with cefoxitin lg and zones £ mm (teicoplanin) and/or £ mm (vancomycin) on mh blood agar, corresponding to mics > lg/ml for both antimicrobials. on bhi agar the zones were £ mm (teicoplanin) and/or £ mm (vancomycin) for the hvisa/visa/gisa strains corresponding to mics > lg/ml for both antimicrobials. conclusion: zones obtained by agar diffusion methods using prediffusion with vancomycin and teicoplanin neo-sensitabs correlated with mics and predicted hvisa/visa/gisa strains. for screening high resistance to cefoxitin (zones < mm) may be used to select strains for further testing in a daily routine laboratory. the system should be evaluated further in the routine diagnostic laboratory. high rate of vancomycin intermediate staphylococcus aureus among methicillinresistant isolates in taiwan from patients in the tri-service general hospital in taiwan were screened for vancomycin resistance and isolates with reduced susceptibility to vancomycin were further studied for their characteristics. methods: brain heart infusion agar plates containing vancomycin ( lg/ml) (bhia-va ) were using for vancomycin resistance screening. minimum inhibitory concentration (mic) of vancomycin was determined on both bhia and the standard mueller-hinton agar (mha) on screened-positive isolates. for strains with mics equaled to lg/ml, characteristics including population analysis, susceptibility to triton x- induced autolysis, van gene and pulsed field gel electrophoresis (pfge) typing were further performed as previously described. background: mrsa isolates with decreased susceptibility to glycopeptides (gisa) have been reported worldwide since . most strains were isolated from catheters and other foreign bodies in patients treated with glycopeptides. we aimed to determine the prevalence of gisa isolates in a belgian hospital where mrsa has been endemic for many years. methods: all mrsa isolates collected between / and / were screened for reduced susceptibility to glycopeptides on mueller-hinton agar and mg/l teicoplanin (mh-teico) as recommended by the casfm. the macromethod etest (bhi agar, mcfarland (mf) inoculum, h incubation) was performed as secondary screening for all isolates which grew colonies on mh-teico. gisa/h-gisa phenotypes were confirmed by e-test mic determination on mh agar with . mf and h incubation as well as by vancomycin (v) and teicoplanin (t) population analysis profiles (pap). all confirmed gisa/h-gisa isolates identified were compared by pfge to other strains identified in belgium in order to delineate their clonality. results: among mrsa strains (from patients), isolates showed growth on mh-teico and of these (from patients) yielded a positive macromethod e-test (mic lg/ml for v and t). seven were confirmed by pap as h-gisa and one as a gisa (t mic: lg/ml; v mic: lg/ml). origins of the isolates were: lower respiratory tract ( ), urine ( ) and wound ( ). all h-gisa strains had a similar antibiotic resistance phenotype by disk diffusion (gentamicin, rifampin, erythromycin, clindamycin, ofloxacin) . by molecular analysis, the h-gisa/ gisa isolates clustered in two different pfge groups a (n = ) and g (n = ). the a pfge group was previously described in h-gisa isolates in belgium. in this study, it was subdivided in types, both including strains. one pfge type resulted in a small outbreak in patients during a stay in icu. all other isolates were sporadic with no apparent geographic or temporal relationship between cases. conclusion: gisa/h-gisa were found in only out of ( . %) mrsa-positive patients. none of them presented with severe infection nor had any previous known exposure to glycopeptides. in view of the low prevalence of gisa/h-gisa in our hospital, screening should not be performed systematically but rather on an individual basis taking into account clinical data and risk factors. whenever the occurrence of gisa is considered, teico-mh agar appears as a suitable first screening approach. a case-control study to evaluate the economic outcome of early po linezolid in patients with methicillin-resistant staphylococcus aureus infections oral therapy with linezolid (lzd) has the potential to decrease hospital costs and shorten length of stay of patients with grampositive infections who would otherwise continue to receive iv therapy. objective: to evaluate the impact of early po conversion to lzd in patients receiving traditional iv therapy for methicillinresistant s. aureus (mrsa) infections. methods: patients with documented mrsa infection between and were evaluated in a matched case-control study. cases included patients who were converted to po lzd from iv table includes comparisons of cost, duration of therapy and los. predictably, patients receiving lzd in the hospital had higher drug costs; however, a decreased los contributed to a $ lower cost of hospitalization. lzd or iv vancomycin (vanc) , and controls were patients who received only iv vanc. patients were paired in a : ratio of cases to controls and matched based on infection type, age and comorbidities. demographics, antimicrobial agents, concomitant infections, and clinical status were assessed daily from the onset of infection through the end of treatment or hospitalization. antibiotic cost, length of stay (los), and clinical success rates were compared between the groups. results: patients were assessed. demographic characteristics were similar between groups. average age (mean ± sd) was ± ; apache ii (median, range) was , charlson comorbidity score [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and % were male. % of patients were treated for skin/soft tissue infection, % pneumonia, % bone/joint infections, % bacteraemia, and % other infections. clinical and microbiologic outcomes were similar between groups, with % clinical success at the end of therapy. objectives: to compare the efficacy and safety of linezolid (lzd) with those of vancomycin (van) for the treatment of infections caused by methicillin-resistant staphylococcus aureus (mrsa) in japan. methods: this was a randomized, open-label study conducted in japan. patients with nosocomial pneumonia, complicated skin and soft tissue infections, or sepsis syndrome, caused by mrsa, were randomized ( : lzd: van) to receive lzd, mg q h, or van, g q h. outcomes were evaluated at end of therapy (eot) and at a follow-up (fu) evaluation - days after completion of therapy. results: patients received study drug ( lzd, van); the treatment groups were similar with regards to demographics and risk factors. at eot, clinical success rates in the mrsa-microbiologically evaluable population were . % and . % for the lzd and van groups, respectively; microbiologic eradication rates were . % and . % in the two groups, respectively (p < . ). at fu, the clinical success rates were . % for both groups, and the microbiologic eradication rates were . %, and . %, respectively. clinical success rates in both groups reflect an automatic outcome of failure for patients receiving prohibited antimicrobials prior to fu. reversible anemia ( %) and thrombocyotopenia ( %) were reported as adverse events more frequently in lzd patients. analysis of laboratory data showed that platelet counts decreased more frequently in lzd patients with recovery by fu. the mean platelet count in lzd patients with an adverse event of thrombocytopenia was , /mm . significantly low platelet counts (< , /mm ) were more frequently observed in van patients ( % vs %). no difference was observed in mean changes in hemoglobin levels between the treatment groups. conclusions: lzd is as effective as van for the treatment of mrsa infections in seriously ill patients, and may be more effective in achieving microbiologic eradication. although hematologic adverse events were reported more frequently in lzd-treated patients, analysis of laboratory data showed only a mild reversible trend toward lower platelet counts. cost-effectiveness of linezolid versus vancomycin in complicated skin and soft-tissue infection due to suspected methicillin-resistant staphylococcus aureus infection in germany - , ; san diego, calif) . the objective of the present analysis was to evaluate the cost-effectiveness of linezolid compared with vancomycin in the treatment of cssti due to suspected mrsa infection from the german perspective. methods: a decision-analytic model was developed to examine the costs and outcomes of using linezolid versus vancomycin in hospitalized patients with cssti in germany. an expert panel of german physicians experienced in treating cssti provided resource-utilization data through structured interviews. costs from published sources (rote liste, dkg-nt, ebm) were applied to clinical and laboratory tests, adverse events, isolation procedures, intravenous (iv) or oral (linezolid only) drug treatment, hospitalization by ward type (medical, intensive care), and outpatient physician consultation. if appropriate, patients could be discharged from hospital and treated in an ambulant setting. the model assumed % of suspected mrsa patients had proven mrsa. outcomes included total costs per patient, cost per death avoided, cost per life-year gained, and cost per cure. results: an additional . % of patients treated with linezolid ( . %) versus vancomycin ( . %) were cured. average total cost per episode was versus for linezolid-versus vancomycin-treated patients, giving a saving per episode. the model was sensitive to the los in hospital, days in isolation, duration of oral versus iv treatment, percentage of mrsa patients, and price of linezolid. conclusion: in this decision analytic model, more patients with cssti due to suspected mrsa achieved clinical cure in the linezolid group compared with the vancomycin group. linezolid was cost saving versus vancomycin for the treatment of cssti. linezolid resulted in a shorter treatment duration and shorter los that offset the higher acquisition cost of linezolid versus vancomycin in cssti in germany. clinical microbiology and infection, volume , supplement , training, scientific output in infectious disease p a bibliometric analysis of worldwide trends in research productivity in microbiology p. vergidis, a. karavasiou, k. paraschakis, i. bliziotis, p. papastamataki, m. falagas (athens, gr) background: microbiology contributes significantly to the understanding and control of infectious diseases and has always been a field of extensive research. however, the literature lacks studies estimating the quantity and quality of worldwide research production. we evaluated the contribution of different world regions in research production in the field of microbiology. methods: using the medline database we retrieved articles from journals included in the ÔmicrobiologyÕ category of the Ôjournal citation reportsÕ database of the institute for scientific information for the period - . the world was divided into regions based on geographic, economic and scientific criteria. using an elaborate retrieval system we obtained data on published articles from different world regions. in our evaluation we introduced an estimate of both quantity and quality of research produced from each world region per year using: ( ) the total number of publications, ( ) the mean impact factor of publications, and ( ) the product of the above two parameters. results: data on the country of origin of the research was available for , out of , retrieved articles ( . %). western europe exceeds all other world regions in research production for the period studied, with usa ranking second (table). the difference in production between these two regions increased gradually from to . however, the mean impact factor for articles published in microbiology journals was highest for the usa ( . ), while it was . for western europe and . for the rest of the world ( regions combined). conclusions: usa and western europe make up a striking % of the world's research production in terms of both quantity and quality. all world regions have increased their research production during the period studied. the highest increase was achieved by asia (excluding japan), latin america, and eastern europe. from conference abstract to full paper: differences between data presented in conferences and journals e. rosmarakis, p. vergidis, a. kapaskelis, k. paraschakis, m. falagas (athens, gr) background: on some occasions, we noted differences between data presented in conference abstracts and subsequent published papers. we studied the frequency and the type of these differences in the fields of infectious diseases and microbiology. methods: we reviewed all abstracts from the first session of out of major research categories presented in the and icaac. for each selected pair of icaac abstract and related full paper published in journals indexed by index medicus, two independent investigators performed a comparison of all data in the abstract and the corresponding information in the published paper. using cox logistic regression models we analysed variables for association with differences of data. results: from abstracts that were reviewed, ( . %) were subsequently published as papers by march . from them, referred to the same study period and population for both the abstract and the paper. differences between data presented in conference abstracts and published papers were found in out of pairs which were analysed further ( . %, % c.i.: . %- . %). the identified differences were related to the numbers and/or rates of the studied patients ( / ), numbers or rates of isolates ( / ), mics values or ki values ( / ), other chemical properties of antibiotics ( / ), odds ratio ( / ), and duration of observation ( / ). the differences were substantial in several pairs. time to publication of full paper was found to be independently associated with presence of differences (p = . , or = . per year), while the research category, type of presentation (oral or poster), number of publications of the presenting and last author, impact factor of the journal, and country of origin were not. conclusions: while there are several explanations for the noted differences between data presented in conference abstracts and full papers, it is likely that the research community may improve the accuracy of presentation of data. worldwide trends in quantity and quality of published articles in the field of infectious diseases i. bliziotis, k. paraschakis, p. vergidis, a. karavasiou, a. kapaskelis, m. falagas (athens, gr) background: trying to confront with the widespread burden of infectious diseases, the society worldwide invests considerably on research. however, the literature lacks studies estimating the quantity and quality of worldwide research production. we evaluated the contribution of different world regions in research production in infectious diseases. divided into regions based on geographic, economic and scientific criteria. using an elaborate retrieval system we obtained data on published articles from different world regions. in our evaluation we introduced an estimate of both quantity and quality of research produced from each world region per year using: ( ) the total number of publications, ( ) the mean impact factor of publications, and ( ) the product of the above two parameters. results: data on the country of origin of the research was available for , out of , retrieved articles ( . %). usa and western europe are by far the most productive regions concerning publications of research articles, as displayed in the table. however, the rate of increase in the production of articles was higher in the developing world regions during the study period. the mean impact factor is highest for articles originating in the usa ( . ), while it was . for western europe and . for the rest of the world ( regions combined). conclusions: usa and western europe make up a striking % of the world's research production in infectious diseases in terms of both quantity and quality. however, all world regions present a steady increase in the production of infectious disease articles with the developing countries displaying the highest rate of increase. . financial support was obtained from a pharmaceutical company. however, the sponsor had no role in organizing the scientific content of the programme. results: physicians including id specialists were selected from several university hospitals or tertiary care government hospitals where running clinical trials has been a common practice. pre-training queries revealed that among the trainee population, . % never obtained an informed consent from a patient, % did not randomize any patients for trials, % did not run any phase i-iv trials, % did not use placebo for trial purposes and % did not report any serious adverse events to local and or international authorities. a -question pre-and post-course tests about basic principles of gcp and local and eu regulations for running clinical trials were applied to determine and compare the knowledge of the trainees on these subjects prior and after the course. the percentage of correct answers ranged - % before, and - % after the course was completed (p < . ). conclusions: training in gcp and other regulations about running clinical trials may increase the awareness and knowledge of physicians. these educational activities will also help to raise the quality of clinical trials. learning appropriate use of antibiotics (pk/pd and guidelines): a cd-rom course for healthcare professionals and students e. ampe, y. glupczynski, p.m. tulkens, f. van bambeke (brussels, mont-godinne, b) objectives: in a context of growing resistance and limited supply of new molecules, a rational use of antibiotics should be a high priority. our objective is to train healthcare professionals and students in pk/pd and in a correct implementation of guidelines, since this could help to improve antibiotic use in both short and mid-terms. methods: we developed a pk/pd -guidelines course on cd-rom, targeted to both physicians and pharmacists but also usable by students. the course was prepared by a team of pharmacists, clinical microbiologist, and pharmacologist. sources of information were (i) textbooks, review papers and primary papers by internationally recognized experts (ii) materials presented at training workshops of the international society for antiinfective pharmacology (isap; www.isap.org) during the last years, (iii) national and, if not available, international guidelines for the management of respiratory tract or urinary tract infections. results: the course is organized as a series of power point presentations covering in a progressive fashion the followings topics: ( ) bases in microbiology (in vitro properties of antibiotics); ( ) pharmacokinetics (definition of the main parameters); ( ) pharmacodynamics, with (a) the concepts, (b) the methods and pertinent models, and (c) the data, including the parameters to take into account to optimize the dosage of the main antibiotic classes; ( ) resistance, including (a) the main mechanisms and (b) the use of pharmacodynamics to avoid the selection of resistance; ( ) the appropriate use (including appropriate dosages) of antibiotics in (a) respiratory tract and (b) urinary tract infections. conclusions: this course promotes continuous education in the pharmacology and pharmacotherapy of antibiotics, in a format easily usable for courses and seminars to both students and professionals. background: although macrolides have no intrinsic antipseudomonal activity, these drugs appear effective in controlling infection by p. aeruginosa through mechanisms such as disruption of quorum sensing or anti-inflammatory effects. objectives: the aim of our prospective study was to evaluate the efficacy of macrolides in patients with bronchiectasis infected and colonised by p. aeruginosa. methods: the study included hospitalised patients with ct evidence of bronchiectasis and presenting an acute exacerbation by p. aeruginosa, isolated in sputum. microbiological findings, clinical data and treatment recommendations were recorded at admission and at the end of therapy. patients completed daily diary cards for symptoms and pef values. patients were followed for year. results: twenty-two patients ( men, mean age . ± . ) with bronchiectasis and p. aeruginosa infection were included. during hospital stay, patients were treated with at least two susceptible antipseudomonal drugs, according to the antibiogram (usually beta-lactam plus aminoglycoside) for a period of . ± . days. an oral macrolide (azithromycin mg x days/week in patients, clarithromycin mg daily in patients) was further administrated for . ± . months. at the end of long-term therapy, ( %) patients showed no evidence of p. aeruginosa in sputum, ( . %) patients still presented p. aeruginosa in sputum and ( . %) discontinued the treatment after less that month because of adverse events. all patients had a significant reduction of sputum volume ( . ml/day before therapy vs. . ml/day at the end of therapy, p = . ) and of the mean number of exacerbations during follow-up ( . in the previous year vs. . in the follow-up year, p = . ). an increase of the mean pef value was also noted, although statistically not significant ( . ± . l/min before therapy vs. . ± . l/ min at the end of therapy, p = . ) conclusion: these observational findings suggest that macrolides may have a role in modulation of p. aeruginosa colonisation in patients with bronchiectasis. objectives: there is little information on cap requiring hospitalization in young adults (£ years). we analysed clinical features, causative organisms, and outcomes of cap in this patient population. methods: prospective observational study of non-immunocompromised adult patients with cap ( cap ( - . patients with hiv infection, transplantation, and neutropenia were not included. for the purposes of the study, patients were divided into age-groups, £ years and > years. results: we documented consecutive patients with cap; patients ( . %) were £ years and were > years. mean patient ages were . years in the former group and . years in the latter group. main reasons for hospitalization in young adults were: hypoxia (po < ) patients, port high severity risk class (iv-v) patients, empyema patients, and/ or shock patients. young patients were more frequently current smokers ( % vs %, p < . ). comorbid conditions were more common in older patients ( % vs %, p < . ), mainly diabetes mellitus, copd, and chronic heart disease. multilobar pneumonia was more frequent in young patients ( % vs %, p < . ). the most common causative organisms were streptococcus pneumoniae ( % vs %, ns) and legionella pneumophila ( % vs %, ns). atypical agents were more frequent in young adults ( % vs %, p = . ), while haemophilus influenzae ( % vs %, p = . ) and gram-negative bacilli ( % vs . %, ns) were rarely identified in this group. the frequency of bacteraemia was similar in both groups ( % vs %, ns). icu admission was more frequent in young adults ( % vs %, p = . ), but no differences were found regarding the need of mechanical ventilation ( % vs %). median los was shorter in young patients ( vs days, p = . ). five young patients died; shock ( patients) and multiorganic failure ( ). early mortality (< hours) was similar in both groups ( . % vs . %). overall mortality (< days) was higher in older patients ( . % vs . %, p = . ). conclusions: cap requiring hospitalization in young adults, mainly smokers, is not uncommon, being respiratory failure the most frequent reason for admission. s. pneumoniae and atypical agents are the most frequent causative organisms in this group. although overall mortality is relatively low, a number of young patients require icu admission and have a complicated course. corticosteroids in patients with severe community-acquired pneumonia: impact on mortality c. garcia vidal, e. calbo, c. ferrer, v. pascual, s. quintana, j. garau (terrassa, e) background: mortality in patients with severe cap has been traditionally related to microorganism virulence and to host characteristics. recently, there has been an increasing interest host-pathogen interaction, and inadequate immunologic response has been shown to be associated with a higher mortality. immune modulation concomitant to antibiotic therapy has been postulated to improve outcome. the aim of our study was to determine the risk factors for increased mortality in patients with severe cap and to evaluate the impact of administration of corticosteroids in outcome. methods: we reviewed the charts of all hospitalized cap patients in our centre between october and december . severe cap defined by pneumonia severity index (psi) categories and were included. data on demographics, comorbidity measured by charlson score, bacterial aetiology, the presence of immunosuppression, copd, icu admission, antibiotic therapy, use of corticosteroids and mortality were recorded. results: of the severe cap patients included, and ,were categories and , respectively. patients( %) were treated with standard antimicrobial therapy and ( %) received also corticosteroids. mean age ( vs ), charlson score ( . vs. . ), neoplasm ( % vs. %), hiv ( . % vs. . %), chronic liver disease ( % vs. %), diabetes mellitus ( % vs. %), icu admission ( % vs. %), monotherapy ( % vs. %), and aetiology (s. pneumoniae % vs. %; l. pneumophila % vs. %; others % vs. %; unknown % vs. %) were similar. patients receiving corticotherapy had been exposed to another immunosuppressive treatment more frequently ( % vs. %; p = . ), copd was commoner ( % vs. %; p < . ) and mortality was higher ( % vs. %; p = . ) than in the group not treated with corticosteroids. in multivariate analysis, severity of disease (or . ; ic % . - . ; p = . ) and the use of corticosteroids (or . ; ic %, . - . ;p = . ) were found to be related to mortality. conclusions: in our experience corticosteroid treatment in patients with severe cap appears to be associated with higher mortality. use of levofloxacin in community-acquired pneumonia j. olalla, i. escot, j.j. garcía-alegría (marbella, e) introduction and objectives: community acquired pneumonia (cap) is a prevalent and important disease, with an important consume of resources at hospitals. our aim is to study the profile of income patients with cap diagnosis, the year of introduction of levofloxacin in our centre and compare the length of stay in order to different antibiotics treatments, adjusting the results by fine's scale. methods: in our hospital (a second level hospital in marbella, spain) all the diagnosis at discharge are codified and included in the hospital database, so, all the diagnosis of cap between september and march were collected, and clinical reports were examined. demographic data were registered, so were all the parameters affecting fine classification, antibiotic treatment, intensive care unit (icu) incomes, deaths and length of stay. results: cases of cap were found ( males, females), with a mean age of years (ys)-ci %: - ), no difference between gender. patients (pts) were living in a residence. pts ( . %) had a previous diagnosis of heart failure -hf-(mean of age vs , p < . ) and other pts had cerebrovascular disease -cvd-(mean of age . vs , p < . ), pts had chronic hepatopathy, - . %-any form of cancer, - . %-pts chronic renal failure (mean of age vs , p = . ), pts ( %) chronic obstructive pulmonary disease (copd). pts were admitted at icu and deaths were registered. blood cultures were collected in % of cases, culture of sputum in %, legionella antigen in urine in . %. in according with fine's classification the patients were stratified in group i ( . %), ii ( . %), iii ( . %), iv ( . %) and v ( . %). in % cases corresponding to fine i and ii levofloxacin was used alone, vs % in fine iii, iv and v (p= . ). when length of stay was analysed in patients fine i and ii, the use of levofloxacin was associated with a non significant reduction (mean + se: . + . days vs . + . days), in people on groups iii, iv and v use of levofloxacin do not showed any reduction of length of stay ( . + . days in people using levofloxacin vs . + . days). no readmissions in relationship with recurrent cap was registered. conclusions: in our experience, use of levofloxacin is associated with less severe cap, in which a non significant reduction in length of stay is observed. telithromycin versus other first-line single-agent antibiotics in the treatment of communityacquired pneumonia: a randomised superiority trial y. mouton, v. thamlikitkul, r.b. nieman, c. janus (tourcoing, f; bangkok, th; bridgewater, usa; paris, f) objectives: macrolides and beta-lactams are commonly recommended for the treatment of outpatients with communityacquired pneumonia (cap). the aim of this study was to demonstrate the superiority of the ketolide telithromycin (tel) to other first-line, oral, single-agent antibiotics (comparators, comp) in achieving clinical cure in outpatients with mild to moderate cap in geographical areas of high pneumococcal resistance (erythromycin resistance rate ‡ %). methods: patients with clinical and radiological evidence of cap were randomised centrally to receive either tel mg once daily for - days or comp (chosen by the investigator in accordance with local treatment guidelines/practices). efficacy was assessed post-therapy (days - ). clinical outcomes in this open-label trial were validated by three independent experts who were fully blinded to treatment assigned. results: of the patients enrolled, were included in the modified intent to treat (mitt) and in the per protocol (pp) efficacy analyses. in the comp group, % of patients had received macrolides, % beta-lactams and % fluoroquinolones. resistance to penicillin and erythromycin among streptococcus pneumoniae isolates was % and %, respectively. clinical cure rates in the tel group were significantly higher than those in the comp group (as shown in the following table). tel and comp were similarly well tolerated. conclusions: in this outpatient cap study, post-therapy clinical cure rates achieved with tel were shown to be statistically superior to those achieved with other usual care first-line antibiotics -in both the mitt and pp populations, and according to evaluations by both the investigators and blinded experts. changing epidemiology, clinical features, and outcome of acute community-acquired pneumonia among adults in india background: acute cap is the third leading cause of mortality in india. two prospective studies from our centre identified common causes of cap in india to be mycoplasma pneumoniae [mp] and legionella pneumophila [lp] by serology in % each, and spn in % by culture of respiratory secretions/blood/ conclusion: although spn is the most common isolate, the rising numbers of gram negative organisms ( %) and atypical pathogens associated with increasing mortality stress the need for review of initial antibiotic choice for adults with higher port classes. in view of enduring susceptibility of spn to pcn and minimization of resistance with narrow spectrum activity, this should be the drug of choice for spn in india. fqr that has a wide coverage against most respiratory pathogens including atypicals, can be considered as an initial choice in both hospitalized and ambulatory setting. continued surveillance of respiratory pathogens is needed. fluoroquinolones in the treatment of community and hospital-acquired legionella pneumonia objective: community-acquired pneumonia (cap) is a major cause of morbidity and mortality worldwide. inability or failure to comply with standard antibiotic regimens, which may last up to days, may result in patients receiving suboptimal antibiotic treatment. treatment with a single-dose antibiotic regimen maximizes compliance with prescribed therapy. a novel microsphere formulation of azithromycin makes it possible to administer more drug in a single dose while maintaining tolerability. the objective of this study was to test the hypothesis that a single . g oral dose of azithromycin microspheres was as effective as a -day regimen of oral levofloxacin mg/day when used to treat adult patients with mild to moderate cap (fine classes i, ii, iii objectives: many guidelines have been issued for the treatment of cap. we wanted to assess how frequently the national guidelines for treatment of cap were followed in spanish acute care hospitals in patients admitted with cap. methods: retrospective review of the charts of patients admitted to the hospital with the diagnosis of cap over a -year period ( nov to oct ) in geographically scattered hospitals in spain. data were available from patients. results: amoxicillin/clavulanate was the most common antibiotic choice ( . % was given as monotherapy and . % in association with clarithromycin). levofloxacin ( . % given only as monotherapy) follows closely. they were followed by third generation cephalosporins (ceftriaxone and cefotaxime) but up to % of them were given in association with clarithromycin. clarithromycin was used mainly as a complement to both amoxicillin/clavulanate and cephalosporins since only . % of its . % was given as monotherapy. other antibiotics were very uncommonly prescribed (cefepime . %, and ciprofloxacin . %). conclusions: ) monotherapy with levofloxacin, followed by amoxicillin/clavulanic acid were the most common first line antibiotic choices in these spanish hospitals. however, amoxicillin/clav alone or in combination with clarithromycin was the most common antibiotic prescribed. ) monotherapy with clarithromycin was relatively uncommon ( . %). ) these choices reflect quite well the recommendations of the different national guidelines issued in our country in an environment of high prevalence of resistance to penicillin and to macrolides in s. pneumoniae in spain and the concomitant coverage of legionella and other atypical pathogens. the microbiology of abecb -potential predictive value of clinical criteria j. kahn (raritan, usa) objectives: to explore the relationship between specific pulmonary disease severity criteria and the microbiology of acute bacterial exacerbations of chronic bronchitis (abecb). methods: this was a double-blind, randomized clinical trial evaluating levofloxacin mg qd for - days in abecb. all potential subjects had to meet the ats definition of chronic bronchitis but only those patients with anthonisen type or exacerbations (n = ) were enrolled. stratification by disease severity was determined using parameters suggested by grossman: fev % of predicted value, defined co-morbidities, and number of exacerbations during the previous months. because different spectra of etiologic organisms were expected on the basis of the stratification, different comparator agents were used for uncomplicated patients (azithromycin for five days) and those considered complicated (amoxicillin/clavulanate for ten days). results: initial microbiology from all intent-to-treat patients revealed notable differences between the two strata. among isolates from uncomplicated cases, % were the ÔtraditionalÕ triad of s. pneumoniae, h. influenzae, and m. catarrhalis. inclusion of h. parainfluenzae raises this figure to %. in the complicated arm the figures were % and %, respectively. gram-negative bacilli, primarily enterobacteriaceae spp. and several pseudomonads, represented % of the uncomplicated and % of the complicated isolates. microbiological eradication rates and the percentage of organism persisting after therapy from the uncomplicated stratum were compatible with the unexpectedly large number of macrolideresistant organisms isolated. in the complicated arm, both levofloxacin and amoxicillin/clavulanate had lower, but comparable, eradication and persistence rates. clinical efficacy in microbiologcally-evaluable patients was consistent with these figures. conclusion: while there was clearly overlap among the flora isolated from the two strata defined by application of the grossman criteria, there was separation of the populations. this predictive approach seems to be of value in identifying the optimal antimicrobial regimen, especially for uncomplicated exacerbations. further work to define and validate predictive clinical parameters may help optimize the choice and duration of antimicrobial agents for abecb. objectives: nosocomial pneumonia is the second most common nosocomial infection and, along with primary bacteraemia, the leading cause of death from infection acquired in the hospital. despite the frequency of ventilator-associated pneumonia (vap) and the threat it poses to patient survival, consensus on an appropriate antibiotic treatment of vap has yet to be established. the uncertainty is compounded by a lack of well-controlled comparisons of specific treatment regimens and their impact on relevant outcomes, such as morbidity, mortality, and cost. the comparable analysis of clinical and microbiological efficacy of cefepime and a combination ceftazidime and amikacin in the treatment of vap in severe trauma patients was the aim of present study. methods: this prospective, randomized study was approved by the institutional review board for human research. thirty adult patients who admitted in icu of an -bed emergency municipal hospital with severe trauma complicated with vap were included. the cpis was used for diagnostics of vap. a combination of ceftazidime ( g tid, iv) and amikacin ( g once daily, iv) was used as initial empiric therapy of vap in patients and monotherapy with cefepime ( g bid, iv) -in patients. these regimes of empiric therapy have been chosen according our local microbiological monitoring and p. aeruginosa was the prevalent pathogen of vap. the cost-effectiveness analysis was performed to compare both costs and outcomes of competing regimes. results: there was no difference between groups in patientsÕ mean age, average time of mechanical ventilation before onset of vap and cpis. mean apache ii score was . in the group of combination therapy and . in the group of monotherapy. the positive clinical outcome was registered in . % patients on cefepime and . % patients on ceftazidime plus amikacin. the rate of eradication was . and . % respectively. the duration of effective treatment with cefepime was from to days (mean . days), with combination therapy -from to days (mean . days). the mean cost of vap treatment with cefepime was . euro in comparison with . euro when the empiric therapy was initiated with ceftazidime and amikacin. conclusions: cefepime in a monotherapy regimen has the same clinical and bacteriological efficacy in comparison with a combination of ceftazidime and amikacin in vap patients with severe trauma but the use of cefepime results in lower costs. influence of antibacterial protocol in multiple trauma patients on incidence and mortality of vap d. protsenko, a. yaroshetskiy, s. yakovlev, b. gelfand (moscow, rus) objectives: the aim of present study was the evaluation of antibacterial protocol in multiple trauma patients on incidence and mortality of vap. methods: a comparable analysis of incidence, attributive mortality (chi-square test) and pathogens of vap in multiple trauma patients was performed during two periods: (before introduction of protocol) and (after introduction of protocol) years. the developed protocol included: . abandoning of antibiotic prophylaxis of vap; . intrusion criteria of early diagnostics of vap; . exclusion of st, nd and rd generation cephalosporines , aminoglycosides and fluoroquinolones as empiric therapy of vap; . cefepime (apache ii < ) or carbapenems (apache ii > ) were used as initial empiric therapy of vap; . efficacy of antibiotic treatment was evaluated within hours; . carbapenems and/or vancomycin was added if initial therapy was inefficient. in the case of suspected diagnosis of vap microbiological analysis of bronco-alveolar lavage fluid (bal) was performed. a widespread use of broad spectrum antibiotics shifted the structure of nosocomial pathogens. we observed decrease of mrsa rate and significant increase (more than times) in rate of klebsiella pneumoniae (from . to . %, most of strains were resistant to rd generation cephalosporines) and in rate of acinetobacter baumanii (from . to . %, most of strains were resistant to ceftazidime). conclusions: introduction of strict antibacterial protocol in patients with multiple trauma and vap results in significant decrease of attributive mortality without any change in incidence of vap. antimicrobial prophylaxis in cardiac surgery and postoperative pneumonia rate objective: the purpose of this study was to assess the postoperative pneumonia rate according to antimicrobial prophylaxis regimens in patients after cardiac surgery with cardiopulmonary bypass. patients and methods: cardiac surgery patients were included (n = ) in the retrospective study. we observed patients with postoperative pneumonia (pneumonia group) and patients without infectious complications in postoperative period. the groups were compared by age, sex, volume of transaction, severity of disease. in a studied period is from to two main regimens of antimicrobial prophylaxis were used: ceftriaxone - g iv once before operation or cefuroxime . g iv before operation and additional dosage . mg in - hours after surgery beginning; then prophylaxis were conducted after operation during - hours. data were compared by fisher exact; we determined about significantly differences between groups when p < . . results: preoperative prophylaxis by cefrtiaxone was administered more often in pneumonia group than in patients without infection complications ( . % vs. . %, ns). ceftriaxone was used significantly more often in comparison to cefuroxime in subgroup of patients with cardiopulmonary bypass less than min who developed postoperative pneumonia ( . % vs %, p < . ). during cardiopulmonary bypass longer than min we did not reveal difference between antimicrobial prophylaxis regimens. conclusion: cefuroxime is more preferable than ceftriaxone for antimicrobial prophylaxis of postoperative pneumonia in patients after cardiac surgery with cardiopulmonary bypass less than min duration. closed versus open tracheal suction system to prevent ventilator-associated pneumonia objective: the aim of this study was to analyze the incidence of ventilator-associated pneumonia (vap) using a closed tracheal suction system ( objectives: dalbavancin is a novel, second generation lipoglycopeptide that has been shown to have clinical efficacy as a once-weekly treatment for complicated skin and skin structure infections (csssi). the dosage used in clinical studies is mg day / mg day . in vitro studies have determined dalbavancin mic s of less than or equal to . mg/l for target bacteria, principally staphylococci and streptococci, including isolates resistant to other antibiotics. the plasma protein binding of dalbavancin is %. as skin infections occur in extravascular space, antibiotic concentrations are assessed in blister fluid as a measure of skin penetration and to obtain pharmacokinetic observations that may correlate with efficacy. methods: a phase i, open label, single-dose study was conducted in healthy subjects. subjects were administered a mg iv dose of dalbavancin. blisters were induced by applying . % cantharidin ointment to the skin. blister fluid was collected prior to dose, and at hours, day , day and day . blood samples were collected throughout the study. blister fluid and plasma were assayed for dalbavancin using a validated lc-ms/ms method and pharmacokinetic parameters were determined. area under the concentration-time curve (auc) was determined for blister fluid (aucbf) and plasma (aucp) through the first week. the degree of penetration into skin was determined by aucbf/aucp (%). results: dalbavancin was well tolerated with no serious adverse events; most adverse events were mild and self-limited. maximum observed dalbavancin blister fluid concentrations were achieved by the first collection ( hours) and concentrations were maintained above . (± . ) mg/l through day . the mean (sd) aucbf and aucp were ± and ± mg h/l, respectively. skin penetration was approximately %. dalbavancin blister fluid concentrations were maintained well above mics throughout the treatment interval, even when considering the possible effects of protein binding. conclusions: blister fluid concentrations are maintained well above mics of csssi target organisms for a week following a single dose of dalbavancin. these data support a once-weekly regimen and are consistent with the efficacy observed in clinical studies. the safety and pharmacokinetics of dalbavancin in subjects with renal impairment or end-stage renal disease j.a. dowell, e. seltzer, d. krause, t. henkel (king of prussia, usa) objectives: dalbavancin is a novel, second generation lipoglycopeptide antibiotic in late stage clinical development for complicated skin and skin structure infections (csssi). the weekly dosage used in clinical studies is mg day / mg day . since dalbavancin will likely be used in patients with various degrees of renal impairment, it is important to determine the safety and pharmacokinetics in this population, as well as to evaluate if a dosage adjustment is necessary. methods: single intravenous doses of mg dalbavancin were examined in subjects with mild (clcr of - ml/min) and moderate (clcr of - ml/min) renal impairment. doses of mg dalbavancin were studied in subjects with endstage renal disease (esrd; dialysis-dependent). single doses of mg and mg dalbavancin were studied in subjects with severe renal impairment (clcr < ml/min). subjects with normal renal function were studied and used as controls. pharmacokinetic data were analysed using non-compartmental methods; parameters included maximum concentration (cmax) and area under the plasma concentration time curve (auc). results: a total of subjects received dalbavancin and were included in the pharmacokinetic analysis ( mild, moderate, severe, esrd, and normal). dalbavancin was well tolerated in each of the renal impairment groups. the majority of adverse events were mild or moderate in severity and unrelated to study drug. there were no related serious adverse events. dalbavancin pharmacokinetics were similar between subjects with mild and moderate renal impairment and subjects with normal renal function. concentrations in subjects with esrd were similar to subjects with normal renal function, indicating compensation in renal insufficiency due to regular dialysis ( times/week). subjects with severe renal impairment had increased concentrations and exposure. concentrations were increased by no more than % through the first week post dose, but differences continued to increase through the rest of the profile. auc was increased almost -fold. background: tlv, a novel lipoglycopeptide antibiotic with multiple mechanisms of action, exerts rapid and concentration-dependent bactericidal activity against clinically important gram-positive bacteria, including methicillin-resistant s. aureus. the kidney is the primary route of elimination of tlv in man. phase studies are ongoing in skin and skin structure infections and hospital acquired pneumonia. the lines represents the model-based predictedprobability of the first occurrence of naused. the boxes represent the th and th percentiles of auc for each dose group. the wiskers extend to the minimum and maximum values. objective: to compare the single dose pk of tlv in subjects maintained on hemodialysis with an aged and sex-matched group of healthy subjects without renal dysfunction. methods: six hemodialysis subjects ( males, female) received a single . -mg/kg dose of tlv intravenously over hour approximately - hours prior to a -hour hemodialysis session. six subjects ( males, female) with normal renal function (mean creatinine clearance ml/min) also received the same dose of tlv over hour. blood and dialysate samples were obtained at specified intervals post dose and assayed for tlv with a validated lc/ms/ms assay. pk parameters were determined using non-compartmental methods. results: average values for plasma pk parameters for tlv in both groups of subjects are shown below.the average (range) clearance of tlv via dialysis was . ml/min ( . - . ) and the per cent of dose removed by dialysis was . % ( . - . ). subjects tolerated tlv well. conclusions: dose reductions of tlv of % are recommended in patients maintained on hemodialysis. supplementation of a tlv dose post dialysis is not needed. pharmacokinetics and tissue penetration of telavancin in healthy subjects background: tlv, a novel lipoglycopeptide antibiotic with multiple mechanisms of action, exerts rapid and concentration-dependent bactericidal activity against clinically important gram-positive bacteria, including methicillin-resistant s. aureus. the mic for s. aureus, including mrsa and gisa, from pooled studies is . lg/ml. phase studies are ongoing in skin and skin structure infections and hospital acquired pneumonia. objectives: to determine the steady-state pk profile of tlv in plasma and skin blister fluid in healthy subjects. methods: subjects ( males, female) received three daily doses of tlv ( . mg/kg) intravenously over hour. cantharidin ointment ( . %) was applied to the forearms to produce blisters/subject beginning - hours prior to the third dose of tlv. blood and blister fluid samples were obtained at specified intervals on days and and assayed for tlv with a validated lc/ms/ms assay. pk parameters in both matrices were determined using non-compartmental methods. the average values for pk parameters for tlv in plasma and in blister fluid are shown below. results: after single intravenous infusions of mg equivalent (eq) ceftobiprole for minutes or mg eq ceftobiprole for minutes, mean cmax-values at infusion endpoint are . lg/ ml and . lg/ml, respectively. the corresponding mean auc -inf-values are lg h/ml and lg h/ml. intersubject variability of the parameters auc and cmax of ceftobiprole is below %. mean systemic clearance is . and . l/h and mean steady-state volume of distribution is . and . l for the mg and mg dosing regimen, respectively. after infusion of mg for minutes or mg for minutes, the Ôtime above micÕ (target mic of lg/ml for methicillin resistant staphylococcus aureus) is and hours for the total plasma concentrations and . and . hours for the corrected unbound plasma concentrations, respectively. with twice-daily administration of mg for minutes or mg for minutes, the proportion of the dosing interval above the mic of lg/ml, would be % and % for the total plasma concentrations and % and % for the unbound plasma concentrations, respectively. conclusions: ceftobiprole infusions of mg eq for minutes or mg eq for minutes, results in similar time above target concentration of lg/ml well above the minimum efficacy requirement - % required for mrsa. methods: the study was a randomized, three-way crossover study with hours wash-out period in healthy volunteers. each subject received imipenem in three regimens : (i) . h infusion of . g every h for doses; (ii) h infusion of . g every h for doses; (iii) h infusion of g every h for doses. conclusion: the h infusion of . or g of imipenem both give greater values for t > mic than a . h infusion and that a h infusion may be a useful mode of administration in tropical countries where drug instability may prevent the use of continuous infusion. pharmacodynamic characterisation of lmb against haemophilus influenzae in an in vitro hollow-fibre system a. meagher, p. smith, a. forrest, a. odundele, p. ambrose (buffalo, albany, usa) objectives: lbm is a peptide deformylase inhibitor with in vitro activity against those pathogens commonly associated with community-acquired respiratory tract infections. the purpose of these studies was to determine which pharmacokineticpharmacodynamic (pk-pd) measure is most strongly associated with drug response and to examine the relationship between drug exposure and response for lmb against haemophilus influenzae (hi). methods: two wild-type hi strains (mic and mg/l) were studied. the hollow-fiber system (hfs) was inoculated with approximately e - e cfu/ml in log-phase growth. simulating human pharmacokinetics (t ½ = hours), bacteria were exposed to escalating free drug lmb exposures (auc ranging from to mg · h/l) using a dose fractionation study design and delivering drug q hours, q hours, and by continuous infusion (ci). serial samples were collected to determine bacterial counts (cfu/ml) and drug concentrations. drug effect was quantified as the log ratio (lr) of the hour area under the bacterial growth/kill curves for drug and growth control (lr = log aucdrug/aucgrowth control). results: overall, the greatest activity (at xmic) was seen with the ci regimen (ci > q >> q ). due to the short half-life, dosing q hours yielded no net kill compared to baseline. neither per cent time above mic, auc:mic ratio, nor peak:mic ratio could co-model the q and q hour regimens with ci results. separating these into datasets (q / vs ci), only the auc:mic ratio could adequately describe the ci results (emax lr of ( at an auc:mic ratio ‡ ). the q / dataset was reasonably fit by the auc:mic ratio and per cent time above mic (emax lr of ( to ( at an auc:mic ratio ‡ and per cent time above mic of ‡ - %, respectively). the peak:mic ratio was not informative. conclusion: for intermittent dosing regimens, per cent time above mic and the auc:mic ratio adequately described drug response. percent time above mic and the auc:mic ratio associated with maximal decline were ‡ - % and ‡ , respectively. ci regimens could not be co-modeled with intermittent regimens, suggesting that neither per cent time above mic nor the auc:mic ratio completely described drug effect. drug effect continued to increase beyond % time above mic. q hour dosing had more effect than q hour dosing, and would probably be an effective lbm regimen in humans for hi. pharmacodynamics of antimicrobials for the empiric treatment of nosocomial pneumonia: a report from the optama program objectives: appropriate empiric antibiotic therapy is vital for maximizing patient outcomes in the treatment of nosocomial pneumonia and is dependent on the drug exposure achieved in a patient and the causative pathogen's mic. the purpose of this study was to compare the probability of achieving bactericidal exposures for commonly used intravenous antibiotics against the bacteria most commonly implicated in nosocomial pneumonia. was high, target attainments for fep, caz, and tzp decreased, but carbapenem target attainments remained the same regardless of pathogen prevalence. conclusions: target attainments were greatest for ipm and mem, followed by higher doses of fep, caz and tzp. because these antibiotic regimens provide optimal bactericidal exposure, they would be most suitable for the empiric treatment of nosocomial pneumonia along with an anti-mrsa antibiotic until pathogen identification and susceptibility results are available. minimal inhibitory concentration effect (sme) of antibacterial agents against two strains of b. anthracis. methods: the pa-sub mic and sme were determined by exposing the bacteria to antibiotic concentrations times the mic for h at °c. following repeated washings and centrifugations to remove the antibiotic, cultures were divided into four tubes. to three tubes, the tested antibiotic was added to make a final concentration of . mic, . mic and . mic to the fourth tube no antibiotic was added. optical density was determined before exposure, immediately after washing and then hourly up to h. for the determination of sme, the same procedure was performed except that the organisms were not pre-exposed to any antibiotic. the ods were converted to cfus by using a standard curve. the pa-sub mic was defined as pa-sub mic = tpa ) c, where tpa is the time for cultures previously exposed to an antibiotic and then reexposed to different sub-mics to increase log above the counts determined immediately after washing and c is the corresponding time for the antibiotic unexposed control. the sme was defined as sme = ts ) c, where ts is the time for the cultures exposed only to sub-mics to increase log above the counts determined immediately after washing and c is the corresponding time for the unexposed control. methods: serum and csf cefepime pk data was obtained from hospitalized patients with pneumonia and external ventricular drains. the concentration-time profiles in serum and csf were modeled using a three-compartment model with zero-order infusion and first order elimination and transfer. the apparent volume of the central compartment (vc), apparent volume in the csf (vcsf), intercompartmental transfer rate constants (k , k , k , and k ) and plasma clearance (cl) were identified in a population pk analysis (npag) [table ]. for cefepime g iv q h ( . h infusion), a monte carlo simulation of , subjects (adapt ii) was performed to estimate the probability of attaining the targets of free cefepime serum concentration (assumed % protein binding) and total cefepime csf concentration - % t > mic for mics . - mg/l (table ) . csf/serum median penetration ratio was calculated. post map-bayesian observed-predicted regression and r for serum and csf were as follows: (serum) observed = . · predicted + . ; r = . . (csf) observed = . · predicted + . ; r = . . the penetration of cefepime as measured by median auccsf/auc serum was . % ( th- th percentiles . - . %). results of serum and csf target attainment analysis for cefepime g iv q h conclusion: in the setting of non-inflamed meninges, cefepime g iv q h does not provide adequate t > mic in the csf for > % of patients for mics ‡ . mg/l. the influence of inflammation on the calculated csf target attainment rates is unknown. the definitive pharmacodynamic target in the csf has not been elucidated and further research is needed. application of microbiological and capillary electrophoresis methods to phenoxymethylpenicillin dissolution assay w. grzybowska, g. pajchel, m. zapasnik, s. tyski (warsaw, pl) objectives: drug absorption from a solid dosage form after oral administration depend on the release of the active substance from the medicinal product. in some cases of drug analysis, especially when the results of tests are out of specification, it is necessary to use another assay, simultaneously with the reference method. in case of phenoxymethylpenicillin tablets, the reference method for dissolution assay is spectrophotometric method. the aim of the study was to apply parallel microbiological and capillary electrophoresis methods and compare the results with those obtained using uv assay. methods: two preparations (tablets), containing iu/mg of penicillin v: taropen and ospen were examined. the dissolution tests were performed at temperature °c ± . °c, in phosphate buffer ph . ( ml for ospen and ml for taropen) using baskets, rotation speed rpm; samples were taken only at: minute or at , and minutes in case of profile of dissolution analysis. the amount of penicillin v dissolved was assayed spectrophotometrically at nm. hanson research dissolution system and ce apparatus: quanta of (waters) were used. pharmacopoeal, microbiological agar diffusion method and staphylococcus aureus atcc p strain, were applied. results: the dissolution of penicillin v from taropen preparation after minutes was %, independently on method applied. in case of ospen tablets these values were different, depending on the analytical assay. the statistical fisher-snedecor test for comparison of dissolution data obtained using three methods was performed. the fisher fcalc. value was lower than theoretical only in taropen case. the dissolution profiles of two examined preparations were also compared and statistically evaluated according to the fda method. the results of these calculations showed that dissolution profiles of phenoxymethylpenicillin from taropen and ospen tablets differ significantly. conclusion: performed analysis proved that capillary electrophoresis and microbiological methods can be used alternatively but only for determination of taropen dissolution. penetration of penicillin g in combination with sulbactam into nasal tissues u. frank, s. wenzler-rö ttele, w. maier, f. knapp, r. trittler, h. egle, k. kuemmerer (freiburg, d) objective: the penetration of antimicrobial agents into target tissues is essential for treatment at the site of infection. we investigated the distribution of penicillin g in combination with sulbactam in human nasal tissues. methods: to determine the pharmacokinetics of penicillin g/ sulbactam, informed written consent was obtained from patients aged between and years scheduled for endonasal operations such as septoplasty and conchotomy. after infusion of m iu of penicillin g and g of sulbactam, the concentrations of these agents were determined in serum and various nasal tissues such as septal mucosa, septal cartilage and bone. serum and nasal tissue concentrations were determined by liquid chromatography-mass spectrometry (lc-ms). results: up to , , , and hours after infusion, the mean serum concentrations of penicillin g were . lg/ml (sd ± . ), . lg/ml (sd ± . ), . (sd ± . ), . lg/ml (sd ± . ), . lg/ml (sd ± . ) and . lg/ml, while the mean serum concentrations were . lg/ml (sd ± . ) . lg/ml (sd ± . ), . lg/ml (sd ± . ), . lg/ml (sd ± . ), and . lg/ml (sd ± . ). mean penicillin g and sulbactam concentrations in septal mucosa decreased from . and . lg/g ( st hour) to . and . lg/g ( th hour), respectively. in septal cartilage, the highest tissue concentrations were observed hours after infusion, with mean penicillin g and sulbactam concentrations of . and . lg/g, respectively. the mean penicillin g and sulbactam concentrations in bone decreased from . and . lg/g ( st hour) to . and . lg/g ( th hour), respectively. the regression analysis of the data showed that h after administration of the drug combination, the levels still exceeded the -fold mic of methicillin-susceptible staphylococcus aureus, streptococcus pyogenes, moraxella catarrhalis and haemophilus influenzae. conclusions: our data support the use of the drug combination in perioperative prophylaxis and the treatment of ent (nasal and paranasal) infections due to common bacterial pathogens. mic vs. kill-curve based pharmacokinetic/ pharmacodynamic modelling of activities of cefpodoxime and cefixime h. derendorf, p. liu, k. rand, b. obermann (gainesville, usa; munich, d) objectives: pharmacokinetic (pk)/pharmacodynamic (pd) modeling of antibiotics usually consists of the comparison between plasma pk and the mic, such as the t > mic, cmax/ mic, and auc/mic. these indices are limited due to the innate inaccuracy of the mic and the fact that it does not reflect the in vivo scenario where concentrations are not static but fluctuate between doses. an alternative is to use time-kill curves that follow bacterial killing and growth as a function of time and concentration. this study provides a systematic comparison of mic and kill-curve approaches to show the potential of both methods for antibiotic evaluation. in an example, we developed a mathematical pharmacokinetic/pharmacodynamic (pk/pd) cefepime population on pk mean (sd) parameter estimates obtained by npag analysis model to integrate the in vitro antimicrobial activity with the pk profile of two oral cephalosporines at the tissue site. methods: kill curves could be described with different combinations of maximum kill rate (kmax), drug concentration at half-maximum effect (ec ) and bacterial growth rate (k ) that resulted in the same mic in each scenario. in the experimental part, bacterial time-kill curves of cefpodoxime and cefixime against four bacterial strains were compared in in vitro kinetic models in which previously measured human pharmacokinetic profiles of unbound antibiotic were integrated. results: different combinations of kmax, ec and k can yield the same mic and consequently the same mic-based index. however, depending on the combination of pd parameters, kill curves may predict quite opposite outcomes in both scenarios. ec values of cefpodoxime and cefixime were consistent with their respective mic values. both antibiotics had similar high potency against h. influenzae (ec : . mg/l) and m. catarrhalis (ec : . mg/l), while the potency of cefpodoxime against s. pneumoniae strains was about -fold higher than that of cefixime (ec s/ sensitive: . vs . mg/l; ec s/intermediate: . vs . mg/ l). simulations showed that cefpodoxime will have higher bacteriological success against s. pneumoniae than cefixime. conclusions: simple comparison of exposure and mic may not be sufficient to evaluate anti-infective efficacy. kill curves provide a more detailed approach in predicting antimicrobial effects. the developed emax model effectively described the pharmacodynamics of cefpodoxime and cefixime. cefpodoxime ( mg bid) has higher tissue penetration and antimicrobial efficacy than cefixime ( mg qd) against s. pneumoniae. bacteriologic efficacy of intravenous piperacillin/ tazobactam and ampicillin/sulbactam for infected diabetic foot ulcers objectives: to test the efficacy of single-agent empiric treatment, either intravenously administered piperacillin/tazobactam (tzp) ( g/ . g q h) or ampicillin/sulbactam (sam) ( g/ g q h), of moderate to severe infected foot ulcers in patients with diabetes. in this open-label trial, adults were randomized to receive tzp or sam for up to days. patients with polymicrobial infections involving methicillin-resistant staphylococcus aureus also received vancomycin g q h. samples for bacteriologic evaluation were taken at baseline from infected ulcers and blood to document causative pathogen(s) and test for antimicrobial susceptibility; samples were taken as clinically indicated at the end-oftreatment and test-of-cure visits ( - days post-treatment). to minimize risk of contamination, samples were to be obtained by aspirate, curettage, or biopsy rather than by swabbing. results: a total of of the patients in the study were bacteriologically evaluable. the most common causative pathogens in monomicrobial infections were s. aureus, streptococcus agalactiae, enterococcus faecalis, and pseudomonas aeruginosa. about % of patients in each treatment arm had polymicrobic infections. the combination of s. aureus plus s. agalactiae was most common. the median duration of treatment was days for both groups. the bypathogen bacteriological success rates were similar in both treatment groups for s. aureus, s. agalactiae, and e. faecalis. tzp had an eradication rate of . % for p. aeruginosa; sam is not active against p. aeruginosa, and patients with p. aeruginosa in the sam group (n = ) were discontinued from the study . conclusions: previous studies have shown that tzp at a dose of g/ . g q h was effective for treatment of diabetic foot infections. our study confirmed that less frequent administra-tion of tzp at g/ . g q h was sufficient to attain bacteriologic success rates of . % for s. aureus, . % for s. agalactiae, . % for e. faecalis, and . % for p. aeruginosa in the bacteriologically evaluable population. this study differed from previous studies of infected diabetic foot ulcers, which found gram-negative enterics to be more common than p. aeruginosa as causative pathogens. however, it is consistent with data showing that p. aeruginosa has recently become the gramnegative pathogen most frequently isolated from soft tissue infections in europe, north american, and latin america. penetration of beta-lactamase inhibitors tazobactam and sulbactam in severe acute pancreatitis objectives: despite of high standard intensive care and surgical management, acute necrotising pancreatitis is still related with an extremely high mortality rate. this is determined by local infectious complications, especially in necrotising areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotising pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of beta-lactamases compared to those of beta-lactam antibiotics alone. some bsp has been shown to penetrate rapidly and efficiently into pancreatic tissue. the penetration of bli into inflamed pancreatic tissue has not been investigated yet. methods: addressing the penetration capability of bli, a clinical trial was designed to investigate the penetration of tazobactam (taz, n = ) and sulbactam (sul, n = ) in patients with severe necrotising pancreatitis undergoing pancreas surgery. samples were taken from blood, necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of . g taz or . g sul. concentrations of bli were determined by hplc/ uv. the aimed concentration for full enzymatic effect of bli should be lg/mg (taz) and lg/mg (sul), respectively. results: mean plasma concentrations at . h after application were . ± . lg/ml (taz) and . ± . lg/ml (sul background: uti is one of the most common infectious conditions treated in general practice and represents a major part of abstracts antibiotic use in the community. it is of major importance to know, how we treat these infections correctly, i.e. maximum efficacy with the least amount of drug in order to reduce the risk of development of resistance. materials and methods: uti was induced in anesthetized female nmri mice via intraurethral inoculation of - cfu of e. coli. one day later, treatment was started with - hour dosing schedules with - different doses of each drug securing a wide variation of time > mic and auc/mic. h after the last dose mice were sacrificed and urine collected, and the bladder and both kidneys removed. bladder and kidneys were homogenized before cfu determination. the drugs used were the cephalosporins, cefuroxime (cef, mic = mg/l) and ceftriaxone (cro, mic = . mg/l), and the penicillin, mecillinam (mic = . mg/l). the pk of the drugs in serum was determined in similar mice as well. the two cephalosporins were studied together (cef has a t ½ of min and cro a t ½ of min, respectively, in mice). pd parameters were calculated from serum total antibiotic concentrations, since protein-binding is a minor issue in the urine, and the relation to cfÚ s estimated by the hill-equation. results: all drugs reduced cfÚ s in urine and kidney tissue by - logs, but little effect was found in the bladder tissue. for the two cephalosporins combined time > mic in % of dosing interval best described the correlation with cfÚ s in urine (r = . , p < . ) and in kidney tissue (r = . , p < . ), respectively, while no such correlation was found in the bladder tissue, neither did auc/mic reveal any correlation with cfÚ s in urine or any organs. the same was found for mecillinam, i.e. no correlation for auc/mic vs. cfÚ s in urine or organs, while time > mic % significantly (p < . ) correlated with cfÚ s in urine (r = . ), and kidney tissue (r = . ), respectively. the time > mic% for maximum efficacy was around - % for all three antibiotics. conclusion: the optimal pkpd parameter for efficacy of betalactam antibiotics in uti is the time > mic. maximum effect is seen for time > mic for - % of the dosing interval, when serum total drug concentration is used as surrogate parameter. in humans this would call for tid dosing of mecillinam and cefuroxime, and od dosing for ceftriaxone. comparative performance of different methods to simulate drug exposure variability in a population v.h. tam, g.l. rosner (houston, usa) objectives: stochastic pharmacokinetic (pk) forecasting such as monte carlo simulations (mcs) are increasingly being used to predict the pk variability of antimicrobials in a population, based on data from relatively few subjects. however, various mcs approaches may significantly differ in the accuracy and precision of the predictions. we compared the performance of different mcs approaches using a dataset with known parameter values and dispersion. methods: concentration-time profiles for subjects after an intravenous bolus of mg were randomly generated using elimination rate constant (k) . ± . h) (mean ± sd) and volume of distribution (v) ± l. normal distribution of parameter values and no correlation between k and v were assumed. system noise was incorporated as a linear function of drug concentration. using these concentration-time profiles, the best-fit parameter estimates were determined by the standard two-stage method. four methods were subsequently used to simulate the auc -inf of the population by mcs, using the central tendency and dispersion of the following in the subject sample: ( ) k and v; ( ) clearance and v; ( ) dose/clearance; ( ) auc -inf. in each scenario, , subject simulations were performed with adapt ii, using normally distributed input parameter(s) with means and variances set to the fitted values. results: reasonably good parameter estimates were obtained (k = . ± . h) ; v = . ± . l). compared to true auc -inf of the population ( . ± . mg h/l), the simulated auc -inf by various methods were: ( ) . ± , ( ) ± , ( ) ± , and ( ) . ± . mg h/l, respectively. conclusions: our results suggest that various mcs approaches may predict pk variability in a population differently. the most realistic approach appeared to be based on the variability of auc -inf in the subject sample. our observations are consistent with statistical principles concerning estimation. this method did not amplify variability of the model parameters and was the least likely to be associated with model misspecification. in objectives: fluoroquinolone treatment of humans and animals can rapidly select for organisms with increased resistance to these antibiotics. animal models are key to investigating in vivo antibiotic concentrations that prevent selection of resistance (e.g. the mutant prevention concentration (mpc) concept), but use of such models requires validated procedures for extraction and analysis of fluoroquinolones from relevant tissues. here, we report the validation of a method to quantify the concentration of enrofloxacin, and its metabolite ciprofloxacin, extracted from chicken liver, caecal contents and serum following treatment with baytril tm (enrofloxacin). methods: the extraction procedure described by wiuff et al. was validated for fluoroquinolone analysis, in pig tissues, by fortification of target matrices with enrofloxacin and ciprofloxacin. norfloxacin was added as an internal standard to all samples. samples were homogenised and extracted with acetonitrile ( ml), centrifuged and the supernatants retained for analysis. the extracts were diluted with distilled water and analysed by hplc equipped with fluorescence detection. the analytes were chromatographed on a c reverse phase column and eluted with an isocratic potassium phosphate buffer/ acetonitrile system. results: liver samples were fortified with . , . and . lg/g enrofloxacin and provided recoveries of . , . and . % respectively. quantification of ciprofloxacin at the . lg/g level was not possible due to interference from sample co-extractives, but the recoveries at . and . lg/g were . and . %. for caecal content samples, enrofloxacin recoveries were . and . % at . and . lg/g respectively, and ciprofloxacin . and . %. the recovery from serum was much higher, samples fortified at . , . and . lg/ml, enrofloxacin and ciprofloxacin yielded recoveries of . , . and . %, and . , . and . % respectively. selected samples were also analysed by zonal microbial growth inhibition bioassay and hplc equipped with ms/ms detection; the data were broadly similar between all methods. conclusion: a valid method for the analysis of fluoroquinolones in chicken liver, caecal content and serum has been established, and will be applied to in vivo mpc studies. where sample matrices interfere (e.g. liver, caeca), bioassay or lc-ms/ ms must be used to provide valid results. the ecological effects of norfloxacin and pivmecillinam (piv) on the periurethral and vaginal flora in women with recurrent urinary tract infection objectives: to compare the ecological effects on the periurethral and vaginal microflora and time to normalisation of orally administered norfloxacin (nflx) and pivmecillinam (piv) in women with recurrent lower uti. methods: women with recurrent lower uti participated in a randomized, single blind parallel multi-centre study. twentyfive women, aged - years, with a positive nitrite test and symptoms (urgency, frequency, dysuria and/or suprapubic pain) of lower uti were included. only patients with an uti caused by e. coli or klebsiella spp were evaluated. key exclusion criteria were; menopause; pregnancy or breast feeding, known hypersensitivity to study drugs, antibiotics within the preceding month, impaired liver or kidney function, known or clinically suspected pyelonephritis, complicated uti and/or gastrointestinal infection. study drugs: seven days treatment of either nflx mg bid or piv mg tid. samples from midstream urine, periurethra and vagina were obtained before start of treatment day and at two follow-ups day - and - . informed consent was obtained. the ethical review committee and medical product agency approved the study protocol. results: nineteen patients ( nflx and piv) fulfilled the inclusion and exclusion criteria. no differences of patient characteristics were seen between the two groups. at the initial visit, more nflx patients were colonized with aerobic bacteria, although no differences were seen for anaerobic bacteria. the e. coli strains were suppressed markedly by nflx and piv in both locations. s. epidermidis increased more markedly following piv treatment compared to nflx in the periurethral location. in the piv group e. faecalis was less frequent initially, increased at visit , and returned to pre-treatment numbers at visit . for the nflx group, more patients were colonized initially with e. faecalis, with a decrease at visit . lactobacilli decreased slightly in the periurethra in the nflx group, whilst no changes were seen for piv. bacteroides spp. decreased more markedly for nflx. restoration of the pre-treatment colonization levels occurred gradually, except for e. coli being markedly suppressed by nflx throughout the study period. the bacterio-logical outcome of the urinary tract infection both at the shortterm and long-term follow up was successful. conclusions: limited ecological effects on the microflora were seen following treatment with nflx and piv, a benefit for antibiotics used for treatment of patients with uti. antibiotics influence release of il- and il- by kb cells and gingival fibroblasts s. eick, m. lausse, s. schwarz, p. wachter, w. pfister, e. straube (jena, d) objectives: in general, antibiotics are used in defense against pathogenic bacteria. nevertheless, side effects such as immunomodulatory activity should be considered. the purpose of this study was to determine the effect of antibiotics normally used in periodontal treatment on the release of il- and il- by kb cells and gingival fibroblasts. methods: kb cells and primary gingival fibroblasts were infected with actinobacillus actinomycetemcomitans y (a.a.) and porphyromonas gingivalis atcc (p.g.). the antibiotics doxycycline, tetracycline, minocycline, metronidazole, and moxifloxacin were added in concentrations ranging from . mic to lg/ml. after , and h supernatants were obtained and the levels of il- and il- were measured by elisa technique. additionally, after h the number of surviving bacteria was enumerated. results: minocycline and moxifloxacin in concentrations predicted in serum as well as lg/ml were effective in killing planctonic a.a. and p.g. as well as adherent and intracellular bacteria. low levels of both interleukines released from kb cells and fibroblasts infected by p.g. were found only after addition of tetracycline up to lg/ml. metronidazole, moxifloxacin and tetracycline in subinhibitory concentrations enhanced the release of il- from non-infected and a.a.-infected fibroblasts (up to pg/ml) after h, contradictory lg/ml tetracycline and minocyline reduced the release of il- . il- in the supernatants of kb-cells was detectable only after addition of lg/ml moxifloxacin. each lg/ml of tetracycline and minocycline suppressed totally the release of il- and il- from non infected and infected fibroblasts, but not from kb cells. metronidazole and moxifloxaxin in high concentrations promoted the release of il- . conclusions: antibiotics influence the release of il- and il- . besides the good antibacterial effect especially minocycline might suppress inflammation. nevertheless, the killing of bacteria as well as a possible inhibition of virulence factors (bacterial proteases of p.g. by tetracycline) might influence the enhanced or reduced release of these cytokines. exceedingly infrequent infectious complications during orthotopic liver transplantation: tubercular peritonitis introduction: tuberculosis (t) is a very infrequent complication in patients (p) who undergo solid organ transplantation: in liver transplant recipients a . - . % rate has been retrieved by a literature update. in these p t occurs within months, and less than % of cases are observed after a repeated transplantation. pulmonary localization predominates ( % of p), followed by disseminated t ( %), while extrapulmonary t is an exceedingly rare event, reported only nu anectdotal p descriptions. pathogenetically, a t peritonitis follows disseminated infection or a primary t enteric localization, and is borne by a % mortality rate. case report: a -year-old female p with a mute t personal and familial history and normal chest x-ray, underwent liver transplantation years ago, because of a hcv-related decompensated cirrhosias. four years later a novel graft was needed, after the development of end-stage hepatic insufficiency due to massive hcv re-infection. one year later (while of cyclosporin treatment), abdominal pain and ascites formation prompted admission, and m. tuberculosis sensible to all tested drugs was repeatedly isolated from an abdominal fluid with an elevated lymphocyte-monocyte count. associated ethambutol, isoniazid, streptomycin and levofloxacin (this one replaced by cycloserine after months), led to a complete clinical and bacteriological cure already achieved after months, although a -drug anti-t treatment was continued for year. conclusionas: in our rare case report, the selected anti-t chemotherapy showed a successful efficacy and tolerability profile, with contained untoward events despite a very critical clinical context, due to the severity of t complication, and the broad spectrum of problems related to graft and all associated issues. in order to obtain an early diagnosis, an elevated clinical suspicion should be maintained for this rare complication too, by recommending microscopy and culture search of mycobacteria on ascitic fluid. methods: from january through december , a prospective cohort study was conducted to determine the rate of bacterial nosocomial infection in renal transplant recipients. the patients were divided in two groups according to the origin of the allograft: cadaver or living donors. enterobacter cloacae strains, the most prevalent multidrug-resistant bacterial organisms isolated from uti were determined using genomic analysis with pulsed-field gel electrophoresis (pfge). results: one hundred sixty-three patients who received renal transplants were reviewed during the hospitalization. one hundred and ten ( . %) renal transplanted were from cadaver and ( . %) from living donor. the median of length of stay in hospital was days (range, - days) from transplant of living donor and days (range, - days) from transplant of cadaver (p < . ). twenty-one ( . %) living donors recipients and ( . %) cadaver donors recipients had bacterial infection episodes (p = . ). post-transplant nosocomial infections diagnosed during the hospitalization were uti ( . %), ssi ( %), pneumonia ( . %), bloodstream catheter-related infection ( . %) and others ( . %). risk factors for uti in the multivariate analysis included cadaver donor recipient; substitution of the initial immunosuppressive regimen; days of urinary bladder catheterization and length of stay in hospital before the infection. substitution of initial protocol of the post-transplant immunosuppressive regimen and the surgeon was also associated with ssi. six different e. cloacae multidrug-resistant to antibiotics dna profiles were detected in uti of our recipients and hospital dissemination was documented. conclusions: uti was the single most important type of hospital infection in renal transplant recipient and a significant difference in the incidence of uti was found comparing living donor and cadaver donor recipient. the high incidence of uti in the early period post-transplant suggested that the operative manipulation of the urinary tract may be an important causative factor for the development of uti. usefulness of cmv antigenaemia assay after liver transplantation recurrence rates were higher (p < . ) in symptomatic infections. underlying liver diseases of the recipients, infectious complications other than cmv, rejection rates, and survival rates were not different between symptomatic and asymptomatic cmv infections (p > . ).conclusion: about an half of the recipients experienced cmv reactivation, mostly within days post-transplantation. thirty percents of reactivation were symptomatic. peak value and duration of cmv antigenaemia were significantly higher in symptomatic infections than those in asymptomatic infections. toxoplasma gondii infection in bone marrow transplant recipients: the polymerase chain reaction-blood sample combination in diagnosis and early detection a. sensini, r. castronari, c. ferranti, t. aloisi, f. aversa, f. bistoni (perugia, i) objectives: toxoplasmosis is a rare and frequently fatal complication in bone marrow transplant recipients. it presents with local brain lesions or disseminated infection. usually, the diagnosis is based on histologic confirmation and observation of typical lesions on radiologic imaging, which resolve after appropriate therapy. the aim of this study was to stress the pivotal role of the polymerase chain reaction (pcr) in diagnosis and early detection of toxoplasma infection. clinical microbiology and infection, volume , supplement , methods: from january to july two hundred and thirteen patients underwent allogeneic peripheral blood stem cell transplantation (pbsct). in the presence of symptoms suggestive of cerebral, pulmonary and disseminated toxoplasma infection, biological samples (blood, serum, csf, pleural fluid, bal) were collected and examined by nested pcr. results: fourteen cases of toxoplasmosis were identified, having an incidence of . %. ten patients manifested cerebral toxoplasmosis, two had pulmonary infection and two disseminated. the overall mortality in this group, not necessarily due to toxoplasmosis, was % ( / ). the pre-transplant recipient serostatus was known in patients: were seropositive, but it is to be noted that were seronegative, whereas were unknown. mri study was performed in patients. typical lesions were observed in patients (sensitivity: %). the pulmonary toxoplasmosis occurred very early (day and ) and cerebral toxoplasmosis from day to day (mean . ) after pbsct. one case of disseminated infection had cns localization (day ) and then pulmonary (day ), whereas the second case had first pulmonary involvement (day ) immediately followed by cns. blood samples demonstrated higher sensitivity ( pcrpositive/ ) than csf ( pcr-positive/ ) in identification of the etiologic agent. all patients with toxoplasmosis were treated with pyrimethamine and sulfadiazine. the study confirms the pivotal role of pcr in early diagnosis of toxoplasmosis after pbsct. in our experience, a positive pcr signal in blood was an early sign of toxoplasma infection, therefore blood appears to be a suitable and reliable biological sample for diagnosis. moreover, the samples were pcr-negative after the start of therapy and therefore pcr can be useful to monitor the effect of treatment. finally, our study indicates that toxoplasmosis is a potential pathology even in pre-transplant seronegative subjects, suggesting primary infection. cryptosporidium associated cholangitis in a livertransplant patient c. denkinger, p. harigopal, p. ruiz, a. tzakis, l. dowdy (wü rzburg, d; miami, usa) cryptosporidium parvum is a parasite of the group coccidia. c. parvum causes self-limiting diarrhoeal illness in immunocompetent hosts and severe long standing diarrhoea in immunocompromised patients. sclerosing cholangitis (sc) caused by c. parvum is frequently found in hiv-infected patients. in the transplant recipients only two case reports exist describing sc due to c. parvum; one in an adult renal transplant patient and the others in three children who underwent liver transplantation. we report herein the first case of c. parvum associated cholangitis in an adult liver transplant patient. this -year-old male patient underwent a liver transplantation three years prior to presentation. his initial transplant failed due to acute rejection requiring retransplant within one year. the second transplant was complicated by sepsis, tacrolimus induced hemolytic uremic syndrome, leaving the patient hemodialysis dependent. in , he presented with pruritus, hypotension, severe dehydration and a three-week history of severe diarrhoea. endoscopic biopsy revealed numerous organisms in the stomach and changes in the colon consistent with cryptosporidiosis. stool cultures were negative. in addition, gamma gluytamyl transferase and alkaline phosphatase were elevated while bilirubin and ast were normal. the patient was diagnosed with colitis due to cryptosporidium and treated with azithromycin. immunosuppression with tacrolimus was continued. the patient continued to have diarrhoea, low-grade fevers and fatigue. two weeks later a cholangiogram and liver biopsy were performed. the liver biopsy revealed c. parvum lining the ductal epithelium. sc was diagnosed. the cholangiogram showed no sclerosing changes in the biliary tree, suggesting an early phase of disease. paromomycin was added to the regimen. the patient improved, and was discharged home. we believe this to be the first case of sc associated with c. parvum in an adult liver transplant recipient. objective: human herpesvirus (hhv- ) infection usually occurs during the first years of life, and its prevalence is higher than % in adult population. negative serostatus for hhv- in solid organ transplant recipients is a risk factor for fungal infection, but the incidence and severity of hhv- infections in seronegative patients has not been evaluated. our objective is evaluate prospectively seronegative hhv- solid organ transplant patients for hhv- infection by means of quantitative pcr for hhv- . methods: from february to august , we prospectly evaluated all solid organ transplant patients. patients must have a minimum follow-up of months. patients underwent basal determination of igg for hhv- . seronegative patients were included for follow-up. blood samples were obtained at , , , , , , , and days post-transplant. for viral dna analysis, following dna amplification, it was determined by hibridation in microplaque (affigene). results: in the study period, patients were evaluated ( kidney, liver, heart, kidney and pancreas, liver and kidney , pancreas , heart and kidney ). nine patients were seronegative ( kidney, liver, and heart). we analysed plasma samples. all patients but one had a negative pcr for hhv- . the patient with primary infection had a maximum of , hhv- dna copies/ml. clinically, the patient suffered from slight abdominal pain and discrete cholestasis in blood analyses; no fever or serious complications were detected. he did not receive pre-emptive treatment with ganciclovir. no fungal infections were detected. conclusion: primary infection by hhv- in seronegative solid organ transplant patients is not frequent, and in patient it appeared with slight symptoms. however, a larger study with more seronegative patients is needed. development and validation of a new real-time pcr assay for hhv- detection and differentiation of hhv- a and hhv- b d. becker, s. ziegelmaier, s. wach, t. laue, t. grewing (hamburg, d) objectives: human herpesvirus (hhv- ) has been identified as the etiologic agent of roseola infantum in infants. in adults hhv- causes complications in immunocompromised patients such as aids patients and transplant recipients. two virus variants can be distinguished: hhv- a and hhv- b. several studies have shown variant specific clinical outcome. the objective of this work was the development of a reliable, sensitive and specific hhv- detection assay, based on real-time pcr technology, which enables the discrimination between hhv- a and b. this assay ought to run with the same temperature profile as the realart lc pcr assays for hsv / , ebv, cmv and vzv (artus gmbh). methods: by sequence alignments, a region of the hhv- genome was defined, which allows the specific amplification by real-time pcr, and discrimination between hhv- a and b by melting curve analysis. assay specificity has been tested by analysing hhv- a/b reference material (abi) and cell culture supernatant from clinical isolates. cross reactivity was excluded by testing dna from related viruses and bacteria. analytical sensitivity was determined by probit analysis. a heterologous pcr system was incorporated, which serves as an internal control. a set of quantitation standards was established, for exact quantitation of the viral load. results: with the realart hhv- a/b lc pcr assay artus gmbh developed a ready-to-use assay for the detection and distinct differentiation of hhv- a and b. quantitation of the viral load within a broad linear range is possible for both variants. simultaneous amplification and parallel detection of internal control and specific sample in one reaction tube excludes false negative results. the realart hhv- a/b lc pcr assay has the same temperature profile as the realart lc pcr assays for hsv / , ebv, cmv and vzv. conclusion: the realart hhv- a/b lc pcr assay allows sensitive and specific detection, as well as subtyping of hhv- . this leads not only to a reliable hhv- diagnosis, but also to a better understanding of the role of hhv- variants in pathogenesis. due to the same temperature profile, the realart hhv- a/b pcr assay completes the realart lc pcr assays for the detection of different herpes viruses. now the parallel detection of hsv / , ebv, cmv, vzv, and hhv- a/b is possible in a single real-time pcr run. thus, clinicians receive a reliable diagnostic tool for rapid detection of herpes viruses, and especially in transplant recipients. quantitative cmv pcr in allogenic stem-cell transplant patients l. cardeñ oso, c. blazquez, r. rodriguez, j. diaz-regañ on, e. lomas, p. sanchez, r. cámara (madrid, e) objective: to assess the clinical value of a commercial quantitative plasma cmv-pcr assay (cobas amplicor cmv monitor test, roche molecular system) in allogeneic sct patients by comparing the results obtained with the pcr with those obtained with antigenaemia. to evaluate the impact of an automatised dna extraction method and a lower cut-off on pcr sensibility. methods: all patients were monitored until day post-sct with weekly antigenaemia and pcr. a total of blood samples from patients ( mieloablative and non-mieloablative; hla identical siblings and from unrelated donors) were tested prospectively. twenty-seven cmv seropositive patients (or negative with a seropositive donor) received high dose of acyclovir as prophylaxis. pcr was considered positive when more or equal dna copies/ml were detected. antigenemia was considered positive when one or more positive cells were detected (in x pmns). positive samples by antigenaemia and/or pcr (n = samples) were tested retrospectively with an automatised extraction method (magnapure). results: all cmv seronegative patients with a seronegative donor (n = ) were antigenaemia and pcr negative. seropositive patients (n = ): had a positive antigenaemia and/or pcr, with a total of cmv infection episodes. none developed cmv disease. pcr detected out of cmv episodes. overall there were positive antigenemias (belonging to patients): were pcr+, pcr negative and pcr inhibited. only patient had a positive pcr with a negative antigenaemia. automatised magnapure extraction increased the sensibility of the pcr. with this method pcr detected out of episodes in contrast with only / with the manual extraction. for samples with less than dna copies/ml a manual calculation of the number of copies was retrospectively done (using a new cut-off of copies/ml). with this lower cut-off, pcr detected out of cmv episodes. none of the negative patients by antigenaemia and/ or pcr gave a positive result with the automatised extraction method or with the lowered cut-off pcr. conclusion: quantitative cmv pcr assay showed a lower sensibility for the detection of cmv infection in allogeneic sct compared with antigenaemia. two technical modifications increased the sensibility without a decrease in the specificity: automatised magnapure extraction, and lowering the cutoff. trichosporon beigelii as a life-threatening pathogen in bone marrow transplantation recipients: the first case report from iran patients undergoing bone marrow transplantation (bmt) are profoundly immunosuppressed as a result of their intensive chemotherapy and are at high risk for opportunistic fungal infections mainly caused by candida spp and aspergillus spp. trichosporon beigelii (t. beigelii) has emerged as a life-threatening opportunistic pathogen in immunocompromised hosts. response to antifungal agents is poor, mortality is high and immunological recovery is the most important factor for a favorable outcome in patients with trichosporonosis. we present the first case of t. beigelii infection in patients undergoing allogeneic bmt in iran. a -year-old female presented with aplastic anemia, cough and fever. she had received cytotoxic drug therapy, broad spectrum antibiotics and was neutropenic. t. beigelii was repeatedly demonstrated by appropriate morphological and physiological characteristics in her sputum, nose and mouth ulcers by direct microscopy and culture, and also isolated and histopathologically recognized in post-mortem from lung and liver. trichosporonosis is likely to be recognized more frequently than apparent from the available reports. objectives: trichinellosis is a well-known problem in greenland. in west greenland a number of large outbreaks took place during the 's and 's, but since then only sporadic cases have been registered. it is unknown whether the decrease in trichinellosis cases reflects the general transition in greenland towards a more western lifestyle with less consumption of meat from wildlife, or whether it reflects insufficient case registration. the objectives of the present study were to determine the prevalence of human trichinellosis from the Õies to present times in nuuk, the capital of greenland, and in ammassalik district on the east coast of greenland, where more traditional food is still eaten, and to evaluate if changes in lifestyle had an effect on prevalence. methods: blood samples from persons collected in and persons collected in in nuuk and ammassalik district were tested for trichinella-specific igg antibodies using elisa and western blot analyses. background information was obtained from questionnaires from the study. results: in the trichinellosis prevalence was . % in nuuk and . % in ammassalik district, while the prevalence in was . % in nuuk and . % in ammassalik district. persons living in the settlements of ammassalik district had higher trichinella-specific antibody prevalence than persons living in the town of tasiilaq. the following risk factors were significant in a univariate analysis: ethnicity, age, place of living, dietary habits, intake of seal meat, and hunting. a multivariate model was constructed consisting of the variables: ethnicity, age, place of living, diet group, intake of seal meat, and hunting. the variables were removed stepwise in the following order: seal meat (p = . ), hunting (p = . ), age (p = . ), and ethnicity (p = . ). the final multivariate model consisted of diet group and place of living, both being significant. thus, living on a greenlandic diet and living in the settlements in ammassalik district implied higher relative risk of being trichinella seropositive than living on a diet dominated by imported food items and living in nuuk. conclusion: as lifestyle in the settlements is more traditional compared both with tasiilaq and nuuk, these results indicate that the decline in human trichinellosis cases in the th century most likely reflects the transition from traditional greenlandic lifestyle to more western dietary habits. study on prevalence of toxocara cati in vagrant cats of sari township, iran, m sharif, p. ziapoor, h. ziaee, s. kholami (sari, ir) objectives: toxocara cati is one of the important and prevalent parasites in cats and common between human and animals. ingestion of larvae of this parasite and lack of it's maturation in human body , the infected persons develop visceral larvae migrans which is known as toxocariasis. considering the significance of transmission of this parasite in human and due to the increase in vagrant cats in sari township, this study was performed in order to determine the severity and prevalence of toxocara cati in these cats in sari township. methods: this cross sectional descriptive study was done on vagrant cats ( males and females) during april to october . sampling was done randomly at different regions by net. the specification of vagant cats were recorded after hunting and were deeply anaesthetized by chloroform to annihilate them. the intestinal content were examined for the presence of parasites and counting of their number was done finally after fixation and staining by recommended kits of reference book for genus and species identification. results: in all, ( %) cats were infected with toxocara cati, of which ( %)were males and ( %)were females. range of infection was - and mean severity of infection was . for each cat. the rate of infection at the west region was more than the other sites, and it was more in the females than males and also it was more in immature than the mature cats. conclusion: considering the merely high prevalence of this zoonotic parasite and it's hygienic significance in causing toxocariasis in human particularly in children, who are in more contact with soil and also lack of control on vagrant cats population, which are the potential risk factors, it is recommended to control personal and dietary hygiene and avoid storing the garbages in unprotected wire boxes on the streets in order to prevent the gathering of vagrant street cats. imaging in patients of pulmonary hydatid disease; review of unusual imaging appearances s. zahirifard, m. bakhshayeshkaram, m. tahbaz, k. kaynama (tehran, ir) objective: to evaluate the chest radiography and ct scan characteristics of pulmonary hydatid disease. material and methods: patients with surgically proven pulmonary hydatid cysts were enrolled for study. we reviewed clinical findings and imaging of patients. the radiological features (localization, diameter, architecture, density and other radiological signs and appearances) were determined. results: on cxr cysts were determined. on available ct scans a total of cysts were detected. no discrete cyst was detected on cxr. on available ct, a total of cysts were detected and in ct scans no cyst was detected. cysts were ruptured and patients with ruptured cysts had hemoptysis. single cysts were in patients while multiple cysts were in . median ct density was hu. respectively, it was seen on ct and cxr waterlily sign in and patients, air-fluid level in and patients and cresent sign on and of patients. inverse cresent sign and calcification were not observed on cxr s but each one was recorded on ct scans. on ct scan % of cysts were smooth and % were uniloculated. % of cysts were infected. conclusion: ct scan should be done to elucidate cystic nature of the lung masses and for accurate localization in the preoperative period. in endemic regions like iran, atypical imaging presentation of complicated pulmonary hydatid disease such as solid masses should be considered in differential diagnosis of pulmonary lesions (abstract truncated at words) the seroprevalence of coxiellosis in farmers and cattle in north-eastern turkey s. seyitoglu, z. ozkurt, u. dinler, b. okumus (erzurum, tr) objectives: coxiellosis is a worldwide zoonosis caused by coxiella burnetii. the aim of this study was to determine the abstracts prevalence of coxiellosis in cattle and farmers in north eastern turkey. methods: a total of cattle and human sera were collected in , and tested for antibodies against c.burnetii by commercial elisa test. results: the antibodies of c. burnetii were detected in ( . %) cattle and ( . %) healthy farmers. seropositivity was found in of ( . %) cattle with abortion history, and of ( . %) cattle without abortion history (p < . ). there was correlation between animals and human seroprevalance in same district. thirteen of farmers who were antibody positive had seropositive animals, and there were seropositive animals in the villages of remaining cases. it was observed that seroprevalance of coxiellosis was higher in north districts (in animals . %, in humans . %), have drier and warmer climate, than in other districts (in animals . %, in humans . %) both for humans and animals (p < . ). conclusions: the study show that coxiellosis was an important health problem both in humans and in animals in our region. an unusual case of gramineae in sublingual salivary gland l. doganci, m. calgurel, y. gungen, g. kiran (ankara, tr) foreign bodies of organic nature and parasitic elements are very rare in oral cavity and in salivary glands in humans because of their structural and functional features. here we report a very interesting case, with sublingual salivary gland involvement of gramineae (brachiaria spp.) which was thought to be a parasitic life cycle in the gland. case: y/o female teacher with a recent history of travel to mountains where she drunk and had an exposure to unsafe spring water. she had a strong stinging pain right after she drunk the water in her mouth, radiating to her neck. she developed fever, pain, lymph node swelling, difficulty of eating, weight lost and remarkable eosinophilia. she also noted a self moving small tail-like object on the orifice of the sublingual salivary gland. removal of the object revealed an interesting unusual living object. then, definitive diagnosis is gramineae (brachiaria spp) after parasitological and pathological consultation. comment: to our best knowledge, this unusual case is the first presented patient related to travel in our region. differential diagnosis has several entities including squid sperm bag sting, marine animal larvae and nematod and trematod life stages. evaluation of echinococcus granulosus prevalence using elisa and abdominal ultrasonography in a group of students staying in a state hostel in turkey objective: cystic echinococcosis, caused by larval form of echinococcus granulosus is one of the most important and widely distributed parasitic zoonotic diseases in the world. echinococcosis is a major problem in all regions of turkey but particularly prevalent in rural areas, where domestic livestock raising is common. the reported epidemiologic findings are usually based on retrospective evaluation pf surgical findings or hospital charts. well planned epidemiologic studies are limited. in this study our aim was to analyse the seroprevalance of cystic echinococosis and prevalance of lesions (with abdominal ultrasonography) in a group of young adult university students who were staying in a state hostel in bornova-izmir-turkey. method: the study group consisted students ( woman, men, aged . ± . , min , max ). informed written consent was received from each student and they were requested to fill a questionnaire form. blood sampling was performed by iv puncture and sera were obtained after centrifugation. anti echinococcus granulosus antibodies were detected by using elisa tecnique. all participants were gven an appointment for abdominal ultrasonography. results: of students ( . %) were seropositive for anti echinococcus granulosus ig g (table ). a total of students ( seropositive- seronegative) were performed abdominal ultrasonography ( . mhz transducer, sonoline, elegra-siemens). two of students ( in liver, in kidney both seropositive) had cystic lesions and were referred to surgery. well designed epidemiologic studies about cystic echinococcosis are lacking in turkey. a previous study showed seropositivity rate of . % for echinococcus granulosus. our findings suggest that cystic echinococcosis is prevalent in turkey and epidemiologic studies combining elisa and abdominal ultrasonograpy are warranted. objective: anthrax is an epidemic zoonosis in eastern part of turkey. this study was aimed to investigate the epizootiology and epidemiology of anthrax in this region. methods: animal and human anthrax cases during - period were included in this study. data were obtained formal records of health directorate, institute of veterinary control and investigation. the diagnosis of anthrax had based on clinical findings and/or microbiological procedures, including gram strains and culture of materials obtained from lesions. results: in a -years period, animal cases and human cases with anthrax were detected. of animal cases, ( . %) were sheep, others ( . %) were cattle. most cases occurred between july and september in both animals and humans. anthrax was seen most frequently in erzurum, which is the important centre for animal commencement. when the first years period ( ) ( ) ( ) ( ) ( ) ( ) was compared with last years period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ) the number of animal cases were vs. and human cases were vs. . an analysis of the yearly case distribution shows that the incidence of anthrax in this region had not been changed. conclusion: anthrax is an important health problem both animals and humans in this region. the preventive measures should be taken for decreasing the incidence of anthrax. objectives: anthrax is endemic in kazakhstan and occurs among humans at an incidence rate of . to per , population per year. during the late s, several epidemics occurred associated with home slaughter of infected animals, and a general increase in incidence was seen among animals and humans. historically, kazakhstan accounted for % of the anthrax cases reported from the soviet union; and the plains of central asia may have served as the historical source of b. anthracis for the rest of the world. in kazakhstan, the geographic distribution of anthrax among animals and humans is focal and generally associated with areas considered to be Ôhigh-riskÕ. the determinants of this focal distribution are poorly understood. in addition, strains of b. anthracis isolated from outbreaks within these foci have not been compared on a molecular or pathogenic basis. methods: work has been initiated to study the geographic distribution and potential environmental preferences of b. anthracis using geographic information system (gis) technologies. results: the gis was used to map a set of b. anthracis strains from several outbreaks between and to evaluate geographic patterns. the gis is also being used to derive a historical database of spatially explicit human and livestock outbreaks from to for spatial analyses on the nationwide distribution of b. anthracis. this database contains information on the location of the anthrax incident, the year and month of the occurrence, whether the incident involved livestock or human, the outcome of the disease, and biological information about the strains. conclusions: although in a preliminary stage of application, the arcgis software can be used to map loci of anthrax occurrence. in the future, these maps will be used to identify factors that influence the occurrence and spread of the pathogen and to prevent infection of livestock or humans. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency (dtra) under the project «kb - -al- » and administered by u.s. civilian research and development foundation (crdf). objectives: the anthrax situation in the country was unfavorable from the s through the s. until the s, anthrax in kazakhstan was an occupational disease in animal husbandry. after , the practice of raising livestock for private use increased. this report shows that, at the same time, the incidence of anthrax in persons involved in this practice increased significantly. methods: in order to characterize the epidemic process, we used methods of descriptive epidemiology, using epidemiological variables (who became ill, where and when, trends of disease by time, sources, transmission factors of b. anthracis, and the age, gender, and occupation of persons who become ill with anthrax). results: anthrax occurs in rural areas among private owners of livestock and their families and persons hired to assist with the slaughter. in the last decade, . % of cases of anthrax illness in humans were caused by the slaughter of domestic livestock. of these cases, . % were unemployed rural residents, . % were shepherds working for cooperatives, . % were blue-collar workers, . % were white-collar workers, and . % were students. a single source, path and factors of transmission of b. anthracis are typical of this type of the disease. in recent years, the majority of cases involve infection of several persons during the slaughter of a single diseased animal. the source of infection is cattle in . % of cases, sheep and goats in . % of cases, horses in . % of cases, and soil in . % of cases. the factors of transmission of b. anthracis to humans are contact with the meat of livestock during slaughter and butchering in . % of cases, vector-borne transmission in . % of cases, and soil in . % of cases. a large number of human cases occur in july through september. conclusion: in kazakhstan today, the prevalent sub-type of anthrax disease in humans involves non-professional activity at home. this requires changes in the tactics of preventing the disease. the research described in this abstract was objectives: severe acute respiratory syndrome (sars) is an emerging infection caused by a novel coronavirus known as sars-cov. during the early phases of the disease, the presence of the replicative intermediates of sars-cov has been shown in pbmc from patients. no information is available on the ability of sars-cov to stimulate ifn induction, although ifn-gamma may be activated in sars patients. we investigate the capability of sars-cov to give rise to a productive infection in normal pbmc, in parallel with the ability to activate ifn-alpha andgamma gene expression. methods: normal pbmc were infected with a moi . . virus progeny formation was measured at subsequent time points, by back-titration of cell lysates on vero cells. cell mortality and apoptosis were detected by trypan blue and propidium iodide staining. in order to detect virus-specific plus-and minus-rna strand, total rna extracted from sars-cov-infected pbmc was retrotranscribed in the presence of the sense and antisense primers, respectively, targeting the replicase gene. measurement of sars-cov genomic rna was performed by quantitative real time rt-pcr. ifn induction was performed by exposing pbmc to sars-cov at different moi, ranging from . to . induction of ifn-alpha and -gamma was analysed by measuring their mrna levels by limiting dilution rt-pcr. sensitivity of sars-cov replication to exogenous ifn-alpha and -gamma was tested in vero cells pre-exposed to the individual cytokines of to a combination of both. results: in unstimulated pbmc, no infectious viral progeny formation was detected up to day post-infection. nevertheless, minus-strand and genomic sars-cov rna peaked at hours post-infection. dose dependent induction of both ifnalpha and ifn-gamma mrna was observed, that was most evident at moi . a combination of these two cytokines was shown to strongly inhibit sars-cov replication in vero cells. our results show that sars-cov is able to infect normal pbmc. however, this appears to be only a transient phenomenon, followed by a progressive disappearance of both virus-specific rna strands, with no infectious virus progeny production. moreover, sars-cov appears to have the intrinsic ability to activate dose-dependently both ifn-alpha andgamma gene expression in pbmc cultures. our results can be pathogenically relevant to the inflammatory events occurring in the diseased tissues that can be mediated by the endogenous activation of inf system. objectives: trichoderma species are potential candidates for biocontrol of plant pathogenic fungi and cellulase producers of biotechnological importance. on the other hand, they are emerging as opportunistic pathogens of immunocompromised and dialized patients. this study was designed to examine the ability of clinical trichoderma isolates to produce extracellular proteases and to screen them for toxicity to mammalian cells. methods: supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with different chromogenic p-nitroaniline substrates. trypsin-and chymotrypsin-like activities were separated by sephadex g- column chromatography and their ph-dependence was studied. sperm toxicity bioassays were performed by exposing boar sperm suspension to trichoderma-methanol extracts, the motility of spermatozoa was estimated by phasecontrast microscopy. mitochondrial membrane damage in spermatozoa was studied by epiflurescence microscopy using the jc- /propidium iodide staining. results: the production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities was common among the examined strains. separation of trypsin-and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. relatively high activity levels were detected between ph and suggesting that the different isoenzymes have different ph characteristics. more than % of the spermatozoa were motile in a non-exposed control sample and in a sample exposed to microliter of methanol. more than % of the spermatozoa were immotile in samples exposed to t. longibrachiatum uamh , atcc , cm and t. harzianum cbs in a concentration of . mg biomass ml- , indicating that these strains produce heat-stable substances toxic to sperm cells. these four strains were found to inhibit the motility of boar spermatozoa even at a low concentration ( % reduction of motility in the case of microgramm ml) of extended boar sperm) and to induce dissipation of mitochondrial membrane potential. other studied strains had no toxic effect. conclusions: production of extracellular proteolytic enzymes and toxic metabolites are among the potential virulence factors of trichoderma strains as emerging fungal pathogens. this work was supported by grants f of the hungarian scientific research fund and of the academy of finland. occurrence of scabies in patients and in the staff of healthcare facilities objectives: scabies is an itching dermatosis which continues to be a frequent health problem. long-term follow-up of the epidemiological situation confirms a thirty-year cycle for which there is no satisfactory explanation. methods: standard methodology of diagnostics is based on a) subjective sensations in the patient b) objective dermatological finding c) positive epidemiological history d) laboratory demonstrations of causative agent e) remission of symptoms upon specific therapy. cases of scabies diagnosed and reported by dermatologists nationwide are included by the public health service in the central system epidat (niph, prague). results: the last two documented epidemic waves of scabies affecting the czech republic peaked in and in . specific morbidity is the highest among persons - years of age (it closely connected with sexual activity-the source of infestation in % of adults was a sexual partner and that is confirmed by an almost identical trend in reported scabies morbidity and fresh cases of syphilis in - years), but the morbidity trend has been falling sharply after the year . however, morbidity in persons over years of age has been gradually rising over recent years. group epidemics ( ) have been repeatedly reported in such facilities as departments of gerio-psychiatry, institutions for long-term chronic patients, old folks homes, social care institutes and charity facilities. that occurs in bed-ridden subjects hospitalized on a long-term basis, the mentally retarded and the superannuated, in whom the scabies is of a nosocomial character. the affected staff of healthcare institution is very often the source of further transmission to others, namely to immobile patients, as well as to especially vulnerable individuals under higher risk of infection. analysis of the reported cases revealed a % share of healthcare workers in this infection. under greater risk are senior nurses ( %), junior nurses ( %), auxiliary paramedical staff ( %)and the last of all physicians ( %). scabies as an occupational disease occurs almost exclusively in the healthcare sector. conclusions: scabies poses on increased risk for the younger population (active scabies) and the elderly population (passive scabies). it poses a great occupational risk in the paramedical healthcare staff carrying out nursing or managing services. it is necessary to consistently observe preventive and repressive epidemiological measures in the population. fire ants, the new public health problem in iran k. akbarzadeh, m. nateghpour, s. tirgari (tehran, ir) ants are probably the most successful of all insects. they are present almost in all countries and all places. a few of them can bite, sting and squirt formic acid. formica rufibarbis and a few the others are secondary hosts of dicrocoelium dentriticum in iran. but the newest public health problem depending on ants in the country is biting and stinging of fire ant pachycondyla sennaarensis (formicidae; hymenoptera) in south and southeastern iran. ecology, biology and morphology of p. sennaarensis were considered in iranshahr county (southeast of iran) where has been much infected because of the ant. according to the survey over % of questioned individuals had bitten at least once with the ant. usually the effects of the stings are mild but this ant is capable of multiple stinging and this can induce annoyance people especially in children. objective: tick play as important role in diseases transmission to human being. different tick-borne diseasea including borreliasis, cchf have been reported from iran. method: an attempt was made to determine the fauna and geographical distribution of soft and hard ticks in this area from october until april . about % of the villages were selected randomly and tick collection was carried out using standard method. ticks were collected from indoor resting and hiding places in villages as well as inside stables, rodent borrows, on sheep, lamps, goat and cattel by monthly in each season. result: ticks were collected and identified using national systematic key. the copmosition and frequencies of them were as follows: ornithodoros lahorensis ( %), argas persicus ( . %), rhipicephalus bursa ( . %), rhipicephalus sanguines ( . %), haemaphysialis concinna ( . %), haemaphysialis sulcata ( . %), haemaphysialis punctata ( . %), hyaloma schulzei ( . %), hyaloma marginatum ( . %), hyaloma dentriticum ( . %), hyaloma asiaticum( . %). conclusion: distribution and ferquency of tlck was differ in various season and location. evaluation of the use of saliva to soothe bloodsucking arthropod bites (dedicoat m et al, , j infect dis : . the conditions for hhv- transmission are met when i) a child's skin responds to the bite of a blood-sucking arthropods, and ii) an hhv- -seropositive mother (or caregiver) attempting to relieve the child's itching and to reduce scratching applies her infective saliva to the bite's site. the use of saliva is a traditional behavioural practice in sub-saharan africa, for healing with herbal leaves crushed and mixed with saliva and for medical practices and in the premastication of food (wojcicki jm, , br j cancer : . the human behaviour associated with the transfer of saliva from parents to infants and the use of herbal leaves treated with saliva in relation to arthropod bite could be a risk factor for hhv- infection and perhaps other infections too (such as the hepatitis b virus). methods: to evaluate to what extent saliva is used to heal insect bites on children's and adolescentsÕ skin, we draw up a questionnaire directed at students of elementary and intermediate school. two groups were tested, one from italy (a school near rome, latium, and schools in veneto) and one from a sub-saharan african country known for high transmission levels (uganda, between kampala and entebbe by lake victoria). results: the frequencies of the use of saliva were not significantly different in latium ( . %) and in veneto ( . %) so that an average frequency of . % ( / ) has been compared with the frequency of . % ( / ) from uganda (p << . ). the frequencies in the two groups are related also to a different cultural and economic development and improvement in hygiene. conclusion: we suggest that blood-sucking promoter arthropods can play a role in the spread of hhv- infection particularly in africa. therefore, a behavioural change limiting the use of saliva on the skin and to prepare herbal leaves, could be an attempt to control the spread of hhv- infection. aim of study -to find out in csf some baseline markers of inflammation and redox status for the estimation of development tendences and clinical course of pathological process in tbe. patients and methods: patients with meningeal and meningoencephalitic form of tbe treated at the infectology center of latvia were included in this study. diagnosis of tbe was confirmed by elisa, enzygnost, anti-tbe igm in blood and/or csf. traditional clinical criteria were used for the characterization of tbe severity degree. some nontraditional lab methods were used: c-reactive protein (crp) (immunoturbidimetrically) and reduced glutathione (gsh) concentration (colorimetrically) detection in csf. results: concentration of crp in csf was significantly higher in tbe patients in acute phase ( . ± . mg/l; n = ) than in reconvalescence ( . ± . mg/l; n = ), p = %. crp and gsh in about % of tbe cases in csf was under detectable values. the second part of tbe patients demonstrated tendency of the increasing of gsh during acute stage of disease ( . ± . mg%; n = ) if compared with reconvalescence ( . ± . mg%; n = ), p = %. no statistically significant differences of crp and gsh concentrations in csf were found in cases of moderate meningitis form of tbe if compared to severe meningoencephalitic form of tbe. conclusion: ) analysis of data obtained from patients with tbe gives evidence of substantial crp and gsh changes in csf in dependence on disease stage, but not on its severity degree. ) these objective biochemical data from csf investigation give proof that there are no separately moderate and severe tbe cases in clinic: they have to be interpreted always as equally severe. ) the origin of crp and gsh in csf is unknown (production in cns and/or from blood due to blood-brain barrier permeability increasing?). background: cholera is a severe disease caused by vibrio cholerae. the natural reservoir of this aquatic pathogenic bacterium is referred to as the Ôaquatic environmentÕ. our group showed that egg masses of the non-biting midge chironomidae spp. (diptera) harbour v. cholerae and provide nutrient source for their multiplication, thereby offering a new natural reservoir for the cholera bacterium. objective: the presence of viable v. cholerae on flying adult chironomids will suggest that v. cholerae can be carried through the air to other water bodies by the adult flying insects and hence a new route of infestation of water sources by the bacteria is suggested. methods: in field studies chironomid egg masses and adults were simultaneously collected from the same environment. in addition fresh, drinking quality water was left exposed or netted in the vicinity of adult chironomid hatching foci. the exposed water was monitored for chironomid egg masses and v. cholerae presence. in the laboratory those experiments were repeated with v. cholerae o tagged with green fluorescent protein. results: to date, more then v. cholerae serogroups have been isolated from chironomids egg masses and adults. in the field experiments, a clear-cut connection between netting and v. cholerae absence was noted implying that a relationship exist, between entrance of large invertebrate to water bodies and presence of v. cholerae in that water. in the laboratory experiments, the gfp tagged bacteria were found on the outer surface of the adult chironomid, in locations prone to microbial attachment. conclusions: the evidence shown here suggests that aerial transport of v. cholerae by chironomids flying adults is feasible and does lead to v. cholerae contamination of unprotected water bodies. objectives: cholera is a severe and potentially life-threatening diarrheal disease caused by certain spices of the gram-negative bacterium vibrio cholerae. many millions of cases of cholera occur annually, in epidemic and pandemic forms due to v. cholerae o and o , and also sporadically due to non-o and non-o v. cholerae strains. in this context, the survival of v. cholerae in nature is of interest from an epidemiological perspective. although the natural reservoirs for survival and multiplication of v. cholerae are far from compeletly disclosed, our previous study has shown that free-living amoebae can be reservoirs for the seventh-pandemic v. cholerae o el tor-inaba strain n , which can survive and grow intracellularly in acanthamoeba castellanii.the aim of this study was to examine the ability of different strains of v. cholerae o el tor to grow and survive in acanthamoeba castellanii, and to examine whether intra-amoebic survival of bacteria alter their pattern of resistance and sensitivity to different antibiotics. methods: v. cholerae o el tor strains were co-cultured with a. castellanii for more than two weeks. the interaction between these microorganisms was followed by viable counts of alone-and co-cultivated microorganisms. intra-amoebic growth and localization of each bacterial strain were estimated by gentamicin assay, viable count, microscopy, and pcr to detect cholera toxin gene and amoebic s rna gene disclosing symbiont-host association. results: the results show that examined v. cholerae o el tor strains multiplied and survived inside trophozoites and cysts of a. castellanii. the bacterial internalization was in cytoplasmic compartment of the amoebae cells. the relation between these microorganisms in co-cultures could be classified as symbiosis, since presence of the amoebae enhanced growth of bacterial strains, and presence of the bacteria did not affect amoebic growth. the intra-amoebic survival of bacteria did not alter their pattern of resistance and sensitivity to different antibiotics such as ampicillin, gentamicin, and tetracycline. conclusions: this study shows a facultative intracellular behaviour of examined v. cholerae o el tor strains, which is in contrast to the general held view, which considers the bacterium to be extracellular microorganisms. the clinical importance of free-living amoebae is their possible role as Ôtrojan horseÕ. the genetic variation of vp and vp antigenic proteins was studied in g strains recovered in palermo from to . vp sequence analysis showed that strains, recovered during - , though cross-reacting with g -st : mab, belonged in fact to genotype g and were % identical to g strains recovered in palermo from . sequence comparison of g strains grouped them into three sublineages (ia to ic) containing respectively viral strains collected in - , and - . amino acid sequences were well conserved within each sublineage and a peculiar single amino acid insertion was observed in the variable region of all sublineage ic strains. both g and g italian strains exhibited p[ ] genotype and their vp -encoding sequences clustered in previously defined lineages following a temporal distribution: strains collected in - fell within lineage p[ ]- , while those recovered in - clustered in lineage p[ ]- . the variety of their deduced amino acid sequences enabled us to describe, respectively, two ( a and b) and six ( a- f) different patterns of substitutions with regard to lineage p[ ]- and p[ ]- reference strains. comparison of vp and vp gene sequences of italian and european g strains indicates that different g p[ ] populations have been circulating in europe over the years and that the latest italian strains are more closely related to recent isolates from other countries than to the reference vaccinal st strain. these results will help increase knowledge about g rotavirus epidemiology and evolution, and might be useful for future rotavirus vaccine formulation. detection of rotavirus, adenoviruses and astrovirus in gastroenteritis cases among young children, first experience with adenovirus identification by pcr and microplate hybridisation assay , adeno (all human serotypes) and astroviruses antigens (seven human serotypes) was performed by enzyme immunoassay method (dako ideia rotavirus, adenovirus and astrovirus tests, respectively). all specimens positive for adenoviruses were confirmed and identified by a pcr-microplate hybridization assay (pcr adenovirus consensus, argene, biosoft), which used adenovirus genus-specific primers and one generic and six species-specific probes defined in the va rna gene. according to our results: rotavirus was detected in cases ( . %), adenovirus in cases ( . %) and astrovirus in cases ( . %). pcr adenovirus consensus assay identified the adenovirus species from all the cases: adenovirus species f (enteric adenovirus serotype / ) in cases ( . %), adenovirus species c in cases ( . %) and adenovirus species a in cases ( . %). rotavirus gastroenteritis presented a seasonal distribution in spring, while adenovirus and astrovirus gastroenteritis presented no seasonality. conclusions: rotavirus gastroenteritis was significantly more common than adenovirus and astrovirus infections in young children in our territory. pcr adenovirus consensus technique was a useful method for the identification of adenovirus in stool specimens. eventhought enteric adenoviruses / (species f) caused the majority of acute gastroenteritis due to adenovirus, non-enteric serotypes (species a and c) were occasionally involved in the aetiology of acute diarrhoea. the study continues with the examination of great number of specimens and patients for the investigation of more useful and specific results. immigrant inpatients from until today: 'pressure' exerted over infectious disease wards r. manfredi (bologna, i) objective: foreign individuals officially living in bologna and province are with predominant origin from northern africa ( . %), eastern europe ( %), and far east ( %). the temporal trend of admissions of patients coming from outside of the european union was examined according to a broad series of variables. methods: through the electronic databases of our hospital located in a metropolitan area of northern italy, informations related to all foreign patients (p) hospitalized or followed by day-hospital centres have been collected from january up to march , . after recording all discharge diagnosisrelated group (drg) codes we focused attention on those regarding infectious diseases. results: overall discharges were , with regarding foreign p:a prevalence of the female sex ( . %) was noticed. in . % of foreign p,the mean age ranged from to years. the rate of foreigners compared with overall admitted p varied from . of year to a. maximum of . % of year . a major involvement was borne by obstetrics-gynecology ( . %), internal medicine ( . %), specialistic surgery ( . %), specialistic medicine and pediatrics ( . % each), and emergency medicine ( . %). when considering infectious disease-related drgs, tuberculosis ( cases), hiv-aids ( ), chronic viral hepatitis and related complications ( ), septicemia, central nervous system infection, and acute viral hepatitis ( episodes each), were the most frequent discharge codes followed by other infectious disease drgs ( cases). the duration of admission of foreign p was very short ( - days) in . % of cases,while it reached - days in . % of p,and - days in . % of cases, while the mean hospitalization time at infectious diseases ward was . ± . days (p < . ). even hospitalizations ended with voluntary discharge which occurred during the first day of admission in . % of p. discussion: our pilot study demostrates that the need for health care expressed by foreign p is increasingly frequent and covers a very broad spectrum of disorders with infectious diseases-associated illnesses gaining a increasing role through time. a significant problem is represented by the tendency to very short duration of hospitalization for foreign p, often ending with voluntary discharge while admissions carried out at our specialized infectious disease ward are significantly more prolonged, although they are expected to give an increased benefit in terms of effective management. change of the characteristics of natural focuses of plague and microbiological properties of plague microbes as a result of consequences of the natural calamity on aral sea enzootics of plague as that of any other transmissible feral herd [feral nidal] infection are determined by the interaction of a disease's causative agent and its warm-blooded carriers under specific ecological environment. introduction and objectives: change of the characteristics of natural focuses of plague and microbiological properties of plague microbes as a result of consequences of the natural calamity on aral sea. methods: conventional microbiological research methods were used, when studying microbiological properties of strains of plague microbe from kyzylkum natural focuses of plague isolated from rodents, ticks and fleas. results: strains from the kyzylkum natural focuses of plague have all determinants of virulence, show characteristics that allow attributing them to the continental variety, i.e. glycerin digestion, do not ferment rhamnose and decompose arabinose; they have nutrient need that is typical for the strains from the central asian desert focus. there are differences in the need for amino acid leucine, whereas strains from ustyurt and gissar focuses are dependant on it. out of strains from the kyzylkum natural focus only turned out to be dependant on luecine. there were identified stains from the kyzylkum natural focus that had essentially different properties as compared to the majority of isolated cultures, in particular, those can not synthesize fraction of the plague agent; have growth factor of different nature, i.e. lower dependence on phenylalanine. plague bacteria strains circulating in rodent populations in the kyzylkum natural focus are characterized by low virulence in white mice and guinea-pig, although they have all the determinants of virulence. when studying resistance to antibiotics, there were identified strains non-susceptible to streptomycin ( %). conclusions: thus, studying the properties of the causative agent of plague is of great importance for solving problems of natural focuses and understanding patterns of fluctuations in the epizootic situation in a certain territory. this understanding is an integral part of determining the epidemic potential of natural focuses, and is used in organizing epidemiological surveillance of the infection. objectives: toll-like receptors (tlr) play a key role in the recognition of products from virtually all classes of pathogenic organisms. amyloid peptides also can stimulate the innate immune system. for this reason, we hypothetized that bacterial compounds and amyloid peptides may jointly stimulate the innate immune system. methods: the interaction of endogenous and exogenous stimulators of innate immunity was examined in primary cultures of mouse microglial cells after application of defined toll-like receptor (tlr) agonists [lipopolysaccharide (lps) (tlr ), the synthetic lipopeptide tripalmytoyl-cysteinyl-seryl-(lysyl) -lysine (pam cys) (tlr ) and single-stranded unmethylated cytosineguanosine (cpg) oligodesoxynucleotide (tlr )] alone and in combination with amyloid beta peptide (abeta) - . supernatants from stimulated glial cultures and unstimulated controls were analysed for nitric oxide (no), tumor necrosis factor-alpha (tnf-alpha) and interleukin- (il- ) production. results: co-administration of abeta - with lps or pam cys led to an additive release of no and tnf-alpha. in contrast, coapplication of abeta - with cpg led to a substantial decrease of no and tnf-alpha release compared to stimulation with cpg alone. cpg was the only compound investigated, which induced the release of the anti-inflammatory cytokine il- . conclusion: the additive effect of lps and pam cys and abeta may be one reason for the clinical deterioration frequently observed in patients with alzheimer's disease during infections. the substantial decrease of no and tnf-alpha release after cpg and abeta application compared to stimulation with cpg alone suggests that not all microbial products enhance the stimulatory effect of abeta on innate immunity. the cause for the divergent behaviour after activation of tlr versus stimulation of tlr and tlr probably lies in differences of the signaling cascade. hla-b is a marker for susceptibility and severity of reactive arthritis after salmonella, shigella, and yersinia infections but not after campylobacter and e. coli enteritis p. schiellerup, h. locht, k.a. krogfelt (copenhagen, dk) objectives: reactive arthritis (rea) is a well-known complication after infections with salmonella, yersinia, shigella and campylobacter. the presence of tissue-antigen hla-b is associated with rea, and hla-b may be a marker of a prolonged disease course. we designed a case-case comparison study of rea after gastrointestinal infections caused by salmonella, yersinia, campylobacter, shigella and e coli, to evaluate the impact of hla-b on attack rate and severity of rea between these bacteria. methods: between january and november consecutive patients were included from the danish registry of gastrointestinal infections, when culture positive for salmonella, yersinia, campylobacter, shigella, or e coli. all patients were addressed by a questionnaire. in this study rea includes reactive arthritis and arthralgia. debut of pain in a previously healthy joint was registered and patients were asked to mark swollen or tender joints on a drawing. a vas-scale was used for assessment of pain caused by rea. all patients were encouraged to donate blood samples for serology and hla-b typing (pcr). results: a total of patients returned the questionnaire (response rate %). only subjects with mono-infections were included. the male/female ratio was / . blood samples were obtained from ( %): campylobacter (n = ), salmonella (n = ), e. coli (n = ), shigella (n = ) and yersinia (n = ). the overall prevalence of hla-b was %, corresponding to the rate within the danish population. odds ratios for contracting rea when the individual was hla-b positive were; campylobacter . (ci: . ; . ), salmonella . (ci: . ; . ), e. coli . (ci: . ; . ), shigella . (ci: . ; . ) and yersinia . (ci: . ; . ). thus, hla-b was significantly more frequent among rea-patients with salmonella, yersinia, and shigella compared to campylobacter and e coli. the vasscore for rea was significantly higher in the groups with yersinia and salmonella. there was a significant positive correlation between vas-score and the fractions of patients that were hla-b + . conclusions: this large comparative study shows that hla-b has a significant impact on the susceptibility to contract rea after salmonella, shigella, and yersinia, but not after campylobacter or e. coli. furthermore the severity of rea as measured by vas differed among bacterial species and was strongly correlated to the presence of the hla-b antigen. objectives: the normal gut microbiota triggers experimental colitis and large intestinal manifestations of human inflammatory bowel diseases (ibd). however, the availability of animal models for the study of ileitis is limited. thus, only little is known hitherto in small intestinal ibd. recently, parasiteinduced ileitis in the mouse, mimicking characteristics of ileitis in human ibd, was proposed as a model for crohn's disease (cd). oral infection of susceptible c bl / mice with toxoplasma gondii induces a severe th -type immunopathology, which is restricted to the terminal ileum. in order to unravel the mechanisms underlying ileitis, we used this model to monitor microflora changes during ileal inflammation and determined contributions of the gut microbiota to ileal disease. methods: c bl / mice were infected perorally with cysts of t. gondii (me strain). ciprofloxacin, metronidazole, ciprofloxacin plus metronidazole (each mg / kg body weight / d, p.o.), piperacillin plus tazobactam ( mg/kg body weight/d, i.p.) were applied from day until day after t. gondii-infection. the ileal bacterial flora was analysed on day p.i. by microbiological and molecular methods ( s rdna-targeted denaturing gel electrophoresis (dgge), sequencing of clone libraries). results: microbiological and molecular approaches revealed that ileal inflammation leads to an increased total bacterial load and to drastic changes in the flora composition. in the acute stage of disease, the grampositive cocci and rods -predominant in the ilea of healthy mice -are displaced by gramnegatives, identified as escherichia coli and bacteroides sp., respectively. antibacterial treatment (ciprofloxacin, metronidazole, a combination of both, or piperacillin plus tazobactam) ameliorated disease symptoms and reduced ileal inflammation. this was also seen in spf-mice colonized by only one grampositive bacterial species. conclusions: the fact that gramnegative bacteria accumulate during acute ileitis and contribute profoundly to intestinal inflammation is well in line with similar observations in experimental colitis and in human ibd. this provides evidence that gut flora modulation is a valuable therapeutical strategy for ibd treatment. finally, the contribution of gut microbiota to ileal disease supports the use of the parasite-induced small intestinal inflammation as a model for ibd research. objectives: pathophysiologic mechanisms that contribute to brain injury in neuroinflammatory disorders include breakdown of the blood-brain-barrier, extravasation of leukocytes, cerebral hypoperfusion and vasculitis. matrix-metalloproteinases (mmps) including the collagenases mmp- and - are crucially involved in all these steps. in this study we aimed to assess the extent of in-vivo collagenolytic activity in cerebrospinal fluid (csf) by determination of the amino acid hydroxyproline, a major and exclusive degradation product of collagen. methods: paired serum and csf samples from patients with bacterial meningitis (n = ), aseptic meningitis/encephalitis (n = ), multiple sclerosis (n = ), and normal csf (n = ) were assessed. degraded collagen was hydrolysed to dissolve its major component hydroxyproline, which subsequently was determined spectrophotometrically. csf levels of mmp- and - were studied by gel zymography. in a rat model of pneumococcal meningitis localization of collagenolytic activity was performed by in-situ zymography with intramolecularly quenched gelatin. results: hydroxyproline in csf from patients with bacterial meningitis was significantly increased compared to all studied groups (p < . ) while serum hydroxyproline did not differ significantly between the groups. the amount of hydroxyproline in csf correlated significantly with the amount of mmp- (r = . ; p < . ). in the rat model in-situ zymography localized gelatinolytic activity to the subarachnoidal and ventricular space inflammation and in association to cortical lesions. the study documents a significant increase of the collagen degradation product hydroxyproline in csf of patients with bacterial meningitis. the close correlation of hydroxyproline and mmp- in the csf validates the assessment of hydroxyproline as an index for csf collagenolytic activity in neuroinflammatory diseases and supports a role for collagenases in the pathogenesis of bacterial meningitis. introduction: toxic shock syndrome (tss), is an acute, noncontagious systemic illness. it is caused by the toxin producing strains of s. aureus and the b-haemolytic streptococci and can occur in any non-immune person exposed to a tss toxin. tss is commonly associated with menstruation and tampon use, however can also be related to skin or soft tissue infections, particularly post surgical, skeletal infections or respiratory tract infection. tss is often non-immunising and recurrent menstrualassociated tss is well-described. literature suggests that tss is extremely rare, but diagnostic difficulties can lead to misdiagnosis and tss can be fatal if left undiagnosed. we report a series of three cases of tss, presenting within a short period of time. case reports: case . year old female, presented with sudden onset collapse, diarrhoea, vomiting and abdominal pains. she gave no history of menstruation and an initial diagnosis of severe gastroenteritis was made. she failed to respond to conservative management and required itu support. she was discharged with no firm diagnosis and re-presented one month later with similar symptoms, when a diagnosis of staphylococcal tss was confirmed. case . year old female, presented during menses with sudden onset rash, rigors, severe diarhhoea, vomiting and abdominal pains. she was diagnosed with staphylococcal tss on admission. case . year old female. presented with sudden onset severe diarrhoea, vomiting, pyrexia and rash. she gave no history of menstruation. she responded poorly to treatment, required itu support, high dose steroids and was eventually diagnosed with streptococcal tss. discussion: diagnostic criteria for tss include high fever, hypotension, erythematous rash and a complicated multisystem disfunction. patients often require aggressive management. the public health laboratory service reports an average of cases of diagnosed tss in the uk per year. however, because of the uncommon and difficult nature of the diagnosis, many cases are misdiagnosed and therefore go unreported. it is essential to maintain a high level of suspicion for patients who are epidemiologically at high risk, but importantly, also the less ill patient with suggestive symptoms who fails to meet all diagnostic criteria but are in an at-risk group. high morbidity and mortality has been reported for undiagnosed and untreated cases. objectives: staphylococcus aureus colonization and infection of burn wounds increases morbidity and delays wound healing of patients suffering from burn wounds. a vulnerable moment in the route of transmission of s. aureus is the exposed burn wound during dressing changes. the aim of this study was to evaluate the effect of dressing changes under laminar flow conditions on burn wound patients with regard to s. aureus colonization. methods: from the first of february to the first of june , patients were included in this study at our -bed burn centre. in of these patients the dressings were changed under laminar flow conditions. all patients were frequently screened for s. aureus in nose, throat and burn wounds. all s. aureus isolates were genotyped with pulsed field gel electrophoresis. results: at admission / ( %) patients had no s. aureus colonization in their nose, throat or burn wounds. dressing changes under laminar flow conditions were carried out on / patients of this group. however, / ( %) patients acquired burn wound colonization with s. aureus during their stay at the centre. six out of patients ( %) whose dressing changes were not carried out under laminar flow conditions acquired burn wound colonization with s. aureus. a total of patients acquired burn wound colonization; these patients also acquired carriage of s. aureus in nose or throat. the same type of s. aureus was carried in the nose or throat as well as the burn wounds in ( %) of these patients. the results of this study suggest firstly that dressing changes under laminar flow conditions does not prevent colonization of burn wounds with s. aureus, and secondly, that the exogenous route plays an important role in the transmission dynamics of this pathogen in burn wound centres. future research should be focused on this mode of transmission of s. aureus in this special circumstance. mupirocin prophylaxis to prevent staphylococcus aureus wound colonisation in patients admitted to a burn centre objectives: staphylococcus aureus (s. aureus) colonisation of burn wounds increases morbidity and delays wound healing. many burn wound colonisations with s. aureus are endogenous in nature. the aim of this study was to evaluate the effect of eradication of nasal s. aureus with mupirocin in patients with regard to s. aureus colonisation of their burn wounds. methods: from september to march , patients admitted to our -bed burn centre were screened for s. aureus in nose and burn wounds. isolates were genotyped with pfge. all patients received nasal mupirocin at the time of admission to the burn centre. fifty-five patients from the same unit who were followed from september till may and had not received mupirocin prophylaxis served as a historical control group. results: at admission / ( %) patients in the mupirocin trial had no s. aureus colonisation in their burn wounds. of this group patients were non-carriers and patients were nasal carriers of s. aureus. seven ( %) non-carriers and ( %) nasal carriers acquired burn wound colonisation with s. aureus during their stay at the centre. of the historical control group, ( %) patients had no s. aureus colonisation of their burn wounds at the time of admission. of this group patients were noncarriers and patients were nasal carriers; ( %) non-carriers and ( %) nasal carriers acquired s. aureus wound colonisation during their stay. the overall probability of wound colonisation in the patients treated with mupirocin was significantly lower than in the historical non-treated group (p = . , chi-square test with yates correction). conclusion: the results of this study suggest that application of nasal mupirocin to all patients upon admission to a burn centre may reduce but not eliminate the risk of subsequent s. aureus colonisation of burn wounds. other measures including improved infection control practices and eradication of exogenous s. aureus reservoirs, including s. aureus carriage among healthcare workers, may be necessary to further reduce the incidence of s. aureus burn wound colonisation. controlling an outbreak of staphylococcus aureus on a surgical ward results: the characterisation of the s. aureus in february showed a cluster of identical strains in three patients and hcw a. the strains of two patients were similar to the strain of hcw b. three patients had a unique strain. in the surveillance period a total of eighteen isolates in sixteen patients were found. in the surveillance period the epidemic-strains were found in other patients but only in patients who have been on the ward during the epidemic period. two patients had an identical new isolate. that indicates that the exchange between patients of s. aureus may occur without notion. another remarkable observation was that some of the patients lost their original strain and acquired a new isolate of s. aureus during hospitalisation. conclusions: a small outbreak of s. aureus could be traced to hcw who proved to be carriers. relieving them from patient contact stopped the outbreak. molecular surveillance is an appropriate way to evaluate the success of measurements taken and to underscore the importance of hygienic measures. risk factors for s. aureus carriage in health care workers a. widmer, m. lang, r. frei (basel, ch) introduction: s. aureus (mssa) carriage is common among health care workers (hcws)and might be associated with type of care provided and other potential risk factors. transmission of mssa has been observed by ''cloud adults''. objectives: to determine risk factors for mssa carriage in hcws, and the percentage of methicillin-resistant s. aureus (mrsa) in a tertiary care centre. methods: retrospective review of all data of staff health department and the microbiology laboratory. all hcws who were working in a high mrsa prevalence country were routinely screened at the time they were hired by the institution. in addition, data were generated from mrsa screenings of hcws if there was evidence for nosocomial transmission of mrsa in patients. mrsa was defined as s. aureus being oxacillin-resistant and expressing the pbp ' and/or being positive for the meca gene by pcr. data of hcws were collected by chart review in a case-report form that included demographic data, work place, skin diseases and other variables. data were compared by univariate and multiple logistic regression analyses. results: a total of swabs were taken from hcws between jan and dec. . data were available from hcws: % originated from switzerland, % from germany, % from france, and % from other countries. the overall prevalence of mssa and mrsa was % and %, respectively. more than one sample was available from individuals: of the repeat samples, % were negative, % were negative, but had one single positive culture, and % were consistently positive. sex, profession, work place, number of years at the institution was not associated with s. aureus carriage (p > . ). the only significant risk factor for s. aureus carriage was young age, defined < years (p = . ). the prevalence of mssa increased from % ( ) to % ( ) . eight hcws were carriers of mrsa: they were young and half of the them reported skin diseases. conclusion: s. aureus carriage among hcws is common: only young age remained a risk factor even after adjusting for other variables. mrsa was rare among hcws. prevalence of nasal carriage of methicillinsusceptible and methicillin-resistant staphylococcus aureus among hospital personnel and outpatients st. fokas, sp. fokas, c. tsamolia, m. skoutari (eghio, gr) objectives: to assess the nasal colonization of staphylococcus aureus among hospital staff in the absence of an epidemic and to compare it with the nasal carrier status among outpatients, focusing on the mrsa rate. methods: the study included hospital personnel and adults admitted to outpatient clinics of our hospital. given that a history of hospitalization is the most important facilitative factor for colonization of staphylococcus aureus we exclude individuals with a history of hospitalization in the preceding year of our study. the specimens were obtained with swabs from the anterior nares-ecological niches of s. aureus-and cultured according nccls guidelines. id staph strips were used for identification and confirmed with slidex staph plus kit (both by biomerieux). mrsa strains detection was achieved with slidex mrsa detection kit of the same company. antibiotic susceptibility of mrsa strains was determined with atb staph method (version ) according to the recommendations of the producer (biomerieux). results: among hospital staff the carriage rates of mrsa and methicillin susceptible s. aureus were . % and % respectively. none of the outpatients was found to be a nasal carrier of mrsa. in addition mssa carriage in this group was %. the prevalence of mrsa nasal carriage rate among personnel working in the different medical wards was also examined. the difference in the rates among personnel working in the surgical ward, operating theaters and the rest of the staff was statistically significant (p < . ). all mrsa strains were susceptible to oral antibiotics. conclusions: although the prevalence of mrsa is low among hospital personnel, education is needed as they are one of most important reservoirs for nosocomial mrsa infections. the low incidence of mrsa infections around the time of the study suggests that with usual infection control practices mrsa spread was avoided. all mrsa carriers were treated with intranasal muripocin for eliminating the nasal carrier status. continuous monitoring of mrsa carriage is needed to avoid nosocomial spread. aids patients are a special population related with high risk of infection due to staphylococcus aureus. previous colonization is a recognized predisposing factor for development of infection due to this organism. however, nasal carriage of s. aureus in hivpatients is still not completely characterized. objectives: to describe the epidemiological features of community-acquired s. aureus nasal colonization in out care hivpatients based on the molecular genotypes. methods: hiv-outpatients without previous hospitalization within the last two years were screened for s. aureus nasal colonization. three samples from each patient were collected, and the variables associated with colonization were assessed. nasal carriage was classified as: absent, transient colonization or persistent colonization. the persistent colonization was assessed according to the molecular profiles performed by pulsed-field gel electrophoresis, in: simple (same profile), multiple (different profiles) or combined (two with the same e and one with different profiles). results: patients were enrolled in the study. seventy patients ( %) had at least one culture positive for s. aureus and patients concluded three samples collection. only one isolate was a community acquired mrsa. the rates of colonization was higher when two or more samples were taken, ranging from . % in the first sample up to . % when samples were collected. hiv-patients with aids were more likely to be colonized than non-aids patients (p= . ). among the patients with s. aureus nasal carriage, % were transient and % were persistent carriers, whose genomic subtype was: . % simple, . % combined and % multiple persistent colonization. previous use of antibiotics was associated with absence of s. aureus colonization (p < . ). conclusions: hiv out care patients had high rates of oxacillinsusceptible s. aureus nasal colonization. regardless, the recent literature, only one isolate was community-acquired mrsa. the most common characteristic of colonization is simple persistent colonization showing the same genomic profile. objectives: s. aureus bacteraemia (sab) continues to be a major problem related to both community-acquired and nosocomial infection. we undertook an evaluation of a large cohort of patients with sab to assess features of the infection and predictors of mortality. patients and methods: cases of sab admitted to our hospital ( - ) were prospectively studied. all patients had at least one positive blood culture for s. aureus and clinical symptoms of bacteraemia. sab was considered to be nosocomial if the first positive blood culture specimen was obtained > h after admission and there was no clinical evidence of infection on admission. microbiological studies were carried under standard protocols. epidemiological and clinical characteristics, antimicrobial treatment and outcome (relapse or mortality) were analysed. results: cases of sab were identified but only were prospectively included in the study protocol. incidence was the highest in year ( . per ). out of the cases, ( %) were male with a mean age of year (sd . have not yet implemented similar guidelines. this study evaluated the detection and reporting of mlsb results in staphylococcus using the bd phoenix tm automated microbiology system and bdxpert system with nccls, sfm or din as interpretative standards. methods: comments regarding mlsb resistance as listed in the nccls m -s were converted into expert rules. special bdxpert rules were also constructed for the detection and reporting of mlsb resistance when applying sfm or din guidelines. a total of strains of staphylococcus ( s. aureus, s. epidermidis, and coagulase-negative staphylococci) were tested in phoenix panels containing erythromycin (e) and clindamycin (cc). nccls, sfm or din breakpoints were used to interpret the phoenix mic results. the double disk diffusion d-test (e and cc) was used as the reference method for the determination of mlsb resistance phenotypes. results: the phoenix e and cc mic values were interpreted based on the standard selected. the bdxpert rules were executed and applicable expert messages were displayed. the phoenix correctly detected out of constitutive mlsb (cmlsb) phenotypes as compared to the d-test results. the remaining cmlsb strains were interpreted by the bdxpert as potential inducible mlsb (imlsb)/efflux phenotypes. a total of imlsb and efflux phenotype isolates were all reported by the bdxpert as imlsb/efflux phenotype and the users were alerted to perform d-test before reporting the cc results. the cc interpretation was suppressed in these isolates. the nccls, sfm, or din showed identical detection and interpretation of mlsb resistant phenotypes by the phoenix and bdxpert systems. conclusions: the phoenix and bdxpert systems can assist laboratories in rapid detection and accurate interpretation of mlsb results for staphylococcus. special messages can be used to communicate timely and accurate information to clinicians for proper therapy of staphylococcal infections with mlsb resistant phenotypes. in vitro activity of new compounds tested against multi-drug resistant s. aureus isolated in latin american medical centres background: oxacillin-resistant s. aureus (orsa) isolated in lamcs shows high rates of co-resistance (r) with most antimicrobial classes used in the clinical setting. we evaluated the mdr patterns of orsa strains collected in lamc by the sentry antimicrobial surveillance program in . we also evaluated the susceptibilities (s) of new, investigational compounds against these important endemic pathogens. methods: among . s. aureus collected, ( . %) were r to oxacillin. these strains were isolated from lamcs ( countries) and tested against > antimicrobials by nccls broth microdilution methods. orsa strains were grouped by the mdr patterns for the primary drugs ( ). conclusions: all newer compounds (lzd, q/d, dap and dal), tei and van were very active against endemic and epidemic orsa in lamcs. these s. aureus having mdr patterns (ave. r to agents) will require greater use of newer compounds particularly in brazil. objectives: coagulase negative staphylococci (cons) represent a part of physiological microflora. however, in recent years, they have also emerged as nosocomial pathogens of infections associated with indwelling medical devices (e. g. pleural drains), mainly in immuno-compromised patients. the aim of this study was to determine dynamics of nasopharyngeal colonization by cons in patients with resectable lung cancer during short-term hospitalization, especially those receiving routinely preoperative antimicrobial prophylaxis. methods: in present study cons species were selected from patients divided into two groups: (i) patients who were undergoing pulmonary resection and receiving preoperative prophylaxis (piperacillin, cefuroxime alone or in combination with amikacin) -ÔprophylaxisÕ group and (ii) control group. throat and nasal specimens were taken from each patient twice, in four-days interval. all isolates were identified by the biochemical microtests api staph (biomerieux) and tested for drugs susceptibility according to nccls reccomendations. results: strains of cons strains were selected from clinical specimens: strains from ÔprophylaxisÕ group and strains from control group. the most often isolated species was staphylococcus epidermidis with the predominated api numerical profile . a detailed analyses of the api numerical profiles and resistance to antimicrobial agents indicated that during four-days interval of hospitalization, changes in phenotypes among cons colonizing nasopharynx were observed in both groups of patients. the changes in drug resistance patterns were found more often in ÔprophylaxisÕ group compared to those in control group - / ( . %) and / ( %), respectively. this difference was statistically significant (p = . -chi test with yates correction). conclusion: our data suggest that preoperative antimicrobial prophylaxis routinely used in patients with resectable lung cancer may be regarded as an important factor predisposing to changes in drug resistance patterns among cons isolates colonizing nasopharynx. the susceptibility of coagulase-negative staphylococci in a teaching hospital compared with a secondary care hospital results: in total, six different species were identified in isolates, % of which were staphylococcus epidermidis. in the lumc the species diversity was greater than in the mca, with five isolates of staphylococcus haemolyticus found in the first. no difference between the two hospitals was found in activity of the antibiotics tested, except for li. the mic s were as follows: v mg / l, t mg / l, c mg / l, m mg / l, l mg / l. of li the mic was mg / l in the lumc and mg / l in the mca. according to dutch guidelines, in total / isolates were found resistant to t and intermediate resistant. all isolates were susceptible for v. conclusion: the first choice glycopeptide for serious cons infections, t or v, has no influence on the susceptibility of cons for these antibiotics. according to dutch guidelines, the activity of t, c, and l against cons is relatively low, whereas the activity of m and le is in the susceptible range. objective: staphylococcus epidermidis is one of the bacterial species mainly implicated in foreign body associated infections. we have characterized several s.epidermidis clinical isolates for their ability to form biofilms and their resistance to antibiotics. methods: s.epidermidis strains isolated from implantable medical devices have been collected from hospitals of central italy. susceptibility to penicillin, methicillin, erythromycin and tetracycline has been determined in vitro by e-test according to nccls guidelines. the specific antibiotic resistance determinants have been checked by pcr (blaz, meca, erma, ermb, ermc, msra, tetk and tetm). the ability to form biofilms has been determined: (i) by pcr, detecting genes specific for attachment and biofilm development (icaadbc operon, aap, and atle); (ii) by congo red agar (cra) plate test to assay the production of polisaccaridic intercellular adhesin (pia); (iii) by crystal violet (cv) stain to determine the biofilm biomass development on polystyrene microtiter plates; (iv) and by cslm microscopy observations to investigate biofilm structure. results: % of the strains under study was resistant to penicillin, % to methicillin, % to erythromycin and % to tetracycline. on the side of biofilm-specific genes detection, % of strains was positive to ica operon genes, % possessed atle gene, and % aap determinant. in % of the population, the cra test confirmed the correlation between the presence of ica genes and slime expression. the cv assay classified the quasitotality of our strains ( %) as biofilm producers on plastic surface. in addition, the distribution of optical density values (od ) obtained after cv stain, showed a significant statistical difference in biofilm biomass development between the ica-adbc-positive strains and the icaadbc-negative ones. finally, a correlation, although not always present, has been observed between ability of the strains to develop in a high-structureted biofilm and specific biofilm-formation determinants. conclusions: analysing the origin of colonization or infection in patients with cons isolated from the operative site, the bacteria could only be traced in one patient who developed an infection. transmission most probably occurred by direct contact. the low level of bacterial contamination in the uca together with the lack of correlation of genotypes between isolates found in the uca and in the wounds suggest that airborne transmission does not play a major role in the development of swi. more than % of staphylococci found in air samples were not traceable to any investigated sources. most probably, they originated from non swabbed parts of the hcw's bodies. since % of the traceable isolates from the uca originated from non scrubbed team members, protection of the operative site by lfv appears to be suboptimal. clonality of endemic methicillin-resistant staphylococcus epidermidis strains isolated in a neonatal intensive care unit n. ben saida, a. ferjani, n. sfar, j. boukadida (sousse, tn) objectives: methicillin-resistant staphylococcus epidermis (mrse) is one of the important pathogen responsible for nosocomial infections particularly in newborns. the aim of our study was to establish if these infections are caused by endemic clones or by incidentally occurring bacterial strains of this ubiquitous species and to evaluate the performance of pcr iner-is as a novel method for typing mrse strains using pulsed-field gel electrophoresis (pfge) as the reference method. methods: twenty strains of mrse (meca+) has been collected during the period of survey (december to september ) from different pathological products of newborns hospitalized in a neonatal intensive care unit of a public maternity hospital in sousse, tunisia. identification of strains was done by conventional procedures and the api staph (api-biomerieux. sa). susceptibility testing to antibiotics was determined according to ca-sfm recommendations. all strains have been characterized by genotypage in pulsed-field gel electrophoresis (pfge) variety chef after smai macrorestriction. genomic profiles have been interpreted according to criteria of tenover (j. clin. microbiol ; : - . strains were also characterized by electrophoretic profiles obtained by pcr-based analysis of inter-is spacer polymorphisms. results: these mrse represent . % of the total strain of s.epidermidis collected from the neonatology ward during the period of survey. majority of these strains come from blood sample ( %), other strains from vascular catheter ( %), pus ( %), sound ( %). antibiotypes and genotypic profiles according tenover criteria were individualized: type a (with subtypes), type b, type c (with subtypes), type d (with subtypes), type e (with subtypes)and type f. a total of four pcr patterns were obtained based on the interpretation criteria that pcr patterns exhibiting more than one band difference were considered to represent distinct types, type , type , type , and type . conclusion: mrse appear to be endemic and multiclonal with a dominant clone in the neonatal intensive care unit. our study demonstrates that a significant proportion of mrse infections may be attributable to transmission among newborns and that certain strain can become endemic over long periods in this setting. clonality of staphylococcus haemolyticus strains isolated in a neonatal intensive care unit n. ben saida, a. ferjani, j. boukadida (sousse, tn) objective: methicilllin-resistant staphylococcus haemolyticus (mrsh) was increasingly important nosocomial pathogens particularly in critically ill neonates. the aim of our study was to establish if these infections are caused by endemic clone or by incidentally occurring bacterial strains of this species. methods: thirteen strains of mrsh according ca-sfm recommendations and meca+ (murakami k. j.clin.microbiol , : has been collected during the period of survey (april to october ). eleven strains come from different pathological products of newborns hospitalized in a neonatal intensive care unit of a public maternity hospital. tow strains come from paediatric and internal medicine wards were used as control to ensure that endemic strains can be differentiated from outbreak strains. all strains were identified with the api-staph method (api biomerieux). strains have been characterized by genotypage in pulsed-field gel electrophoresis (pfge variety chef) after smai macrorestriction. genomic profiles have been interpreted according to tenover criteria (j. clin.microbiol. ; : - . results: s. haemolyticus was quantitatively the second staphylococci ( . %) after s. epidermidis isolated in neonatology ward. mrsh represent . % of the total strains of s. haemolyticus collected from the neonatology ward during the period of study. the majority of the strains come from blood sample ( . %), ( . %) from pus and ( %) from csf. eight resistant antibiotyps profiles and genotypic pattern according tenover criteria were individualized: type a, type b (with subtypes), and type c. the strains coming from paediatric ward present pattern type b , but strains coming from internal medicine ward present distinct pattern. conclusion: mrsh is a major bacterium of nosocomial infection in neonatal intensive care unit. this bacterium is responsible as shown in our study, of endemic multiclonal nosocomial infections with a predominant clone. results: s. haemolyticus was isolated from % of all positive blood cultures and represented the . % of the coagulase negative staphylococci. twenty-nine cases of shb occurred during the study period ( . episodes per patients-days). all cases were hospital-acquired and occurred after a mean hospitalisation of . days. the mean age was . years (range - ) and patients ( . %) were males. twenty-eight patients ( . %) had a central venous catheter (cvc). all patients received previously antimicrobial therapy. thirteen patients ( . %) had polymicrobial bacteraemia. in cases ( . %) the source of bacteraemia was the cvc. all isolates were resistant to oxacillin and gentamicin, . % and . % were resistant to ciprofloxacin and teicoplanin, respectively; all strains were susceptible to vancomycin. sixteen patients ( . %) had sepsis, ( . %) severe sepsis, and ( . %), all polymicrobial, septic shock.; in patients ( . %) shb had no clinical significance. twenty patients died ( %) and deaths were directly related to shb in patient ( . %), and partially related in ( . %). nine patients ( %) survived.the patient who died for shb, developed a persistent cvc-related bacteraemia after a -week course therapy for streptococcal endocarditis. s. haemolyticus become resistant to teicoplanin during a teicoplanin-based therapy and the patient died days after the onset of bacteraemia. the patients, in which death was partially related to shb, had a polimicrobial bacteraemia due to c. tropicalis and enterococcus spp, respectively, and died after and days respectively, for septic shock, during adequate therapy. among the patients in which death was not related to shb, ( . %) received adequate therapy that cleared the blood cultures, patients improved after the removal of cvc without antimicrobial therapy. conclusions: s. haemolyticus is an emerging pathogen, responsible of sepsis, frequently cvc-related; most of strains are resistant to many antistaphylococcal agents currently used as empiric therapy, thus constituting a clinical concern. nevertheless, this pathogen shows a low virulence and is associated with a low related mortality, probably representing a marker of severe underlying diseases. staphylococci from the skin of patients: changes associated with treatment by isotretinoin a. thomas, r. williams, n. hepburn, t. watts, r. dixon (lincoln, uk) objectives: isotretinoin, a retinoid has been used to treat patients with moderate to severe acne despite the adverse effects of mucosal surface drying and the contraindication of use during pregnancy. in clinical practice, patients are frequently prescribed systemic isotretinoin because they have not responded to previous treatment with oral or topical antibiotics. broad-spectrum antibacterial therapy has nevertheless promoted changes in the diversity and antibiotic resistance status of the patientsÕ skin microbiota and we are studying the effect of isotretinoin on these changes. methods: the present study investigated the recovery and analysis of skin organisms from patients (mean age y range - y) before, during and after treatment with a -week course of isotretinoin ( mg / kg body weight). the number of aerobic bacterial isolates (presumptive staphylococci) recovered from baird-parker agar from each of three specific sites per patient were compared with age-matched controls of healthy volunteers. results: both patients and controls had generally similar numbers of organisms on the nares, cheek and toe webs before treatment, whereas all patients showed a significant reduction in staphylococci recovered from one site during and after weeks of isotretinoin treatment. the majority of the patients had a greater reduction ( - log) for the cheek site than either nares or toe webs both of which showed minimal reduction. conclusions: preliminary results from this study show a pronounced reduction in the number of presumptive staphylococci recovered from the treated group immediately following isotretinoin compared to untreated controls. since isotretinoin has little known antibacterial activity against the staphylococci the observed reductions would suggest that the effect is mediated through changes in the skin nutritional micro-environment. objectives: emergence of mutational resistance to linezolid (lin) in staphylococcus aureus has been associated with loss of erythromycin resistance methylase (erm) genes, both in vivo and in vitro. we tested the general validity of this claim, and examined whether the emergence of lin resistance is delayed in the presence of ery. methods: six lin-susceptible s. aureus strains were used: genetically-related, ery-resistant (ermc) and susceptible pairs of emrsa- , also the isogenic pair rn and its hypermutable rn muts mutant, which harbours ermb. in replicated experiments, ery-susceptible and ery-resistant isolates were grown on agar containing increasing concentrations of lin. in addition, ery-resistant isolates were grown with mg / l ery and increasing concentrations of lin. the number of days for isolates to develop the ability to grow in the presence of mg / l lin was recorded. the absence of erm genes was checked by pcr; mics of lin and ery were determined by agar dilution, and pfge profiles of mutants were verified. results: thirty-three mutants were obtained from the experiments; from the clinical emrsa- isolates, from rn and from rn muts. lin-resistance emerged more slowly in ery-resistant clinical isolates grown with lin and ery than with the same isolates grown with lin alone: mean days (sd . ) to emerge on mg / l lin versus days (sd . ) in the presence of lin and ery. growing the hypermutable strain, rn muts, in the presence of lin and ery did not delay the emergence of lin-resistance ( days with sd . , in either case). in the absence of ery selection, loss of erm with the emergence of lin-resistance was only noted for one emrsa- isolate in of experiments. all isolates grown in the presence of ery retained erm determinants. ery mg / l did not supress growth of ery-resistant strains and linezolid was stable within the ambit of the long-selection experiments. conclusions: growing the clinical isolates in the presence of ery and lin slowed the emergence of lin-resistance. this merits further study with lower, clinically-relevant concentrations of ery. in vitro selection of linezolid resistance in hypermutable and wild-type staphylococcus aureus objectives: resistance to linezolid (lin) is rare, but can arise via mutations in the s rrna genes. we hypothesised (a) that resistance would emerge preferentially in staphylococci with a hypermutable phenotype engineered by mutations in the muts gene, and (b) that hypermutability might be co-selected with linezolid resistance. muts is part of the dna mismatch repair (mmr) system, which identifies and corrects genome alterations post-replication, thereby influencing the net mutation rate. method: linezolid-resistant (linr) mutants of staphylococcus aureus rn , its hypermutable muts deletion mutant rn muts, harbouring an erythromycin resistance marker gene (ermb), and a genetically-related pair of clinical isolates were selected by repeated passage with increasing concentrations of lin. mutant parentage was confirmed by pfge and susceptibility was tested by agar dilution. an amplified -bp fragment of s rrna genes was studied by sequencing and by rflp analysis. frequencies at which linr mutants yielded variants resistant to fusidic acid and rifampin were calculated in triplicate experiments. results: seventeen linr mutants (mics - lg/ml) were raised. ten mutants had a g t mutation in their s rrna genes; , all from rn muts, had g t, typically seen in linr gram-positive cocci from the clinic; had t c; had no identifiable mutations. curiously / rn muts mutants lost ermb during the course of the experiment, reverting to macrolide susceptible. linr mutants had mutation frequencies for rifampicin and fusidic acid resistance comparable with those of their parents ( - to - ), implying no co-selection of stable hypermutability. conclusion: more linr mutants were obtained from the hypermutable (rn muts) strain than from the wild type strain, and g t mutants were only obtained from the hypermutable organism. these data suggest hypermutability facilitates the emergence of linr, nevertheless, linr mutants derived from wild-type parents did not have elevated mutation frequencies. objectives: the mechanism of resistance in glycopeptide intermediate staphylococcus aureus (gisa) and hgisa isolates is unknown. however the inactivation of certain genes has been shown to confer an increase in glycopeptide resistance. two genes, tcaa and mprf, when inactivated in gisa strains cause a reduction in glycopeptide mic. overexpression of tcaa causes an increase in teicoplanin susceptibility and so both genes may play a role in the development of glycopeptide resistance. this study aims to investigate the expression levels of tcaa and mprf in a set of gisa, hgisa and glycopeptide-susceptible s. aureus (gssa). methods: rna was extracted from the following: set of clinically related (cr) gssa and hgisa (lla and lle), sets of cr hgisa and gisa (pc and pc , lim and lim ) harvested during exponential phase growth (ex) and during exponential phase growth in the presence of sub-mic levels of vancomycin (exv). rna was extracted from a further hgisa, mu and gisa, mu as well as clinical gssa strains. rt-pcr was performed with customised primers and the products visualised by electrophoresis. band intensity was taken as indicative of mrna quantity, and hence expression level, at time of cell harvest. results: the expression of tcaa is similar in all strains tested. each strain within the related strain sets all show similar band intensities, despite differing glycopeptide resistance phenotype. both mu and mu also exhibited similar expression levels to those found for all other gisa, hgisa and gssa strains. levels of expression were not affected by the presence of sub-mic levels of vancomycin in any strain tested. sequence analysis of tcaa in these strains showed no mutations present as previously thought. expression levels of mprf were found to be strain specific. no difference in gene expression was found in the cr related strain sets. conclusions: no reduction in expression levels of tcaa and mprf were found to correlate with clinical strains exhibiting higher glycopeptide resistance. thus it it uncertain what role tcaa ot mprf play with mediating glycopeptide resistance in staphylococcus aureus. objectives: gisa and hgisa strains all have a characteristic thickened cell wall with reportedly fewer cross-links. fewer peptidoglycan cross-links create increased d-ala-d-ala pentapeptide termini and hence more vancomycin targets. pbp has been shown to have both transpeptidase and d,d-carboxypeptidase activity, cleaving terminal d-alanine residues from un-cross-linked muropeptides. reduced levels of pbp have been reported in laboratory-generated gisa mutants and a few clinical gisa strains. this study aims to investigate the expression levels of pbp in a set of gisa, hgisa and glycopeptide-susceptible s. aureus (gssa). methods: rna was extracted from the following: set of clinically related (cr) vssa and hgisa (lla and lle), sets of cr hgisa and gisa (pc and pc , lim and lim ) harvested during exponential phase growth (ex), after overnight growth (on) and during exponential phase growth in the presence of sub-mic levels of vancomycin (exv). rna was extracted from a further clinically unrelated (cu) gisa (including mu ), cu hgisa (including mu ) and gssa (ex). rt-pcr was performed with customised primers and the products visualised by electrophoresis. band intensity was taken as indicative of mrna quantity at time of harvest and hence gene expression. results: the expression of pbp varies according to strain. the band intensities of the related strain set lla (gssa) and lle (hgisa) reduce slightly with increasing intermediate vancomycin resistance. however in two other cr hgisa and gisa (pc / pc , lim /lim ) strain sets no variation in expression was observed. both mu and mu exhibited lower pbp expression than any other strain, with the presence of sub-mic levels of vancomycin reducing expression further. pbp expression was found to be reduced in other gisa and other hgisa, which showed lower band intensities than gssa. conclusions: the reduced levels of pbp suspected to be associated with reduced susceptibility to vancomycin in s. aureus is not exclusive. the expression of pbp is reduced in mu and clinical gisa, as reported previously. however other gisa strains exhibit pbp expression levels similar to gssa isolates. therefore pbp expression levels appear to be strain specific and a lowered expression level is not an essential requirement in the development of the gisa phenotype. objectives: the prolonged use of vancomycin is known to be the most important factor in inducing vancomycin resistance to s. aureus. methicillin-resistant s. aureus (mrsa) colonizing in respiratory tracts might be chronically exposed to subtherapeutic concentrations of vancomycin because the level of vancomycin within pulmonary lining fluids was much lower than the concentration of serum. despite the continuous use of vancomycin mrsas were still found to be presented in respiratory specimens of some patients. methods: all mrsas which had been continuously isolated in respiratory specimens during vancomycin therapy were collected from jul -mar at seoul paik hospital. mrsas were screened on brain heart infusion plates containing vancomycin lg / ml (bhi- ) and lg / ml (bhi- ) for vancomycin resistance. minimal inhibitory concentrations (mics) were determined by e-tests, agar dilution methods, and broth dilution methods. the patterns of pulsed field gel electrophoresis were analysed. results: nine patients and isolates were assessed. five patients had pneumonia and four patients had other mrsa infections. twelve mrsas were isolated from tracheal aspirates and from expectorated sputum specimens. the duration of vancomycin use ranged from - days. five isolates and one isolate were cultured on bhi- and bhi- , respectively; the vancomycin mics of these isolates revealed lg / ml by e-tests. the mics from agar dilution methods and broth dilution methods showed from . - lg / ml. conclusion: vancomycin resistance during the treatment of patients did not develop. objectives: fusidic acid resistance (fusr) in staphylococcus aureus usually results from acquisition of the fusb resistance determinant, or from mutations in the fusa gene encoding the drug target (elongation factor g). following a recent report of fusr in s. intermedius, and the detection in our own studies of fusr s. lugdunensis (unpublished), we have examined whether resistance in these strains results from fusa / fusb-type mechanisms. methods: standard methodology was employed for strain identification, susceptibility testing, southern hybridization (sh), pcr amplification and dna sequencing. primers for pcr amplification of the previously-unsequenced s. intermedius fusa gene were based on portions of the flanking genes (rpsg, tufa) that are conserved between b. subtilis and s. aureus. results: three fusr s. lugdunensis (fus mic = ug / ml) and two fusr s. intermedius (fus mic = ug / ml) were probed for the presence of fusb by sh alongside their wild-type, fuss counterparts (fus mics of . and . ug / ml, respectively). the fusr s. lugdunensis all carried fusb, but this gene was not detected in s. intermedius, or in the fuss strains. furthermore, fusb appeared to be chromosomally-located, since no hybridization was observed with purified plasmid dna. pcr analysis mapped fusb to a chromosomal region upstream of the groel gene, the same locus at which this gene is carried in the epidemic fusr european s. aureus clone. to examine whether mutations in fusa are responsible for fusr in s. intermedius, pcr amplification and sequencing of fusa from the resistant and sensitive strains was performed. relative to fuss s. intermedius nctc , the resistant strains carried six coding polymorphisms in fusa, although none of these correspond to reported fusr mutations in the fusa gene of s. aureus. conclusions: fus resistance in recent strains of s. lugdunensis results from carriage of the fusb determinant on the chromosome, probably acquired horizontally from fusr s. aureus strains. the fusb gene was not detected in the s. intermedius strains, although multiple fusa polymorphisms were identi-fied which may be responsible for the observed fusr phenotype. objectives: methicillin-resistant s. aureus (mrsa) and methicillin-resistant coagulase-negative staphylococci (mrcns) are the most important pathogens that cause nosocomial infections worldwide. one of the few antibiotics which is still effective against mrsa is mupirocin. but mupirocin-resistant s. aureus has been increased since first reported in . and more than - % of s. aureus isolated in tertiary hospitals in korea was reported to be high level mupirocin resistant. in this study, we investigated the restriction fragment length polymorphism (rflp) type for mupa gene loci of plasmid dna and sequenced mupr plasmid containing mupa gene of high level mupirocin resistant (muh) staphylococci from tertiary hospitals. methods: susceptibility to antimicrobial agents including mupirocin was tested by the disk diffusion and mic of mupirocin was determined by agar dilution method. the plasmid-encoded mupa gene was detected by pcr. mupa polymorphisms of plasmid dna digested by hind iii, ecor i, and cla i restriction enzymes was investigated with southern hybridization using mupa probe. s. aureus isolate showing the most prevalent rflp type was conjugated by filter mating method and selected the transconjugant showing only mupirocin resistant phenotype. the mupr plasmid of selected transconjugant was purified and analysed the plasmid dna sequence. results: muh-staphylococci were isolated among the staphylococci from tertiary hospital in korea. muh-mrsa ( isolates), s. haemolyticus ( isolates), s. hominis ( isolates) and s. epidermidis ( isolates) were isolated. . kb mupa pcr product was detected in plasmid of muh isolate. all isolates contained more than two plasmid molecules that were repeatedly purified with different efficiences. three different polymorphs of the mupa loci were investigated. the most prevalent mupa polymorph was . kb ecori- kb hindiii- . kb clai hybridizing fragment. the mupirocin resistant plasmid dna was about kb and is -mupa-is sequence was located. conclusion: the high-level mupirocin resistant staphylococci had the multiple plasmids of various size and the diverse rflp type suggested the variation of mupa gene loci. the mupr plasmid contained transferable element such as is with mupa gene. genetic basis of macrolide, lincosamide, streptogramin resistance in coagulase-negative staphylococci s. gatermann, t. koschinski, s. friedrich (bochum, d) objective: in s. aureus erythromycin resistance is almost exclusively caused by erma or ermc genes whereas export of macrolide antibiotics by msra or inactivation of lincosamides by lina is rare. resistance may be inducibly (clindamycin appears susceptible) or constitutively (clindamycin appears resistant) expressed. during therapy of inducibly resistant strains with clindamycin mutations may occur that render them objectives: lysostaphin is an antibacterial enzyme which is specifically capable of cleaving the cross-linking pentaglycine bridges in the cell walls of staphylococci. s. aureus cell walls contain high proportions of pentaglycine, making lysostaphin a highly effective agent against both actively growing and quiescent bacteria. relationship between lysostaphin susceptibility, vancomycin susceptibility and cell wall thickness of passage-selected vancomycin -resistant staphylococcus aureus isolates (vrsa) and their parent strains were studied in order to investigate characterization of isolates with decreased susceptibility to vancomycin. methods: the susceptibility of lysostaphin for six vrsa and their parent strains were determined using the spectrophotometric and the macrodilution methods. spectrophotometric determination of turbidity was measured as a decrease in a during incubation of the isolates samples at °c. mics were determined by a broth macrodilution method in cationadjusted mueller-hinton broth according to standards of the national committee for clinical laboratory standards. cell wall measurement were determined using transmission electron microscopy. results: determination of lysostaphin activity revealed that there are significant decreases in per cent reduction in turbidity (activity of lysostaphin) of the vrsa. vrsa shown to be more resistant to lysostaphin than parent strains. the mics of lysostaphin for the vrsa strains also increase. there were either a or fold increases in the mic to lysostaphin. electron microscopy of the vrsa showed enhanced cell wall thickness, uneven surfaces and irregular shape in contrast of the thin and regular cell wall morphology of the parent strains. those strains acquiring the highest thickness in cell wall demonstrating the highest resistant to lysostaphin. conclusion: the mechanism of lysostaphin resistance and its relationship to vancomycin resistance remains unclear. it is possible that the increase in cell wall thickness we observed prevented lysostaphin access to all pentaglycine targets or increased the number of cross-linkers requiring cleavage before the strain could lyse. typing and epidemiology of mrsa methods: creation and distribution of a consensus document detailing the needed harmonization of sequencing technology in diagnostic microbiology will be followed by a certification program for sequence based typing, using methicillin-resistant s. aureus (mrsa) as a prototype organism. as typing method spatyping will be used. five strains, dnas, and forward and reverse chromatogram files of well characterized mrsa strains will be distributed to all participating laboratories. an annual proficiency testing for sequence based typing of mrsa is planned. staphylococcus aureus is an important human pathogen related to its ability to develop resistance to many antimicrobial agents. methicillin resistance of s. aureus (mrsa) has become a major public health problem especially in nosocomial infectious. more over community acquired mrsa is a growing concern, the rate of mrsa vary importantly within countries, the highest rates were reported especially in southern european countries. a multicentric study was done to establish antimicrobial resistance of s. aureus isolated from clinical specimens of hospitalized patients over five years period ( ) ( ) ( ) ( ) ( ) . s. aureus was identified by conventional methods and antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. a total of strains of s. aureus were collected, they were recovered mainly from pus ( %), blood cultures ( %), respiratory samples ( %) and urines ( %). % of the strains were from in medicine, % from surgery, % from orthopaedics, % from paediatrics and % from intensive care units. the rates of resistance to different antibiotics was % to oxacilline, % to gentamicin, % to amikacin, % to ofloxacin, % to erythromycin and % to clindamycin. all isolates were susceptible to vancomycin. according to this data the rate of mrsa remained relatively low (< %) with no significant change during this time period, however, mrsa presented high rates of associated resistance to gentamicin ( %), to amikacin, ( %) to erythromycin ( %), to clindamycin ( %), to ofloxacin ( %) and to trimethoprim-sulfamethoxazole ( %).mrsa were not of great concern in our country and meticillin remains an effective antibiotic for treatment of s. aureus infections. characterisation of gentamicin-susceptible strains of methicillin-resistant staphylococcus aureus in two spanish hospitals c. potel, m. alvarez, c. lopez-melendez, i. otero (vigo, e) objectives: characterize gentamicin-susceptible methicillinresistant staphylococcus aureus(gs-mrsa)by two molecular typing methods. find out whether some gs-mrsa clones are related to gr-mrsa. describe the spread of these clones. methods: one hundred and twelve samr were isolated in two hospitals between - . the epidemiological relatedness was studied by repetitive element sequence-based pcr and coa gene restriction fragment length polymorphism. the genes ant( ) and aac( )-aph( Õ), that codify two aminoglycoside modifying enzymes, were detected by pcr.we analysed the gentamicin consumption and its relation with the emergence of gs-mrsa. results: thirty nine strains were gs-mrsa. the % of these gs-mrsa belonged to two epidemic clones, the clone a was tobramycin sensitive and the clone b was tobramycin resistant. the clone a was only isolated at hospital-i and was replaced by an epidemic gr-samr clone (clone c). the clone b was only isolated at hospital-ii and displaced the clone c. objectives: the long-term care facilities (ltcfs) patients are those with serious underlying disease, poor functional status, wounds such as pressure sores, invasive devices of urinary catheters. there is increasing concern about the emergence of resistance to antimicrobial agents including methicillin resistant s. aureus in ltcfs and mupirocin has been used in ltcfs to prevent the occurrence and spread of mrsa. for inducible mls-resistance. nitrocefin discs were used for beta-laktamase detection after induction with cefoxitin and/or oxacillin. the oxacillin ( mg / l) agar method was used for mrsa screening. mrsa was confirmed by meca and nuc pcr and further analysed by smai-pulsed-field gel electrophoresis (pfge). results: a total of strains ( %) were beta-lactamase positive. other resistance prevalence rates were: tc ( %), em ( %), cm ( %), ci ( %), gm ( %), ts ( %) and fu ( %). thirteen strains ( %) were mrsa-screen positive, both with the oxacillin-agar test and the cefoxitin disc, and confirmed mrsa by meca and nuc pcr. all mrsa strains were isolated from hospitalized patients and expressed multiple resistances. pfgeanalysis revealed that the mrsa-strains belonged three different pfge-types. type consists of strains from the department of general surgery and icu. the type group consisted of strains from general surgery department. finally, one strain from a patient at the department of pulmonary medicine belonged to the type group. we were not able to type one strain. mlsttyping of mrsa-strains will be performed. results: a total of infants were included in this study. mrsa colonization was detected in infants ( %) during nicu stay and % were detected by the first samplings. naris ( %) and umbilicus ( %) were the most common sites of colonization. clinical mrsa isolates were identified from infants ( %). among the infants, mrsa colonization, either preceding or later acquiring, was noted in infants ( %). clinical isolates from infants with episodes of possible mrsa infection were available for genotyping analysis. prior colonization was detected in episodes ( %), among which both the colonized and clinical isolates were indistinguishable in episodes ( %), highly related in episodes and distinct in episodes. among the episodes without prior colonization, colonization was acquired following infection in episodes ( %), among which indistinguishable or highly related colonized strain was acquired in episodes. conclusion: surveillance of mrsa strains as a basis for active antibiotic policy has become of increasing concern to both health care providers in hospitals and community general practitioners. there is a need for better awareness of mrsa infections among both health care professionals and the public. the incidence of mrsa infections in last years in the czech republic shows a slightly increasing trend. very interesting is local distribution of mrsa strains. the development of the incidence of mrsa infections in their spread will be the subject of further study. comparison of phenotypic typing methods and pfge of methicillin resistant s. aureus isolated from two university hospitals in thailand c. chomvarin, k. chaicumpar, s. srigulbutr (khon kaen, th) objectives: comparison of phenotypic typing methods and genotypic typing method to differentiate mrsa isolates obtained from the two university hospital in thailand. methods: seventy-four mrsa isolates were randomly collected from the two university hospital (central and northeastern) in thailand. they were confirmed with the presence of meca gene. antibiograms, phage typing and enterotoxin productions were used for the phenotypic typing analysis. pulsed-field gel electrophoresis (pfge) with smai digestion of chromosomal dna was used for the genotypic typing analysis. results: we found distinct profiles by phenotypic typing methods and pfge types designed as major types (a to e) and subtypes. the most frequently types and their related subtypes found in both hospital, were a and c with represented . % and %, respectively. all isolates resistant to penicillin, cephalosporin, erythromycin, gentamicin, kanamycin, tetracycline and oxacillin; and variably resistant to cotrimoxazole, lincomycin, chloramphenicol, ciprofloxacin. all isolates were susceptible to vancomycin and fosfomycin. ten ( . %) mrsa isolates produced enterotoxin a. forty-five ( . %) isolates were nontypable by phage typing method. no significant correlation between the phenotypic typing methods and the genotypic typing method were found. conclusions: the phenotypic typing methods were not significantly correlated with the genotypic typing (pfge). our results demonstrated that pfge types a and c were the common endemic mrsa clone of both hospitals in thailand. objective: to investigate the molecular characteristics of methicillin-resistant staphylococcus aureus (mrsa) strains isolated from neonates transferred from primary care obstetric clinics. methods: twelve mrsa strains were isolated from neonates in . ten mrsa strains were also isolated from nurses and facilities in corresponding primary care obstetric clinics. molecular features of mrsa strains were analysed by using multilocus sequence typing (mlst, arcc-aroe-glpf-gmk-ptatpi-yqil), spa typing, and sccmec typing. presence of panton-valentine leukocidin (pvl) gene was investigated by pcr method. results: all mrsa strains analysed in this study contained sccmec type iva. of six isolates from neonates of clinic a, five showed st ( - - - - - - ) and resistance to only gentamicin and tetracycline. the remaining one showed novel st ( - - - - - - ), shared with one isolate from nurse of clinic a. four mrsa strains from neonates of clinic b showed st ( - - - - - - ) and resistance to erythromycin and gentamicin, which is the same characteristics with three isolates from nurses and environment of clinic b. the other two mrsa strains from neonates of clinic b showed st ( - - - - - - ), which is shared by six strains from nurses and environment of clinic b. spa type of these mrsa strains was identical as ujebkbp and all strains were negative for pvl gene. objectives: over the last years there has been a dramatic increase in the prevalence of methicillin-and multiresistant staphylococcus aureus (mrsa) leading to a major health problem, especially in the nosocomial setting. to support infection control measures typing of mrsa is essential. the present Ôgold standardÕ for strain typing is pulsed field gel electrophoresis (pfge), but several sequence based methods including spa typing have recently been introduced. therefore this study was initiated to compare typing results obtained from pfge with those from spa typing. methods: well characterized s. aureus strains of different evolutionary origin including methicillin sensitive and resistant isolates were included. strains were selected from a variety of clonal groups prevalent in germany and middle europe during the last ten years. the collection comprised hospital derived strains as well as isolates from the community, including the recently emerging community acquired mrsa. all isolates were subjected to pfge with subsequent cluster analysis; additionally the polymorphic x-region of the protein a gene was sequenced and a spa type was assigned using the ridom staphtype software. the newly developed algorithm burp (based upon repeat patterns) was applied to the spa sequences to cluster the resulting spa types into different groups. clustering results were compared to those obtained from pfge and mlst. results: overall the results obtained from the different typing approaches were in agreement. in some cases where pfge results were inconsistent despite uniform epidemiological background spa typing was able to group the corresponding strains together. only two strongly related epidemic clones could not be separated by spa typing. conclusion: spa typing in combination with burp analysis is an easy, rapid and reliable method for epidemiological analyses in s. aureus. it is superior to pfge in cases where pfge might be ÔoverdiscriminatoryÕ. in rare cases a second discriminatory locus (for example a single mlst locus) might be helpful for an ultimate classification. the antibiotic susceptibility of methicillinresistant staphylococcus aureus methicillin-resistant staphylococci (mrsa) are often multidrug resistant and represent a major problem for the antimicrobial therapy. glicopeptides are the golden standard for these infections but resistance and toxicity concerns limit their usage. mrsa antibiotic resistance may be divided into two distinctive profiles: multidrug resistance (probably nosocomial infections) and variable resistance, usually to or non-betalactamic antibiotics (often correlated with community-related mrsa infections results: in total, . % ( ) of s. pn was resistant to erythromycin and . % ( ) of invasive and . % ( ) of non-invasive s. pyo were resistant to erythromycin. none of the isolates were resistant to clindamycin. among the ery-res s. pn, the most frequent gene was mef(a) . %, while erm(b) was found in . %. in two isolates no resistance genes were identified. out of mef(a) carrying isolates had serotype .erm(a) and mef(a) was present in . % and . % of the invasive ery-res. s. pyo isolates. none of the isolates were positive for erm(b). in non-invasive ery-res. s. pyo isolates %, %, % were pcrpositive for erm(a), erm(b) and mef(a) respectively. conclusion: in contrast to s. pyo, ery-res. s. pn is dominated by one serotype, carrying mef(a). the overall resistance profile of s. pn and s. pyo is still favourable, but the resistance to macrolides is of growing concern in denmark. resistance in streptococcus pneumoniae: auc/ mic breakpoints differ between gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin k. laplante, m. rybak, b. tsuji, g. kaatz (detroit, usa) objective: the potential for resistance development in streptococcus pneumoniae (sp) secondary to varying exposure to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin was examined at high inoculum ( . - log cfu/ml) over hours in an in vitro pharmacodynamic model. results: the molecular analysis of one hundred and forty serotype a dspn isolates revealed six clonal types. however, the vast majority ( %) belonged to a single clone. the prevalence of this dominant clone during the study ranged from . % to . % and it was disseminated in all day-care centres. conclusions: serotype a is not a capsular type with an important representation in drug-resistant collections which are the most commonly studied worldwide. however, it seems to be common among dspn strains. this serotype a collection showed little genetic variability. in fact, one genotype was found to be dominant and disseminated successfully in all day-care centres studied. these findings suggest that this serotype a clone is frequent in colonization and thus its monitoring is of importance particularly after the introduction of the sevenvalent pneumococcal conjugate vaccine. diseases. an abrupt antimicrobial resistance increase and clonal spread of resistant pneumococcal strains has resulted in serious therapeutic problems in recent years. the aim of this study was to analyze resistance patterns and genetic diversity of s. pneumoniae resistant strains isolated in our region during three years ( ) ( ) ( ) . methods: using e-test method and the nccls criteria for benzylpenicillin (p), erythromycin (e), clindamycin (l), tetracycline (t), cotrimoxazole (s), ceftriaxone (c), chloramphenicol (h), vankomycin (w), imipenem (i), fifty nine resistant or intermediate to at least one drug strains were obtained. strains were submitted to molecular characteristic by pfge with smai restriction enzyme and computer analysis using molecular analyst software application. for each strain resistance pattern and pfge profile was determined. results: resistance to out of determined antibiotics (except vancomycin) was described. strains showed different resistance patterns and tsh ( %strains), psi ( . %) and elts ( . %) were the most often. resistance degree reached drugs ( % strains) and % strains were mdr. we have found pfge profiles: of them (u - ) were unique single isolates, clusters (a-k) were represented by - strains, which were more than % of similar. the most numerous clusters (a-k) consists of strains that were isolated over three years of study and showed the same or very similar resistant patterns: a-tsh ( of strains), th ( ); b-psi ( of ), s ( ); c-pelshi ( of ), ptshi ( ), ptschi ( ); d-elts ( of ), els ( ); e-sh ( of ), ts ( ), t ( ). all strains of tsh and psi resistance patterns were found in one cluster a(a -a ) or d (d -d ) respectively but strains with elts and pelshi patterns belonged to different ( - ) clusters and unique pfge profiles. another clusters (f-k) were represented by two strains with different resistant patterns each. conclusions: population of s. pneumoniae resistant strains in our region presents high genetic diversity and numerous different resistance patterns. the majority of strains with the same resistance pattern showed different pfge profiles but there were observed some strains of the same resistance pattern, which belonged to only one cluster and were isolated over three years of study. ) hlpr strains showed a profile identical or related to two epidemic clones: spain f- and spain v- . the remaining hlpr pneumococci belonged to unique or rare clones. llpr isolates were characterised by more variable backgrounds in terms of pfge patterns and serotypes than hlpr strains. in fact, . % showed an identical or related profile (arbitrarily called d, serotype a) previously not described in italy and ( . %) exhibited the pfge patterns typical or related to the spain f- and spain v- clones. the remaining llpr isolates ( . %) belonged to different profiles and to distinct serotypes. in this study, the f Ôitalian cloneÕ circulating in - was not found. conclusions: the recent increase of total penicillin-resistance in italy can be ascribed to the emergence of a new clone and to the diffusion of two well known international clones, whose ability to spread is higher than that of the autochthonous italian clone described in . changing antibiotic prescribing habits, including the recent strict limitation to the consumption of parenteral drugs, may also explain the variations described. results: during the study period, strains of s. pneumoniae were isolated: ( . %) in csf and ( . %) in blood. total incidence was cases/ , inhabitants/year, with maximum incidence in winter and spring. the largest number of cases, ( %), were in the over s. in children, ( . %) were detected, all in the under s. the total number of strains whose sensitivity to penicillin fell was ( %), of which ( . %) had an mic mg/ml. . % of the penicillin-resistant strains were also resistant to erythromycin. ( %)/ had reduced sensitivity to cefotaxime, and most of these ( %) had intermediate sensitivity. resistance to erythromycin was detected in ( %). during the study period, resistance to antibiotics decreased gradually in such a way that the resistance percentages each year were: penicillin %, %, %, %; cefotaxime %, %, %, %; erythromycin %, %, %, %. no differences were observed in the resistance percentage in the different age groups. conclusions: the prevalence of csf and blood strains of s. pneumoniae with reduced sensitivity to penicillin in this study was %. the treatment of choice in s. pneumoniae invasive disease was third-generation cephalosporins, due to their low level of resistance. resistance to the main antimicrobials from this type of strain has fallen. during the study period, resistance to penicillin fell by . %, . % for cefotaxime . % for erythromycin. the fall in resistance among invasive s. pneumoniae could be due to vaccination in children and the elderly from onwards, since the vaccination includes the most common and resistant serotypes. the low number of isolates in children could be due to the low number of samples processed. change in distribution of macrolide-resistant streptococcus pneumoniae genotypes over years -data from protekt - objectives: this analysis of data from countries over the first years ( ) ( ) ( ) ( ) ( ) of the protekt global surveillance study was undertaken to investigate longitudinal and regional trends in the distribution of macrolide resistance mechanisms among streptococcus pneumoniae, and the susceptibility of these isolates to antibacterials, including the ketolide telithromycin. objectives: the -valent formulation of the pneumococcal conjugate vaccine (pcv ) was introduced in the usa in february . there is a general recommendation for its use in all children < years of age, and the vaccine is licensed for children up to years of age. in europe, the vaccine is licensed for children up to years of age, but most countries do not have a general recommendation for use. protekt -a global, longitudinal study of the antimicrobial susceptibility of bacterial respiratory tract pathogens -has now completed its fourth year. a comparison was made between serotype distribution and pcv coverage between y and y . methods: analysis was performed only on streptococcus pneumoniae isolates obtained from paediatric patients at the centres common to both years (y , n = ; y , n = ). serotyping was performed by neufeld's quellung reaction using statens serum institute antisera (ssi, denmark). results: pcv coverage decreased from . % and . % in y to . % and . % in y in the usa and latin america, respectively (chi-squared; p = < . and . , respectively). serotypes that showed the greatest increase (y , y ) in the usa were a ( . %, . %), a ( . %, . %), ( . %, . %), ( . %, . %), and ( %, . %). in brazil, a similar pattern was seen although there was no change in the proportion of isolates with the a serotype. in contrast, no change in serotype distribution was observed in canada, western europe, and the far east, where the pcv vaccine is not routinely used for paediatric vaccination. conclusions: results from this analysis of protekt data indicate that the proportion of s. pneumoniae isolates from paediatric patients covered by the pcv vaccine has decreased significantly over years in regions where the vaccine is routinely used. other serotypes, such as a, which is known to be highly resistant to antimicrobials and is not covered by pcv , have increased in these regions. this study demonstrates the importance of serotyping antimicrobial surveillance study isolates in order to monitor such changes and any potential future implications for therapy and vaccine formulations. background: sequential alterations in pbp sequences constitute the classical mechanism to acquire penicillin resistance in s. pneumoniae, that is reflected in changes in the cell wall peptidoglycan structure. murm gene controls the addition of the first amino acid of the dipeptide bridge of the pneumococcal muropeptide. mutations in this gene are apparently required for high penicillin and cefotaxime resistance. the aim of this study was to compare the murm gene sequence within the earlier and the latest highly penicillin resistant pneumococcal populations recovered in spain. material and methods: a total of s. pneumoniae isolates ( of them from - and from - ) with resistance to penicillin (mic - lg/ml) were included. susceptibility to different b-lactams, macrolides, tetracycline, levofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole (sxt) were determined by the agar dilution method. all isolates were grouped according to serotype and pulsotype (pfge-smai pattern) and the murm gene was sequenced. results: a high percentage of resistance (intermediate plus resistant) was observed for cefotaxime ( %) and sxt ( . %). all isolates remained susceptible to telithromycin but not to levofloxacin ( % of susceptibility). all isolates recovered during - were genetically unrelated and mainly belonged to serotypes f ( %) and b ( %). in contrast, the most frequent serotypes during - were ( %), v ( %), and b ( %). in this period, a predominant spain v- clone was detected in out of isolates of serotype , and in out of isolates of serotype v (capsular switch). all isolates of serotype b belonged to a single clone (spain b- ). the analysis of murm sequences of isolates from both periods revealed the existence of different alleles. homologous sequences to the murma (identical to that described for r penicillin susceptible strain), were replaced in part by murmb , and murmb (different variants of these alleles were detected in our collection). conclusion: early high penicillin resistant s. pneumoniae isolates in spain were genetically unrelated and corresponding to f and b serotypes. on the contrary, recent isolates showed dissemination of selected clones, particularly spain v- . many s. pneumoniae isolates with high-level resistance to penicillin retain (or reacquired) the murm gene of susceptible strains, but different allelic variants was also detected. objectives: to determine among s. pneumoniae clinical isolates recovered from different spanish hospitals during three different periods ( - , - and - ) the prevalence of erythromycin resistance phenotypes and resistance genes associated to this resistance. methods: susceptibility testing was performed by the standard microdilution technique (nccls). a pcr assay was carried out to identified erythromycin resistance genes (erm or mef) and a multiplex-pcr was designed to distinguish between mef(a) and mef(e) genes within mef positive isolates. clonality was studied using the sma-i-pfge technique. during the studied period ranged from . % (first studied period) to . % (third studied period) for penicillin, % (first studied period) to . % (third studied period) for erythromycin, and % (first studied period) to . % (third studied period) for clindamycin. levofloxacin resistance was only found in . % of isolates and were recovered during the third studied period. m phenotype was observed in . % of all tested isolates. considering non-susceptible erythromycin isolates, ermb and mef genes was found in . % and . % of isolates, respectively, as a sole erythromycin resistance gene. the concomitant presence of both determinants was found in . % of isolates. interestingly, among the isolates with mef pcr positive result, only one ( . %) carried the mef(a) gene whereas the mef(e) gene was detected in the others ( . %). an increase in mef prevalence was observed between the first and the second studied periods ( % to . %) and a slightly decrease ( . %) in the third period. pfge analysis revealed a polyclonal structure of mef positive isolates. conclusions: prevalence of mef positive isolates among erythromycin-resistant strains remains low in our country ( . %) being the mef(e) gene the most prevalent mef determinant among these isolates ( . %). results: among young children, single ery resistance was most prominent in all countries, (from % in sweden to % in belgium), except for spain where the proportion of dual resistance was highest ( %). for the other countries dual resistance among young children ranged from % in denmark to % in belgium. except for ireland ( %) and spain ( %), single pen resistance remained below %. conclusion: between countries, large differences in the patterns of s. pneumoniae resistance were found among young children. ery resistance was most common among young children, which may indicate greater use of macrolides in this age group. in order to assess the effectiveness of interventions like vaccination, resistance and serotype data should be monitored carefully. evolution in the pattern of sensitivity to penicillin and cefotaxime of streptococcus pneumoniae background: the increase in resistance to penicillin, and more recently to cefotaxime, means that it is necessary to study the prevalence of resistance to these antibiotics, giving them huge clinical importance. this study compares the sensitivity of s. pneumoniae to penicillin and cefotaxime during , , , , , and . methods: a total of strains of s.pneumoniae from significant respiratory tract isolates were studied. these corresponded to the years ( ) conclusions: in , ( ) . % of the isolates were erythromycin-resistant and clindamycin-and miocamycin susceptible (m phenotype), a smaller percentage than in previous studies; ( ) there was a significant increase in of isolates with the mlsb constitutive phenotype; ( ) there was a high prevalence of resistance to telithromycin ( . %) in the strains with mlsb constitutive phenotype. patterns of macrolide resistance determinants among s. pyogenes and s. pneumoniae isolates in saudi arabia objective: to characterize the macrolide sensitivity of recent isolates of s. pyogenes and s. pneumoniae collected from different hospitals around saudi arabia and to investigate the resistance determinants carried by macrolide-resistant isolates. methods: susceptibility testing was performed using standard nccls methodology on s. pyogenes and s. pneumoniae isolates. macrolide resistance mechanism phenotypes were identified using double disk diffusion. results: all s. pyogenes were penicillin sensitive, while . % were macrolide resistant, the main mechanism of which was of m-phenotype ( %). approximately % of s. pneumoniae was penicillin non-susceptible. macrolide resistance in s. pneumoniae accounted for . %, the majority of which was m phenotype ( %). low-level resistance mediated by mef-bearing strains predominated. conclusions: efforts should focus not only on antibiotic resistance surveillance and the development of guidelines but also on appropriate use of antibiotics. strategies have been proposed which include restricting access, compliance promotion and reduction in our prescriptions and inappropriate use of antibiotics. newer macrolides, including azithromycin, are still considered drugs of choice for empirical treatment of respiratory infection in such circumstances. objectives: mlsb phenotype erythromycin resistance (eryr) is often associated with tetracycline resistance (tetr) and in streptococci is mediated by the methylase genes erm(b) or erm(tr). in streptococcus pneumoniae, erm(b) is carried in transposons such as tn which also carry the tetr gene tet(m). m phenotype eryr is associated with the efflux genes mef(a) or mef(e). these genes are carried in genetic elements that do not commonly include tetr genes, but the mef(e) element in s. pneumoniae can be inserted in a transposon that carries tet(m). our objective was to investigate tetr in eryr beta-haemolytic streptococci of groups a, b, c and g. methods: eryr beta-haemolytic streptococci of groups a ( ), b ( ), c ( ) and g ( ) were collected over two years. tetr was determined by disc diffusion and mics by e-test. pcr was performed for tet(m), tet(o) and tet(l); eryr genes had been characterised previously. results: the prevalence of tetr amongst eryr mlsb isolates was high in groups a ( %, of ), b ( %, of ) and c ( %, of ) but lower in group g ( %, of ). the range of mics in tetr isolates was ‡ (mg/l). the tet(m) gene was responsible for most tetr in groups a ( %, of ), b ( %, of ), c ( %, of ) and g ( %, of ); tet(l) and tet(m) were both found in the same isolates in groups b ( ) and g ( ). at least % of erm(b) isolates and % of erm(tr) isolates of groups a, b and c carried tet(m). in contrast, only % of erm(tr) isolates in group g carried tet(m). amongst m phenotype isolates, the prevalence of tetr was high in groups b ( %, of ) and g ( %, of ) and lower in group c ( %, of ). tetr was not detected in m phenotypes of group a. in m phenotypes, tet(m) was responsible for % tetr in group b ( of ) and % in group g ( of ); isolate of group c carried tet(o). tet(m) and tet(o) were found in mef(e) isolates but not in mef(a) isolates. there were tetr isolates in groups b ( ) and g ( ) in which no tetr gene was identified. conclusions: ) the prevalence of tetr in eryr beta-haemolytic streptococci was high in mlsb isolates of groups a, b and c and in m isolates of groups b and g. ) tet(m) was the predominant tetr gene. ) tet(m) was strongly associated with erm(b) and to a lesser extent with erm(tr). ) tet(m) was found in association with mef(e) but not mef(a). results: reduced susceptibility (mic > mg/l) was detected in ( . %) s. pyogenes isolates. a total of ( %) s. pyogenes isolates demonstrated a mlsb phenotype resistance. the dominating gene was erm(a) (n = ), encoding resistance in strains, whereas erm(b) was detected in only five of the strains isolates. while one of the cmlsb strains carried both erm(a) and the mef(a) gene, another cmlsb strain was reproducibly negative in erm and mef pcr analyses, and will be further investigated. the remaining ( %) isolates all carried the mef(a) gene and showed a consistent with their m phenotype resistance. thirty ( %) strains were resistant to tetracycline and carried the erm-positive strains (n = ) genes, while all the mtype strains were susceptible to tetracycline. typing analyses revealed t-type and st dominated in nine out of with mtype phenotype, whereas t-type was associated with st in of erm(a)-positive and tetracycline resistant strains. results from emm typing supported these clonal observations. conclusions: (i) the prevalence of macrolide resistance among s. pyogenes in norway is low, and the (ii) mlsb resistance, erm(a) or erm(b) was the most prevalent resistance type and co-resistance to tetracycline was frequently observed. rates of resistance to macrolides vary from region to region and these rates are important in defining the role of macrolides in empiric therapy. macrolide and lincosamide resistance are mediated by distinct mechanisms, ( ) mefa encodes resistance to erythromycin but not clindamycin, ( ) ermb encodes resistance to erythromycin and clindamycin, ( ) erma encodes resistance to erythromycin and inducible resistance to clindamycin. methods: consecutive pharyngeal isolates of streptococcus pyogenes isolates were collected between january and april . isolates were confirmed as s. pyogenes by lancefield grouping using the pastorex strep rapid agglutination kit. erythromycin susceptibility testing was performed by nccls disc diffusion. erythromycin resistant isolates were tested for susceptibility to erythromycin and azithromycin by etest. the mechanisms of resistance was assessed using the erythromycin/ clindamycin disc approximation test and by pcr, with primers specific for the erma, ermb and mefa genes. positive controls were kindly provided by ralf r. reinert. results: four ( ) of consecutive isolates ( %) were resistant to erythromycin. erythromycin resistance was confirmed by etest and these isolates were also resistant to azithromycin. all erythromycin-resistant study isolates and of previously collected erythromycin-resistant isolates were confirmed as erma positive by pcr and exhibited inducible resistance by disc approximation. one previously collected isolate was susceptible to clindamycin and was mefa positive by pcr. conclusion: these data show that macrolide resistance in s. pyogenes is very uncommon in this area and is encoded primarily by erma. objective: aim of this study was to evaluate antimicrobial prophylaxis (ap) practice and its cost in intensive care unit of heart surgery depertment in our hospital. method: study was performed prospectively between january-december . ap lasting > day was considered inappropriate, unless the patient was in high risk group (heart transplantation or repeated by-pass). in addition ap with two beta lactam agents; beta lactam+aminoglycoside (only beta lactam+gentamicin was allowed); rd generation cephalosporins; quinolones or glycopeptides were considered inappropriate. in high risk patients, ap lasting up to days and ap with glycopeptides were considered appropriate. cost of inappropriate ap was calculated by using august- prices in euro. cost of inappropriate ap in patients who received cefazolin or beta lactam/beta lactamase inhibitors (ampicillin/sulbactam or amoxycillin/clavulanate) was calculated by substraction of cost of day lasting ap from total ap cost. in case of inappropriate glycopeptide or quinolone or aminoglycoside usage, one day lasting cefazolin cost was substracted from total ap cost. in high risk patients cost of inappropriate glycopeptide usage was calculated by substracting days lasting ap cost from total cost. infections were diagnosed according to the criteria of center for diseases control and prevention (cdc). data were analysed with chi square and student's t-tests. objectives: the aim of the present study was to investigate the frequency and the type of wound infections, as well as the bacteria involved, after posterior spinal fusion in children, especially with spinal deformities, over a -year period ( ) ( ) ( ) ( ) ( ) ( ) . materials and methods: a total of spinal fusions were performed on children, with or without instrumentation, because of spinal scoliosis ( ), spondylolisthesis ( ), fractures ( ), and for other reasons ( objectives: therapy of chronic wounds with lucilia sericata presents a promising alternative to the classical approach with antibiotics. larvae are placed in wounds free-roaming or in biobags. the therapy often stimulates the process of wound healing. this effect is based on different mechanisms. maggotsÕ saliva contains collagenases, trypsin and chymotrypsin-like enzymes which dilute necrotic tissue. furthermore, an antibacterial effect has been described. in our study, we focused on the question whether this effect could be explained by the ingestion of bacteria by larvae. methods: free roaming maggots were placed on agar plates with escherichia coli. these bacteria had been transformed with green fluorescent protein (gfp)-containing plasmids. gfp-free escherichia coli xl blue served as a control. two days old maggots were put on agar plates in groups of ten at room temperature. every thirty seconds, a maggot was displaced from the agar plate and washed with sterile saline ( . %). dead larvae which could not have taken up bacteria served as a control. maggots were tested for flourescence under a fluorescence microscope [zeiss axioskop; hpo /ac, axiocam mrmzeiss]. larvae were examined by an independent observer as well. results: maggots that had been placed on gfp-labelled escherichia coli showed an intensive green fluorescence, especially at the back of the head and in the intestines. the controls did not show any fluorescence. after three to four minutes gfp-labelled escherichia coli could be detected inside the maggots' body. at this early stage of digestion gfp-fluorescence could mainly be examined in the area of the head. this fluorescence results of the ingestion of gfp-labelled escherichia coli by larvae. we have proven that the uptake of bacteria represents a further mechanism of antibacterial activity of larvae. not only diluting necrotic tissue, but also minimizing the rate of infections, maggot therapy which is also well-known as biosurgery could be a beneficial alternative to the use of antibiotics. as the mechanical uptake is a non-specific process, there might not be a risk of resistance. antibiotic resistance: nosocomial pathogens p antimicrobial resistance patterns of bacterial pathogens from blood culture of cancer patients in a single cancer institution m. faiz, h. aziz, t. bashir, s. asghar (lahore, pak) objective: the widespread emergence of resistance to antimicrobial agents among bacterial pathogens is well known and has an impact on our ability to treat patients effectively. blood stream infections (bacteraemia) among cancer patients that develop during the course of disease are potentially life threatening because of suppression in their immune systems. the changing spectrum in the incidence and epidemiology of microbial pathogens has resulted in an increase in resistance to many antibiotic compounds emphasizing the need to monitor the prevalence of resistance in these strains. methods: susceptibility and resistance pattern of clinically significant bacterial isolates from positive blood cultures collected during - was studied. the isolated strains were tested against a wide range of antibiotics belonging to cephalosporins, aminoglycosides, carbapenems and quinolones derivative groups by disc diffusion method and the results were interpreted according to british standard for antimicrobial chemotherapy. results: a total of bacterial pathogens were isolated with % gram positive and % gram negative bacteria. the dominating pathogens were staphylococcus aureus, streptococci, pseudomonas , enterobacter and klebsiella. among gram negative strains, highest level of resistance ( %) to third generation cephalosporins was observed followed by carbapenems and penicillin ( %, %) respectively. similarly, high resistance to aminoglycosides were found ( % to ampicillin, % to tobramycin and % resistant to quinolone derivates group of antibiotics. however only % were resistant to ciprofloxacin. in gram positive bacteria, high resistance to ciprofloxacin ( %) was observed as compared to gram negative bacteria. a still higher resistance level ( %) was observed for aminoglycosides and third generation cephalosporins ( %) respectively. conclusion: the spectrum of isolates among our patients were shifting towards gram positive bacteria with high resistance to different groups of antimicrobial agents limiting few choices for alternative therapies for infection control. antimicrobial resistance continues to increase and ongoing surveillance of microbial pathogens is essential. this study also warrants the need of infection control measures, rational antibiotic policies and rapid laboratory detection of resistance to prevent the spread of resistance among these strains. ( ), and epidemiological swabs from throat and rectum ( ). we found two susceptibility groups: group ( patients) resistant to amox/ clavul.acid, pip/tazobactam, aztreonam, cefuroxime, and group ( patients) susceptible for these antibiotics. rapd profiles corresponded with the susceptibility testing results. drug resistance profile in group could be attributed to overproduction of chromosomal encoded k beta-lactamase. group was from ru and group from nicu, pu and su. analysis of sociodemographic variables and potential risk factors showed differences between analysed groups in mean birth weight, length of hospital stay, antimicrobial treatment and invasive therapeutic procedures usage. the outbreak analysis revealed concurrent isolation of two k. oxytoca clones, normal and k overproducer. rapd results were supported antibiotyping results. results: a total of isolates was analysed. . % of these isolates were non-susceptible to at least one agent, the highest proportion is due to mono-drug resistance to piperacillin ( . %). . % of the strains were classified as multi-resistant, the most frequent patterns were pip/caz/ctx/gen ( . %) and pip/caz/ctx/gen/cip ( . %). two strains were resistant to all six antibiotics. significant differences in multi-drug resistance rates were associated with ward type with highest rates for icu-patients ( . %). furthermore, multi-resistance rates varied significantly between the centres involved with a range from . % to . %. the centres differed not only in respect to the multi-drug resistance rates, but also in respect to the dominating phenotype. conclusions: the relevance of multi-resistance in k. pneumoniae as a major clinical problem is proved by an overall rate of almost five per cent for german university hospitals and an even higher proportion for icu-patients. among the agents tested piperacillin plays an eminent role in regard to its mono-resistant rate as well as a component of the most frequent resistance patterns. resistance to the combination of pip with tazobactam however is rare. the development of antimicrobial resistance in german hospitals objectives: quinolones (q) are among the most frequently used agents for treating ltcf-acquired infections. consequently, bacteria exhibit increasing resistance to q. a multicentre survey was conducted in order to determine (i) the prevalence and risk factors for colonization with qrgn in greek ltcf (ii) the corresponding antimicrobial resistant rates (Á r). methods: a total of ltcf were randomly selected from the public sanitation list of attica province. urine, nasopharyngeal and wound samples were collected from elderly residents. we chose randomly % of the existing population from each ltcf (minimum sum residents, age above years). gram negative bacteria were identified by api strips and underwent antimicrobial disk susceptibility testing, following the nccls guidelines. data were also collected on resident factors and institutional variables. univariate and multivariate analyses were performed. odds ratios (or) and p values were calculated. results: gram negative (gn) strains were isolated from samples (prevalence rate . %). the majority of them were recovered from urine samples ( . %). % of the residents had been taking systemic antibiotics during the preceding month. q were the leading class ( %). Á r of gn to ciprofloxacin (cip) were estimated (table) . multi-drug qrgn accounted for . % of the isolated strains. the most prevalent phenotype was resistance to ampicilline, cip and trimethoprim-sulfamethoxazole. in the multivariate analysis, only prior exposure to antimicrobial agents (p < . ; or = ), specifically to q (p < . ; or = ), and the presence of a urinary catheter (p < . ; or = ) were significantly associated with colonization with qrgn. objectives: colonization and resistance dynamics of gramnegative bacteria in the intestinal and oropharyngeal microflora of patients admitted to intensive care units (icu) and general wards (gw) were investigated during and after hospitalization. methods: specimens were obtained on admission, once weekly during hospitalization, at discharge from the icu, at discharge from the hospital and one and three months after discharge from the hospital. five colonies per specimen were selected for identification and susceptibility testing by the vitek system. results: a total of specimens from patients were collected. in both patient populations, the gram-negative colonization rates in oropharyngeal specimens increased during hospitalization and did not decrease in the three months after discharge. in rectal specimens, colonization rates decreased during hospitalization and increased after discharge. there was a change in species distribution among the dominant microflora during hospitalization. klebsiella spp., enterobacter spp., serratia marcescens and pseudomonas aeruginosa were more often isolated, whereas the frequency of e. coli declined. the percentage of icu patients colonized with ampicillin and/or cephalothin resistant faecal e. coli was significantly increased at discharge from the hospital and did not change in the three months after discharge. emergence of multi-drug resistance was observed in many gram-negative species during stay on the icu. resistance frequencies in e. coli significantly increased with length of stay on the icu. for the gw population, no significant changes in resistance frequencies were found during hospitalization. conclusion: from a population perspective, the risk of dissemination of resistant gram-negative bacteria into the community through hospitalized patients appears to be low in gw patients, but is noticeably higher among icu patients. association of antibiotic resistance phenotypes and the presence of class i integrons in a sample of bacterial isolates from u.s. hospitals objective: we assessed the association between class integrons and patterns of resistance to aminoglycosides, beta-lactams, quinolones, and chloramphenicol in escherichia coli and klebsiella species isolates from u.s. hospitals participating in project icare. methods: a convenience sample of e. coli and klebsiella species isolates was collected between october and december from u.s. hospitals as part of phase iv of project icare and tested for susceptibility to a variety of antimicrobial agents. these organisms were submitted in response to a request for organisms resistant to rd generation cephalosporins (ceftazidime, ceftriaxone, cefotaxime) and/or fluoroquinolones. the isolates were screened for the presence of class i integrons using sets of primers specific for ¢ and ¢ conserved segment of the integrase gene. we obtained measures of association between the presence and absence of integrons and resistance phenotypes. results: of the e. coli and klebsiella species isolates screened, ( %) contained a class i integron. a significant association was seen between the presence of the class i integron and resistance to amikacin, gentamycin, tobramycin, ampillicin, chloramphenicol, aztreonam, ceftazidime, cefotaxime, and cefpodoxime. resistance to ciprofloxacin and cefepime was not significantly associated with the presence of an integron. the presence of class i integrons in % of our convenience sample of e. coli and klebsiella species isolates, along with the differential association of those integrons with resistance to some drugs, but not others, suggests that integrons may play an important role in determining how the epidemiology of bacterial resistance results from antimicrobial agent selective pressure. objectives: the main purpose of this study was to investigate the mechanisms of resistance to fluoroquinolones in two isogenic citrobacter freundii clinical isolates, which led us to characterize the acra and acrb genes of this microorganism. methods: two c. freundii strains ( . and . ) sequentially isolated from the same patient were characterized. the relationship was determined by rep-pcr and pfge. the susceptibility to ciprofloxacin and chloramphenicol was determined by e-test. mutations in the quinolone resistance determining region of the gyra and parc genes, the outer membrane protein profile and the accumulation of ciprofloxacin was also investigated. the expression of genes in both strains was analysed using dna microarrays for e. coli and the expression of the acra and acrb genes was verified by rt-pcr using the gapa gene as the control. the acra gene was cloned and dna sequenced. results: both c. freundii belong to the same clone by both rep-pcr and pfge. the mic of ciprofloxacin was of (strain . ) and mg/l (strain . ). the mic of chloramphenicol was of and mg/l, respectively. the two strains showed the same substitutions in the gyra and parc (thr- -ile and asp- -tyr in gyra and ser- -ile in parc). no major differences were found between the outer membrane protein profiles. however, differences were observed in the amount of ciprofloxacin accumulated, with strain . showing less accumulation. eleven genes were overexpressed in strain . compared to strain . . among these genes the acra was overexpressed. this result was further corroborated by rt-pcr. the nucleotide similarity between the partially sequenced acra ( bp) and acrb ( bp) genes of c. freundii and e. coli was of . % and %, respectively. conclusion: the acra and acrb genes of c. freundii are similar to those described in e. coli and in collaboration with mutations in the gyra and parc genes, their overexpression may play an important role in modulating the final mic of fluoroquinolones. high prevalence of aac ( ) objectives: multidrug resistance of gram negative bacilli (gnb) has become a major public health problem in tunisia. surveillance of both antimicrobial resistance and antimicrobial consumption has now been recognised as essential for planning future strategies to control resistance. the aim of our study is to examine for the whole hospital and wards with risks the correlation between resistance of gnb and the consumption of third generation cephalosporins ( rd gc) and imipenem for the period from january to september . methods: a surveillance of antibiotic resistance and antibiotic consumption was established respectively by the laboratory and the department of pharmacy. data on antimicrobial resistance and antibiotic consumption were collected quarterly and antibiotic consumption was calculated in defined daily doses (ddd) using abc calc. results were expressed per bed-days (bd). statistical analysis was done using spss software for the calculation of the spearman rank correlation (rs) with an alpha risk of %. results: for the whole hospital, resistance to rd gc ranged from . to . gnb/ bd. the highest rates were observed in intensive care unit (icu: . - . gnb/ bd) and the lowest rates in orthopaedic ward ( - gnb/ bd all mrsa isolates were resistant to multiple classes of antimicrobials whereas mssa were sensitive (p < . ). all esbl+ bacteria were resistant to multiple classes but sensitive to meropenem, colistin, amikacin ( %), cotrimoxazole ( %) and chloramphenicol ( %). resistance in non-esbl+ bacteria was detected to ampicillin ( %), cefalexin ( %), nd/ rd generation cephalosporin ( %), chloramphenicol ( %), cotrimoxazole ( %), ciprofloxacin ( %), trimethoprim ( %), gentamicin ( %), netilmicin ( %) and tobramycin ( %). potentially community acquired isolates showed lower rates of resistance ( % to trimethoprim, ciprofloxacin, gentamicin, amikacin, tobramycin, nd/ rd generation cephalosporin).duration of hospital stay was not a risk for acquiring either esbl+ bacteria or mrsa whereas having a surgery was associated. exposure to quinolone was a risk factor for mrsa but not esbl+ bacteria. aminoglycoside and cephalosporin were risks for esbl+ bacteria. acquiring both mrsa and esbl+ enterobacteriacae together did correlated with the duration of hospital stay in addition to exposure to aminoglycoside and cephalosporin. conclusion: majority of patients remained free from resistant bacteria implying that cross-transmission alone was not sufficient in absence of risk factors, particularly exposure to antimicrobials. good hand-hygiene and prudent use of antimicrobials are realistic options for resource poor countries to reduce the burden of resistant bacteria. there is a unique opportunity to sequentially introduce specific infection control measure and evaluate its effectiveness. antimicrobial resistance in an intensive care unit before adopting an antibiotic rotation programme for empiric therapy of ventilator-associated pneumonia results: among gram-negative aerobic bacilli, we found p. aeruginosa strains to be meropenem and piperacillin/tazobactam-resistant in % and . % of cases, respectively; p. aeruginosa, e. coli and k. pneumoniae were resistant to ceftazidime in . %, . %, and % of cases, respectively, and resistant to ciprofloxacin in . %, . %, and . % of cases, respectively. neither e. coli nor k. pneumoniae were resistant to either meropenem or piperacillin/tazobactam. among gram-positive aerobic cocci, oxacillin-resistant s. aureus and cns were . % and . %, respectively; s. aureus isolates were very susceptible to cotrimoxazole (resistance . %) and rifampin ( %), whereas resistance exceeded % for clindamycin, gentamicin, and norfloxacin. ampicillin-and penicillin-resistant e. faecalis were . % and . %, respectively. neither s. aureus nor e. faecalis were resistant to vancomycin, whereas one cns strain was resistant to it ( . %) and out of to teicoplanin ( . %). conclusion: in our icu, where a careful policy of antibiotic use with no predetermined restrictions has been applied for years, ar among both gram-negative and gram-positive microorganisms is generally lower than in the icus of italy and other mediterranean countries. we expect that the institution of an arp for empiric therapy of vap can further minimize ar in our icu. background: although newer antibiotics have been introduced to the market during the last years, they have not solved the problems arising in the management of infections due to multiresistant gram-negative bacteria. colistin, an antibiotic almost forgotten for decades has proved itself helpful, when used parenterally in patients where a lot of the classic and newer antibiotics fail. methods: we report our experience with the management of the case of a young patient who, after head trauma, had five episodes of meningitis due to multidrug-resistant gram-negative microorganisms. results: a -year-old patient was admitted in our hospital because of coma. three months prior to his admission, he sustained acute brain injury due to a fall and was hospitalized elsewhere for this entire period. a computerized tomography scan of his brain at his admission in the first hospital, showed multiple cerebral contusions, and an intracerebral hematoma causing deviation of the midline. he underwent a decompressive craniotomy with removal of the hematoma. during his long-standing hospitalization, he developed episodes of meningitis, i.e. on day , , , , and after head trauma. the pathogens isolated from each episode of meningitis were multiresistant strains of pseudomonas aeruginosa ( st and nd episodes), acinetobacter baumannii ( rd episode), enterobacter cloacae ( th episode), and acinetobacter baumannii ( th episode). the two episodes of acinetobacter baumannii meningitis were managed only when colistin and amikacin were given by the intraventricular in addition to the intravenous route for long period of time (six weeks for the last episode) and in high doses ( , iu of colistin and mg of amikacin results: eight patients received aerosolized colistin. all were admitted to the icu with a mean acute physiological and chronic health evaluation ii (apache ii) score on the day of icu admission and on the st day of aerosolized colistin administration of . and . , respectively. six of the patients had ventilator-associated pneumonia. the responsible pathogens were acinetobacter baumannii ( / cases) and pseudomonas aeruginosa ( / cases) strains. half of the isolated pathogens were sensitive only to colistin. daily dosage of aerosolized colistin ranged from . to million iu (divided into or doses) and the mean duration of administration was . days. seven out of patients received concomitant intravenous treatment with colistin or other antimicrobial agents. clinical response of pneumonia was observed in out of patients [ cured, improved (they were transferred to another facility)]. one patient deteriorated and died due to septic shock and multiple organ failure. aerosolized colistin was well tolerated by all patients; no bronchoconstriction or chest tightness was reported. conclusions: aerosolized colistin may be a beneficial adjunctive treatment in the management of nosocomial pneumonia (vap or not) due to multidrug-resistant gram-negative bacteria. to date, susceptibility rates to imipenem and colistin of nosocomial strains are reported as high as % and > %, respectively. in particular, resistance to colistin has been rarely described and the mechanisms of this resistance are poorly understood. we report a case of colistin resistant a. baumannii infection in a patient without previous administration of colistin. case report -methods: a colistin resistant (mic > mcg/ml) a. baumannii was isolated from sets of blood cultures in a patient admitted in a burn unit without previous colistin exposure. the patient was treated with ampicillin/sulbactam and colistin, despite the documented resistance to all antibiotics. mdr a. baumannii was repeatedly isolated from different sites and the patient died after days of hospitalization. strain identification and susceptibility tests were performed using phoenix automated microbiology system Ò . resistance to ampicillin, ampicillin/sulbactam, ceftazidime, imipenem, meropenem, cotrimoxazole, amikacin, ciprofloxacin and colistin was confirmed by disk diffusion test and broth microdilution, in accordance with the nccls guidelines. the susceptibility results were confirmed by a reference laboratory. conclusion: colistin is one of the few effective drug available against mdr acinetobacter infections and acquired resistance is exceptional. our report points out that colistin resistant a. baumannii may be found in hospitalized patients without previous exposure to colistin. nosocomial infections by colistinresistant a. baumannii could represent a new therapeutic challenge. programmes of active surveillance cultures and contact precautions should be implemented to control the spread of this mdr microorganism. intrathecal colistin treatment of central nervous system infections due to multi-resistant acinetobacter baumannii c. suárez fernández, p. fernández-viladrich, f. tubau, l. corral , a. mateu, j. ariza (barcelona, e) objectives: the optimal therapy of cns infections due to multi-resistant acinetobacter baumannii (mrab) is not established. in we published the first two cases of ventriculitis due to mrab treated with intrathecal (it) colistin. one patient received mg colistin base/ hours after delayed csf sterilization was documented with initial dosage of mg/ hours. here we describe our extended experience with this kind of treatment . methods: we retrospectively reviewed all documented cns infections due to mrab diagnosed in our terciary -bed hospital from to . mrab was defined as those strains with only susceptibility to colistin (mic < mg/l). cns infections were defined as those cases with suggestive clinical features plus ventricular or lumbar csf with both characteristic cytochemical alterations and positive culture. results: there were cases with documented nosocomial cns infection due to mrab. six of them were acquired after neurosurgical procedure through surgical wound infection or local catheter infection. one case was caused by hematogenous spread after urological manipulation. intrathecal therapy was administered through a ventricular catheter in five cases, lumbar catheter in one case and both ventricular and lumbar catheter in one case. the usual doses administered was colistin base mg (equivalent to colistimethate sodium mg) every hours with csf continuous drainage being interrupted for about hours. length of treatment were - days with any apparent adverse effect. six of these patients received simultaneous intravenous colistin therapy. culture sterilization was documented in all patients. two patients died (one from unclear cause after initial favorable evolution; one after receiving only two doses of it colistin). the rest of them cured with sequela attributable to their previous neurological diseases. conclusions: in our experience, combined treatment with both intrathecal and intravenous colimicin seems safe and effective. when administered by local route to patients with continuous csf drainage we suggest a dosage of colistin base of mg every hours with temporary interruptions of the drainage. bacillus cereus -the forgotten pathogen. an unusual infection in an orthopaedic patient s. thomas, a. pillai, j. arora, a.c. ogden (dumfries, uk) background: infection with bacillus cereus has been well documented in the literature for over a century. infection is generally associated with the gastrointestinal effects of food poisoning and linked to the consumption of infected rice dishes. however, the bacillus genus is extremely heterogenic. it can occupy a wide variety of ecological niches and bacillus spores are found ubiquitously in the environment. even so, bacillus cereus isolated from the hospital and clinic setting in any material other than vomitus or faeces is commonly dismissed since it is a known contaminant of blood cultures and most bacillus bactereamias are transient and usually insignificant. case report: we report a case of bacillus cereus wound infection in a previously healthy -year-old male admitted to the orthopaedic ward with a comminuted fracture of the right distal tibia. he developed compartment syndrome one day later and was taken to theatre for fasciotomy. within hours, he developed chest complications and was admitted to the high dependency unit. after three days he was taken back to theatre for wound inspection and change of dressing. seven days later external fixation was applied. subsequently, the wound site became red, inflamed and tender and was associated with an acute rise in inflammatory markers. microbiological reports showed bacillus cereus growth from the suture sites, sensitive to ciprofloxacin. no source was identified. discussion: this report merits concern, because it highlights the risk of wound cross infection with this unlikely pathogenic species. bacillus cereus has been known to be a contaminant of dressings, intravenous catheters, theatre scrub suits and linens pointing to an array of possible infective sources within the hospital. it emphasizes the need for theatre and hospital sterility and stresses the importance of vigilance against this infrequent cause of a potentially serious non-gastrointestinal bacillus infection. this is of particular importance because alcoholbased cleaning solutions are known to be ineffective against bacillus spores and infections dismissed as contaminants may lead to rapid clinical deterioration. antibacterial effect of phenytoin in wound healing objectives: phenytoin is an antiseizure medication, has since been reported to promote wound healing when applied as a topical agent. this effect is due to rapid infiltration of fibroblasts, collagen deposition, new vessel formation as well as antibacterial activity. this study was undertaken to evaluate it's effect on chronic skin ulcers with different causes and compare it with normal saline. methods: fifty inpatients with chronic skin ulcers were included in this case-control study: diabetic foot ( %), fracture and surgery wounds ( %), decubitus ulcers resulting from warrelated ( %). the patients were matched for age, sex, severity and size of wounds and randomly assigned to two group: phenytoin treated (pht) and control (ctl). each group included men and women, age range was between and . surface areas of the ulcers was - cm . the ulcers were debraded of necrotic tissue (if required). cultures of ulcers were taken at the beginning of treatment and on day , . in pht, thin dusting of phenytoin powder and dry gauze dressing were applied daily after washing with normal saline. ctl group received only daily washing with normal saline and plain dressing. results: bacteriologic culture in both group confirmed some strains (staphylococcus aureus, kelebsiella, proteus and psudomonas). at the beginning of treatment, there was culture positive in pht. case became negative by day and case became negative by day . in ctl, we had culture positive that they didn't become negative by day and (seventy-six per cent of pht had negative cultures by day compared to % of ctl). the mean time for appearance of granulation tissue was . days in pht compared with ctl that was . (p = . ). the average time to complete healing in pht was . days compared to . in the ctl (p = . ). in pht only patients required to analgesics (mean /day) compared with ctl that all patients required to analgesics about - /day (p = . ). age, sex and kind of wounds didn't effect the healing time in both group. conclusions: this study with statistical analysis demonstrated good improvement and efficacy of phenytoin in treatment of chronic ulcers compared with normal saline. . prompt pain relief, . reduction in ulcer size, . changing bacterial culture to negative, . more rapid granulation and healing time, characterized the pht group. we recommend wider use of this safe, inexpensive, readily available and easy to use agent because of it's positive effect on wound healing especially in our country that a lot of patients suffer from decubitus ulcers resulting from war-related wounds and limited access to more expensive wound care therapies. recurrent osteomyelitis secondary to different bacteria i. uckay, m. assal, l. legout, p. rohner, r. stern, d. lew, p. hoffmeyer, l. bernard on behalf of the osteomyelitis group study objective: recurrence of osteomyelitis due to the same bacteria from a prior site of infection without evidence of trauma or bacteraemia is an entity well-known in the literature and clinical practice. the objective of this study is to try and determine if all these reactivations are actually due to the same bacteria, as has been assumed in previous case reports. case reports: we report three cases of osteomyelitis recurring in the same bone in otherwise healthy patients. in all three patients, there was no history of illness or trauma to assume another origin. surprisingly, the strains were different from the two infectious episodes. conclusion: reactivation of osteomyelitis can be caused by different strains of bacteria, many years after the initial episode without trauma or evident bacteraemia. former infected and therefore altered bone surface might be a region of diminished resistance for a new infection during silent transient bacteraemia, reminiscent of the clinical entity of (recurrent) infectious endocarditis on altered valve surfaces. objectives: vascular graft infection proved to be the most dangerous complication in vascular surgery patients. the aim of our study was the identification of microorganisms causing vascular graft infections and the evaluation of their antimicrobial susceptibility. patients with infected vascular grafts, treated in vascular surgery department, took part in our research. in % of patients, the late type of infection was recognized, in % of patients the infection was qualified as early. methods: purulent discharge obtained from the fistula was inoculated on the bacteriological media. antimicrobial susceptibility was assessed by disc-diffusion method. results: staphylococcus aureus and pseudomonas aeruginosa, present in , % of patients, proved to be the most frequently isolated microorganisms. staphylococcus epidermidis and e. coli were isolated in . % and % of patients, respectively. mixed infection, caused by two distinct bacteria, occurred in % of patients; in all cases one species belonged to gram-positive, and the second one to gram-negative bacteria. in % of patients with early type infection different species of gram-negative rods were present, in . % of patients, staph. aureus and staph. epidermidis were isolated, enterococcus faecalis occurred in . % of patients. in late type infection gram-negative rods were isolated from . % of patients and gram-positive bacteria from . % of patients. the most frequently isolated species appeared to be pseudomonas aeruginosa. in one patient candida crusei was isolated. the isolated species of bacteria varied depending on the degree of infection (according to shilagy and samson). in iiia degree of infection staph. epidermidis and e. coli were present in . % and . % of patients, respectively. pseudomonas aeruginosa and staph. aureus were isolated in , % of patients with iiib degree of infection. various species of gramnegative rods were isolated from % of patients with iiic degree of graft infection. conclusions: most isolated bacteria appeared to possess the resistance patterns typical for hospital flora; among them methicillin-resistant staph. aureus (mrsa) and methicillin-resistant coagulase-negative saphylococci (mrcns) strains, gramnegative rods producing extended spectrum of beta-lactamases (esbl) or ampc beta-lactamases, and enterococcus faecalis with high level of aminoglicosides resistance (hlar). treatment failure due to emergence of resistance to imipenem during therapy for shewanella algae bacteraemia objective: we describe here a case of bacteraemia caused by s. algae, which was initially susceptible to imipenem, but the bacteria later became resistant during the treatment. in addition, we investigated the propensity of s. algae to develop resistance to imipenem by using a serial passage technique. method: in vitro test for resistance induction . bacterial strains resistant variants selection (a) single step resistant variants were obtained from clinical isolate on mueller-hinton agar containing increasing amounts of imipenem ( xmic, xmic, xmic, xmic), (b) serial passage experiment s. algae strain and p. aeruginosa atcc were grown overnight in mueller-hinton agar and then grown overnight in mueller-hinton agar and then swabbed onto mueller-hinton agar plates containing one-half the mic of imipenem. the surface growth at h was swabbed to mueller-hinton agar containing twice the prior concentration of imipenem. results: single step resistant variants were selected from isolate at up to times the mic, whereas the resistant variant from p. aeruginosa atcc could be selected at up to times the mic. all the resistant variants of s. algae selected either by a single step or by a sequential stepwise passage exhibited at up to - lg/ml of mic, whereas those of p. aeruginosa atcc showed up to lg/ml of mic conclusion: we documented that s. algae, which was initially susceptible to imipenem, subsequently became resistant to imipenem during the treatment. we also demonstrated in vitro that the initial isolate of s. algae could easily develop resistance to imipenem when the organism was exposed to imipenem, suggesting that s. algae organisms have a propensity toward resistance to imipenem. objective: peritonitis remains a common and serious complication of peritoneal dialysis. in this study, we evaluated the frequency of microorganisms isolated from peritoneal fluids of patients on continuous ambulatory peritoneal dialysis (capd). methods: during a . year period, peritoneal fluid samples were collected from capd patients with peritonitis symptoms. the specimens were inoculated on appropriate media after -min centrifugation. gram stained smears and aerobic, anaerobic and broth cultures were performed. the isolates were identified by commercial id panels. mics were determined with a microdilution method according to nccls guidelines. results: fifty two out of samples were positive ( . %), six of the positives were polymicrobial. the following microorganisms were obtained: pseudomonas aeruginosa ( ), staphylococcus epidermidis ( ), escherichia coli ( ), staphylococcus aureus ( ), streptococcus viridans ( ), klebsiella pneumoniae ( ), enterococcus faecalis ( ), stenotrophomonas maltophilia ( ), enterobacter aerogenes ( ), acinetobacter baumannii ( ), enterococcus faecium ( ), proteus mirabilis ( ), pseudomonas fluorescens/putida ( ), acinetobacter lwoffii ( ), salmonella group d ( ), bacteroides spp. ( ). fungi were isolated in three patients: candida tropicalis ( ), candida krusei ( ) and candida parapsilosis ( ). three out of of s. aureus strains and three out of of s. epidermidis isolates were found resistant to methicillin. all saphylococci and enterococci were susceptible to vancomycin and linezolid. gram-negative pathogens were sensitive to used antibiotics. conclusion: thirty five patients ( . %) developed peritonitis the most prevalent etiologic agents of peritonitis on capd patients were pseudomonas aeruginosa, staphylococcus epidermidis, escherichia coli, staphylococcus aureus and streptococcus viridans. since capd patients are commonly outpatients, the antimicrobial resistance in the gram-negative strains is low compared to the nosocomial isolates. appropriate antibiotic therapy based on microbiologic results needs for the management of peritonitis on capd patients. objectives: cefepime is a fourth generation cephalosporin with a broader spectrum of activity against gram-negative and gram-positive pathogens in comparison with all other cephalosporins. due to the excellent antibacterial activity including pseudomonas aeruginosa and enterobacter spp. together with low rates of resistance and favourable pharmacokinetic and clinical properties, cefepime is a drug of choice for initial empiric treatment of severe nosocomial infections. nevertheless, in addition to recommendations in guidelines, the surveillance of local resistance data is important for empiric treatment decisions in hospitals. methods: in this study, the in vitro activity of cefepime (cep), imipenem (imi), piperacillin tazobactam (p/t), ceftazidime (caz) and cefotaxime (cft) has been investigated against bacterial strains, isolated in the year . mics have been determined by microdilution method according to din . reference test strains were e.coli atcc , s. aureus atcc , p.aeruginosa atcc , and s.pneumoniae atcc . results: highest in vitro activity against gram negative enterobacteriaceae were determined for cep and imi with susceptibility rates for enterobacter spp. %, %, m.morganii %, %, proteus spp. %, % and citrobacter spp. %, % whereas the susceptibility rates for p/t, caz and cft were much lower (e.spp. %, %, %; m.m. %, %, %; p.spp. %, %, %; c.spp. %, %, %). for pseudomonas aeruginosa (n = ), susceptibility rates were % (cep), % (caz), % (imi) and % (p/t) with lowest resistance rates for cep ( %) followed by caz ( %), p/t ( %), imi ( %). the study confirmed excellent cefepime activity against gram positive isolates (pneumococci, streptococcus viridans and b-hemolytic streptococci) and high cefepime susceptibility rates for staphylococci (mssa % and msse %) on contrary to ceftazidime (mssa % and msse %). conclusion: regarding our in vitro data, cep is a reliable treatment option for empiric therapy in patients with severe nosocomial infections, demonstrating high activity against gram positive isolates, comparable activity in gram negative enterobacteriaceae to carbapenems (imi) and lowest resistance rates in pseudomonas aeruginosa. antimicrobial activity and spectrum of cefepime tested against , clinical strains from north american medical centres: report from the sentry antimicrobial surveillance program, program, - h. sader, d. biedenbach, t. fritsche, r. jones (north liberty, usa) objectives: to evaluate antimicrobial spectrum and potency of cefepime (cpm) and selected comparators against clinical bacterial strains collected in north america (na) over a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) . methods: isolates were consecutively collected from bloodstream ( %), respiratory tract ( %), urinary tract ( %) and skin/soft tissue ( %) infections in medical centres. % of isolates were from hospitalized patients. isolates were susceptibility (s) tested by reference nccls broth microdilution methods in a central laboratory. oxacillin-resistant (r) staphylococci (ors) and enterococci were excluded. results: the activity of cpm against the key organisms tested is summarized in the table. overall, . % of gram-positive cocci (gp) tested were s to cpm. imipenem (imp; mic , mg/l; . % s) was the most active compound tested against ent, followed by cpm (mic , . mg/l; . % s) > amikacin (amk; . % s) > ceftriaxone ( . % s) > aztreonam ( . % s). the lowest s rate for ent was observed with ciprofloxacin (cipro; . %). imp was also the most active compound against esbl-producing ksp and e. coli ( . and % s, respectively), followed by amk ( . and . % s) and cpm ( . and . % s). cpm activity against psa ( . % s) was similar to that of imp ( . % s). against ossa, cpm was -fold more potent than ceftazidime (caz; mic , mg/l, . % s) and showed higher activity than cipro ( . % s). cpm was the most active compound against spn after gatifloxacin and levofloxacin ( . % s). against vgs, cpm was -fold more potent than caz and -fold more potent than piperacillin/tazobactam. regionally, mdr ranged from . % in los angeles to . % in south florida. r to pen, ery, and sxt was the most common mdr phenotype in all regions except baltimore/dc (r to ery and sxt was most common). by specimen source, . % of blood, . % of lr, and . % of ur isolates were mdr with r to pen, ery, and sxt being the most common phenotype regardless of specimen source. for the strains tested, the fluoroquinolone mic ranges were: . - . mg/l (gem), . - . mg/l (gat), . - . mg/l (mxf), . - mg/l (lfx). conclusions: mdr among sp is a phenotype that is widely dispersed geographically and is likely to be encountered regardless of the site of infection. fluoroquinolones show activity against most mdr isolates. as the use of fluoroquinolone compounds increases, surveillance to monitor the prevalence of mdr and track the in vitro activity of agents such as gem used for these resistant strains must be continued. background: multi-drug resistant (mdr) s. pneumoniae strains are increasing at an alarming rate worldwide. the therapy of respiratory tract infections due to these strains is challenging with an urgent need for antimicrobials with reliable activity against the mdr strains. comparative activity of oral cephalosporins and parenteral cephalosporins against various pneumococcal mdr phenotypes was analysed in a large, multi-year international collection of clinical strains of s. pneumoniae. methods: s. pneumoniae strains ( , ) collected during - from worldwide participants of the sentry program were tested and interpreted using nccls (m -a , m -s ) guidelines. the antimicrobial agents analysed included penicillin (pen), erythromycin (er), clindamycin (cm), tetracycline (tet) and trimethoprim/sulfamethoxazole (ts); cephalosporins monitored included oral (cefpodoxime, cefuroxime) and parenteral (ceftriaxone, cefepime) agents. results: the rank order of occurrence rates of the various resistance phenotypes were: pen only ( . %) > pen and er ( . %) > pen, er and cm ( . %) > pen, er, cm and tet ( . %) > pen, er, cm and ts ( . %) > all five drugs ( . %). the susceptibility rate of all strains to the orally administered cephalosporins, cefpodoxime ( . %) and cefuroxime ( . %), dropped to only . % and . % respectively, for the five-drug mdr phenotype. the parenteral cephalosporins retained excellent activity for all mdr phenotypes, with resistance rates being lower for cefepime than ceftriaxone (cefepime, . - . %; ceftriaxone, . - . %) or the oral cephalosporins (cefpodoxime, . - . %; cefuroxime, . - . %) using respiratory infection breakpoints (nccls). conclusions: our in vitro findings confirm that the parenteral cephalosporins, cefepime and ceftriaxone, retain excellent activity against the mdr phenotypes analysed, and remain useful drugs in the armamentarium to treat mdr pneumococcal respiratory tract infections. antibiotic resistance surveillance over a -year period in spain: results of the mystic programme results: three icus, two neutropenia units and two general wards provided a total of . gram-negative and grampositive isolates during the period - . the most common species tested were escherichia coli ( . %), methicillin-susceptible staphylococcus aureus ( . %) and coagulase-negative staphylococci ( . %), pseudomonas aeruginosa ( . %), enterobacter cloacae ( . %), klebsiella pneumoniae ( . %) and a.baumannii ( . %). in general, the carbapenems (mem and ipm) were the most active antimicrobial agents tested against the common organisms (range of %s: % to % and % to %,respectively). among enterobacteriaceae, % of enterobacter spp, citrobacter spp and serratia spp were susceptible to carbapenems. e. coli and k. pneumoniae susceptibility to carbapenems were % and > % respectively. the %s of mem was the same as or higher than ipm against every organism tested. cip resistance in e. coli was around %. caz and taz were the least active antimicrobial agents against enterobacter sp ( % and % s, respectively) and citrobacter spp ( % and % s, respectively). mem and taz were the most active agents against p. aeruginosa ( % and %s, respectively). a. baumannii were - % susceptible to carbapenems, but only - % susceptible to cip. in this period, around % of methicillinsusceptible staphylococci were susceptible to mem. conclusion: there was no significant decrease in susceptibility to the carbapenems throughout the five-year period. mem and imp appear to remain reliable options for the treatment of serious nosocomial infections. the clinical use of mem did not increase bacterial resistance to this agent in the spanish centres evaluated. surveillance of antimicrobial susceptibility of anaerobes in a belgian university hospital y. glupczynski, h. nizet, c. berhin (yvoir, b) background: antimicrobial resistance is becoming a growing concern among anaerobes involved in human infections. since there are only scarce data on the in vitro susceptibility of anaerobes in our country, we aimed to determine the susceptibility and resistance profiles of anaerobic bacteria isolated in our hospital. table. conclusion: b-lactams-b-lactamases inhibitors, carbapenems, and met remain highly active against anaerobes including the more resistant bfg of organism. resistance in cli actually almost reaches % and is observed among almost all different species of anaerobes. mox, as a representative of the newer fluoroquinolones exhibits a broad spectrum and potent activity in vitro against all anaerobes tested and looks promising for the treatment of mixed infections involving anaerobes. pharmacokinetics/pharmacodynamics of quinolones objective: garenoxacin (grn) is a novel des-f( )-quinolone effective against a broad spectrum of pathogens, including anaerobes. some of the fluoroquinolones have the potential to prolong qtc. to determine the effect of grn on qtc, a retrospective analysis was performed on manually read ecgs from five phase i studies. methods: serial ecgs were collected in randomized, doubleblind, placebo-controlled studies in healthy adult subjects administered oral (po) or intravenous (iv) grn -to mg doses (therapeutic dose £ mg) with dosing duration to days (therapeutic duration £ days). the qt interval was corrected for heart rate using bazett's (qtcb) and fridericia's (qtcf) formulas, and the effect of grn was assessed by counts of outliers and linear regressions. single-and multiple-dose pharmacokinetics of po and iv grn were derived from plasma concentration versus time data. results: a total of subjects received grn and received placebo. grn plasma exposure (auc and cmax) increased with increasing dose. no subjects experienced a prolonged qtcb interval (> msec for males, > msec for females). only one subject experienced a prolonged (> msec) qtcb change from baseline which occurred on day , but not on subsequent days despite continued dosing. the incidence of borderline qtcb (change between - msec) was comparable between placebo and grn. means for qtcb max, qtcb avg and qtcb at tmax and their changes from baseline for grn were similar to those for placebo, with the exception of qtcb max, qtcb at tmax, and their changes for mg iv grn on day . although mg po and mg iv grn produced higher plasma exposures than mg iv on day , the means for the derived qtcb values were comparable to placebo. all % confidence intervals for the linear regression slopes of derived (delta)qtcb on cavg( - h) were equal to or less than , except for (delta)qtcb at tmax on day , indicating that grn had no effect on qtcb. results obtained for qtcf were similar to those for qtcb. conclusions: grn does not appear to have dose-, route of administration-or concentration-dependent effects on qtc interval in healthy subjects. objective: garenoxacin (grn) is a novel des-f( )-quinolone being developed for a variety of indications because of its efficacy against a broad spectrum of pathogens, including anaerobes. the objective of this study was to assess the tissue or fluid to plasma ratios of grn following a single oral dose. method: an open-label study was conducted in subjects ‡ years of age with a body mass index £ kg/m undergoing abdominal surgical procedures that would permit the removal of tissue or fluid without increased risk to the subject. a single mg oral dose of grn was administered based on the scheduled operative time. the tissue or fluid (with corresponding plasma sample) was collected to hours post-dose. concentrations of grn in biological fluids and tissues were determined using validated lc/ms/ms assays. safety was assessed by measurement of vital signs, physical examination, and electrocardiographic and clinical laboratory evaluations. adverse event (ae) monitoring was performed from time of consent until discharge from the study. results: thirty-one subjects were enrolled in and completed the study. mean fluid or tissue grn concentrations greater than that found in plasma (mean fluid or tissue to plasma ratio > ) occurred in large and small bowel tissue, gallbladder, and liver. mean fluid or tissue grn concentrations less than that found in plasma occurred in adipose tissue, bone, sinus mucosa, striated muscle, incisional skin, and subcutaneous tissue. mean grn concentrations in bile and lymph node tissue were roughly similar to that found in plasma. tissue and fluid concentrations of grn exceeded the mic of most target organisms involved in skin and soft tissue, bone, and intra-abdominal infections and sinusitis ‡ -fold. no treatment-emergent aes or serious aes were considered to be related to grn. conclusion: a mg oral dose of grn penetrates well into most of the tissues and fluids studied, with concentrations exceeding the mic of most pathogens causing sinusitis, skin and soft tissue infections, bone infections, and intra-abdominal infections. these results suggest that adequate concentrations of grn can be achieved to treat infections at these sites. penetration of garenoxacin into lung tissues in patients undergoing lung biopsy or resection objective: garenoxacin (grn) is a novel des-f( )-quinolone effective against a broad spectrum of pathogens, including those commonly found in the respiratory tract (rt). this study was conducted to determine the penetration of grn into lung parenchyma (lp) and bronchial mucosa (bm) following a single mg oral dose. penetration of grn into bone was also assessed. this was an open-label, nonrandomized study in subjects undergoing an invasive procedure to the lung (other than percutaneous lung biopsy) which facilitated the removal of macroscopically normal healthy lung tissue without exposing the subject to undue risk. penetrance of grn into bone was determined when removal of rib bone was part of the normal surgical procedure. a single mg oral grn dose was administered based on the scheduled operative time. lp and, if possible, bm and/or bone samples and corresponding plasma samples were removed at to , to , to , or to h post-dose. concentrations of grn in these samples were determined using validated lc/ms/ms assays. safety was assessed based on the occurrence of adverse events (aes) and changes in physical examinations, vital signs, clinical laboratory results, and electrocardiographic results. results: twenty-seven subjects were enrolled; samples were taken from a minimum of patients at each time interval. mean grn concentration in lp increased between the - and - h post-dose, suggesting rapid penetration into lp, then declined similar to the decrease seen in plasma concentration. concentrations of grn in lp exceeded the mic of organisms associated with rt infections by -to -fold over a h period. grn concentrations in bm over a h period exceeded the mic of respiratory pathogens -to -fold. concentrations of grn in bone exceeded the mic of the organisms associated with bone infections (except pseudomonas aeruginosa, mrsa, fusobacterium species, and enterobacter species) by -to -fold over a h period. across the time intervals, mean ratios of tissue to plasma grn concentration in lp, bm, and bone reached . , . , and . , respectively, suggesting adequate penetration. no grn-related aes were reported, indicating that a single mg oral grn dose was well tolerated in subjects undergoing invasive procedures. conclusions: grn penetrates rapidly into lp, bm, and bone tissue, producing sustained concentrations that predict adequate coverage of grn-susceptible pathogens at these sites. bioavailability and safety in healthy volunteers unaltered when crushed garenoxacin tablets are administered via a nasogastric tube with or without enteral feeding r. noveck, r. vargas, g. krishna, d. grasela (new orleans, kenilworth, princeton, usa) objective: the purpose of this trial was to assess, in healthy volunteers, the bioavailability and safety of single doses of the novel des-f( )-quinolone garenoxacin (grn) administered as crushed tablets with and without concomitant enteral feeding compared with intact tablets. methods: in this randomized, crossover, single-dose study, healthy adult volunteers (aged to y) received grn mg (one mg and one mg tablet) orally in regimens: a) intact tablets; b) crushed tablets suspended in water and delivered via nasogastric (ng) tube; c) regimen b plus concomitant enteral feeding (osmolite, ml at ml/hr). subjects were randomized to receive all regimens in of crossover sequences (abc, acb, bac, bca, cab, cba) separated by -day washout intervals. for each treatment, subjects entered the facility day before and were confined for hours after drug administration. subjects were discharged from the study after final assessments on day of the third treatment. post-screening assessments included vital signs; plasma drug levels for pk analysis; and physical, laboratory, and ecg examinations for drug safety. results: male subjects were enrolled ( white, black; mean age, y). pk analysis for grn administered as crushed tablets with and without concomitant enteral feeding vs intact tablets showed no differences in the adjusted geometric means for cmax ( . and . lg/ml vs . lg/ml) or auc(inf) ( . and . lg*hr/ml vs . lg*hr/ml). the % ci for logtransformed cmax and auc(inf) for b and c compared to a were contained within the protocol-specified Ôno effectsÕ limit of % to %. mean grn half-life was similar among the groups (mean range of - h). tmax was hour following administration of intact tablets compared with . hour in the other groups. a single oral mg dose of grn was well tolerated whether administered as crushed tablets suspended in water and delivered via ng tube with or without enteral feeding, or as intact tablets. three adverse events (aes) were reported; (nausea) was deemed probably related to study drug. there were no serious aes, meaningful changes on safety examinations, or discontinuations due to aes. conclusions: the bioavailability of grn was similar for crushed versus intact tablets, regardless of whether an enteral feeding was given with crushed tablets. these results show that grn may be administered as crushed tablets with or without concomitant feeding. garenoxacin pharmacokinetics in subjects with severe renal impairment objective: garenoxacin (grn) is a novel des-f( )-quinolone effective against a broad spectrum of pathogens. the absolute bioavailability of grn after oral intake is % and approximately % of grn is excreted unchanged in the urine. the objective of this study was to evaluate the pharmacokinetics of grn in subjects with severe renal impairment. methods: this non-randomized, open-label study enrolled patients into of groups: healthy control subjects (hc) with normal renal function (clcr > ml/min); subjects with severe renal impairment (sri) (clcr < ml/min) not requiring dialysis; subjects with sri receiving hemodialysis (hd); and subjects with sri receiving continuous ambulatory peritoneal dialysis (capd). a single mg oral dose of grn was administered on day for all groups except hd patients. hd patients received a single mg oral grn dose both before (hd ) and after (hd ) hd, with dosings separated by a -day washout. blood, urine, and dialysate samples were collected up to h post-dose and concentrations of grn were determined using validated lc/ms/ms assays. safety was assessed by physical examination, vital signs, and electrocardiographic and laboratory evaluations. results: twenty-five subjects received grn (hc, n = ; sri, n = ; hd , n = ; capd, n = ). six subjects in hd received grn. mean grn exposure [auc(i)] was similar in hc and hd but was %, %, and % higher in sri, hd and capd, respectively, than in hc. however, many of the individual values were within the range observed for hc, and these average increases in auc(i) did not exceed values previously shown to be well tolerated. decreases in cmax were observed in sri, hd , capd, and hd , but were not considered clinically relevant. approximately %, . %, and % of grn dose was removed in the dialysate for hd , hd , and capd, respectively. a total of treatment-emergent adverse events (aes) were reported, all mild or moderate in severity; were considered to be probably or possibly related to grn. one treatment-emergent but not treatment-related serious ae was reported. conclusions: a single dose of mg grn was well tolerated in patients with sri, including those requiring hd and capd. there was no clinically significant increase of grn exposure in patients with sri based on the broad therapeutic index of grn. therefore, no grn dosage adjustment is recommended in subjects with sri. the effect of omeprazole on the bioavailability and safety of garenoxacin in healthy volunteers j. kisicki, g. krishna, s. olsen, d. grasela (lincoln, kenilworth, princeton, usa) objective: the novel des-f( )-quinolone garenoxacin (grn) is more soluble in acidic conditions than at neutral ph. therefore, this study was designed to determine if omeprazole affects the bioavailability of grn. methods: this non-randomized, open-label pharmacokinetic study was conducted in healthy adult subjects. on day , the single-dose pharmacokinetics of mg of oral grn (one and one mg tablet) were determined in fasting subjects, with serial blood samples obtained through hours post-dose. omeprazole mg was administered once daily on days to to achieve steady-state inhibition of gastric acid secretion. on day , single doses of grn and omeprazole were administered concomitantly. omeprazole treatment was continued on days and throughout the period of pharmacokinetic blood sampling. study assessments included vital signs and physical, laboratory, and electrocardiographic examinations for safety. the gm cmax value for grn and grn/omeprazole co-administration was . and . mcg/ml, respectively, with % ci of % to %. half-life (mean range . to h) and tmax (median range . to . h) were similar after administration of grn either alone or concomitantly with omeprazole. concomitant administration of garenoxacin and omeprazole was well tolerated. nine of subjects ( %) experienced a total of adverse events (aes). the majority of aes were mild, and only were deemed possibly or probably related to the study drug. the most frequently cited aes, headache, nausea and abdominal pain, were mild to moderate in severity. two subjects withdrew from the study; neither discontinuation was due to aes. conclusions: the concomitant administration of omeprazole had no effect on garenoxacin bioavailability. these findings indicate that garenoxacin can be administered with omeprazole or other agents that affect gastric ph to a similar or lesser extent. the effect of maalox on bioavailability and safety of garenoxacin in healthy volunteers j. kisicki, g. krishna, s. olsen, d. grasela (lincoln, kenilworth, princeton, usa) objective: garenoxacin (grn) is a novel broad-spectrum des-f( )-quinolone antibiotic. quinolones are known to chelate with cations such as aluminum, impairing antibiotic absorption. this study was therefore designed to assess the effect of a -ml dose of maaloxÒ (containing mg aluminum hydroxide and mg magnesium hydroxide) on the pharmacokinetics of grn when administered concomitantly with, and hours before, and and hours after, maalox. methods: this was a randomized, open-label, -treatment, control-balanced, residual effects design pharmacokinetic study. the oral treatments consisted of mg grn (one and one mg grn tablet) given alone, with concomitant maalox, h before maalox, h before maalox, h after maalox, and h after maalox. each healthy adult subject received of the above treatments, with a -day washout period between treatments. in each treatment, serial blood samples for pharmacokinetic analysis were collected before and up to h after grn dosing. study assessments included vital signs and physical, laboratory, and electrocardiographic examinations for drug safety. results: twenty subjects ( male, females; mean age, years) were enrolled. exposure to grn [auc(inf)] was reduced by % when coadministered with maalox. exposure to grn was also reduced when administered or hours after maalox, by % and %, respectively. in contrast, when administered hours before maalox, grn exposure was not affected. when grn was administered hours before maalox, a small reduction ( %) in grn exposure was observed that is unlikely to be clinically relevant. half-life (mean range . to . hours) and tmax (median range . to hours) were similar across treatment groups. a single oral mg dose of grn was well tolerated. mild adverse events were reported by subjects; subject discontinued due to mild abdominal pain and blood in the stool. conclusions: maalox does not affect grn bioavailability when grn is administered at least h prior to maalox. however, a reduction in grn exposure is observed when grn is administered concomitantly or up to h after maalox. therefore, grn can be administered h before or h after administration of maalox or other products containing a high content of cations, particularly aluminum. objectives: it has been demonstrated that fluoroquinolone treatment of humans and animals can rapidly select for bacteria with reduced susceptibility to fluoroquinolone antibiotics. in recent years, there has been considerably interest in the concept of the mutant prevention concentration (mpc). the mpc has been defined as that concentration of antibiotic at which no mutants are selected from , , , organisms. in previous studies mpcs of ciprofloxacin and enrofloxacin were determined in vitro against salmonella enterica serovar typhimurium dt . here the use of a chick model to investigate the mpc of enrofloxacin in vivo is reported. methods: chicks were experimentally infected with s. typhimurium and treated with baytril (enrofloxacin) at ppm/ mg/kg for days (recommended therapeutic dose) or days or ppm/ mg/kg for days or ppm/ mg/kg for day. chicks received the dose in the drinking water (ppm) or by oral gavage (mg/kg). during and hours after dosing, birds were killed and tissue samples (caecal contents, liver and sera) were taken and the levels of enrofloxacin and ciprofloxacin were determined in these tissues by hplc or bio-assay. caecal contents were also monitored for the presence of salmonella during dosing and for up to weeks after dosing. selection of resistance was monitored by replica plating of colonies to media clinical microbiology and infection, volume , supplement , containing x the ciprofloxacin and nalidixic acid mic of the strain before treatment.results: the only conditions where the antibiotic treatments did not select for reduced susceptibility (e.g. x mic of parent strains) were in birds that received the antibiotic at . x the dose for days by oral gavage or x the dose for day in the water. this oral gavage treatment also significantly reduced the counts of salmonella compared to all other oral gavage treatments. for the oral gavage study, concentrations of fluoroquinolones in caecal contents were above the mpc for at least hours after dosing for all treatment regimes. conclusion: it was of interest to note that whilst the fluoroquinlone antibiotics are regarded as concentration rather time dependent antibiotics, the day at . x the dose was more effective at reducing the counts of salmonella in both experiments than the day at x the dose. these results would suggest that length of treatment as well as dose and route of administration, is critical in minimising the selection of fluoroquinolone resistance in vivo. effect of levofloxacin coadministration on the international normalised ratios during acenocumarol therapy j. de otero, m. payeras, c. carratala, a. artigues, m. sanz, p. nadal (manacor, e) levofloxacin (l) showed no effect on warfarin pharmacokinetics in healthy volunteers but several recent descriptions reveal elevated international normalised ratios (inr) in patients receiving concurrent l and warfarin. to our knowledge no cases of l-acenocumarol (a) interaction have been reported. objectives: to report cases of hypoprothrombotic response resulting from addition of l therapy to chronic a therapy. methods: a -month retrospective analysis of adult patients receiving a who were admitted to our ward under l prescription on the basis of clinical diagnosis and judgement. descriptive statistical measurements were performed. results: six patients, - years old (median . ), were included. four were men ( . %). they suffered from a median of (range - ) concurrent chronic medical problems (including coronary heart disease, atrial fibrillation, hypertension, obstructive pulmonary disease, renal insufficiency, cardiomyopathy and heart failure) and took a median of (range - ) different kind of drugs (including digoxin, furosemide, antihypertensives and inhaled bronchodilators). types of infection for which the patients were treated with l included acute bronchitis ( cases) and pneumonia ( cases objectives: cip is substrate for an mrp efflux pump in j mouse macrophages (michot et al, aac : - ), which reduces its cellular accumulation. we have recently shown that cip-ÔresistantÕ macrophages can be selected upon chronical exposure of these cells to cip, in which cip accumulation becomes negligible because of an increased activity and/or expression of the efflux transporter (icaac (icaac , abstract a- . we have now examined whether this reduced accumulation affects cip intracellular activity using a model of intracellular infection by l. monocytogenes (l.m.) and comparing resistant cells to the wild type parent cell line. methods: infection was carried out using a bacteria: macrophage ratio of : , according to the procedure described in seral et al (jac : - ) . cfu/mg cell protein was determined after h exposure to increasing concentrations of cip ± mm probenecid (inhibitor of mrp transporters). cip cellular concentration was measured by fluorimetry (aac : - ) . results: in wild cells, an extracellular concentration corresponding to x the mic was sufficient to obtain a static effect, and this value rose to x in cip-resistant cells. this value was decreased to x and . x the mic, for wild and cip-resistant cells respectively, in the presence of probenecid. in cells incubated with mg/l cip ( x mic), cip accumulation was . ± . and . ± . in wild and cip resistant cells, but increased to . ± . and . ± . in the presence of probenecid. conclusions: increase in expression and/or activity of the cip efflux transporter causes a reduction of the antibiotic efficacy against intracellular infection, which can be reversed upon inhibition of the efflux transporter. objectives: quinolones can cause tendinitis and tendon ruptures. retrospective studies and case reports reveal that an additional therapy with glucocorticoids increases the risk of quinolone-induced tendon disorders. methods: we investigated possible combination effects of quinolones and glucocorticoids in an in vitro model with human tendon cells. tenocytes were cultured for , , and days as monolayers and incubated with (a) ciprofloxacin or levofloxacin ( mg/l), (b) . nm dexamethasone, and (c) ciprofloxacin or levofloxacin ( mg/l) plus . nm dexamethasone. immunoblotting was used to quantify changes in the amount of beta -integrin, activated src homology collagen (shc) and of activated caspase- (casp- ). furthermore, ultrastructural alterations were analysed by electron microscopy. results: at mg/l, ciprofloxacin caused a significant decrease of the amount of beta -integrin from day onwards, while no effect was seen with mg levofloxacin/l medium or . nm dexamethasone compared to the untreated control. the combination of both quinolones ( mg/l) with . nm dexamethasone resulted in an earlier onset of the beta -integrin reduction: for ciprofloxacin + dexamethasone from the first day of incubation, for levofloxacin + dexamethasone from day onwards. quinolone-induced changes in signalling proteins, such as activated shc, are not fortified by dexamethasone at the concentration tested. interestingly, the time and concentration dependent increase of the apoptosis marker activated casp- was intensified with dexamethasone. the results of immunoblotting with respect to the induction of apoptosis were confirmed by electron microscopy. conclusions: our results show that quinolone-induced changes in the amount of the beta -integrin as well as of the apoptosis marker activated casp- are enhanced by dexamethasone in vitro. the addition of the glucocorticoid caused an earlier onset of the quinolone-induced changes. our results support the clinical observations that glucocorticoids are an additional risk factor for quinolone-induced tendopathies. pharmacokinetics and pharmacodynamics of a novel extended release formulation of ciprofloxacin as compared to levofloxacin against extended spectrum beta-lactamase producing enterobacteriaceae s. schubert, h. stass, u. ullmann, a. dalhoff (kiel, wuppertal, d) background: an extended release formulation of ciprofloxacin (cip xr, mg qd, p.o.) has been developed, delivering peak concentrations which are % to % higher than following the administration of the conventional formulation (cip ir, mg bid, p.o.) while the areas under the curve (auc) were comparable. objectives: first, cip is used for the treatment of infections due to enterobacteriaceae, frequently being esbl producers. thus, the pk/pd characteristics of cip xr vs. ir against esbl strains was examined. second, cip is used in urinary tract infections (utis). thus, the pk/pd profiles of cip xr vs. ir in serum and urine were studied. third, the pk/pd characteristics in either bhi or artificial urine were compared. methods: genotypically characterised esbl producing e. coli (ec) and k. pneumoniae (kpn) and their wild type counterparts were exposed to the geometric mean plasma and urine concentration profiles following cip mg xr qd, mg ir bid and lev ( mg qd). the cip and lev urine concentrations were fitted from phase i study data by compartmental modeling using topfit . . bacteria were cultivated in a modified grasso model at °c in brain-heart infusion broth as well as artificial urine acc. to griffith et al.; viable counts were quantitated at . , , . , , , , , and h. the areas under the bacterial kill curves normalised to the inoculum were calculated. results: first, irrespective of the pk profiles simulated, the wt were eliminated rapidly. the esbl producing ec and kpn were moderately to negligibly affected upon simulation of serum pk profiles of both cip and lev. second, simulation of urine concentrations by using artificial urine ir and xr resulted in an elimination of the esbl producers from the test system. lev eliminated the esbl producing k. pneumoniae but not the e.coli strain, which regrew. cip xr killed the esbl kpn strain more rapidly than cip ir or lev. third, pk/pd surrogates derived from studies in conventional media or artificial urine are significantly different. conclusions: this model and the use of artificial urine predicts a more rapid and pronounced effect of cip xr as compared to cip ir or lev. objectives: the antibacterial spectrum of moxifloxacin includes all the major respiratory pathogens and its pharmacokinetics demonstrates high peak concentrations in plasma as well as at respiratory sites. nevertheless, the tonsillar tissue concentrations have never been investigated. in the present study we determined the moxifloxacin concentrations in plasma and tonsils after the administration of three doses of mg to adult patients with chronic or recurrent tonsillitis undergoing tonsillectomy. methods: this was an uncontrolled, open-label, randomised, parallel group study including patients randomly assigned to groups of patients each, depending on the time between the last dose of moxifloxacin and that of plasma and tissue sampling ( , , , and h). moxifloxacin was given orally od for days. moxifloxacin concentrations were measured by a validated hplc assay and fluorescence detection. each sample was analysed twice and the mean value obtained used for statistical analysis. pharmacokinetic data were analysed by presenting descriptive statistics of moxifloxacin levels in plasma and tonsillar tissue. results: cmax occurred at h in plasma (mean value . mg/ l) and in tonsillar tissue (mean . mg/l). tonsillar tissue/ plasma ratios (mean values) were constantly > mg/l at any collecting time, ranging between . mg/l (after h) and . mg/l (after h), which indicates a prolonged maintenance of moxifloxacin level in tonsillar tissue compared to plasma. variability among pts was present at h, the tonsillar tissue / plasma ratio ranging between . and . mg/l. conclusions: moxifloxacin achieves a good penetration in tonsillar tissue, which favourably compares with that reported for other fluoroquinolones. the moxifloxacin concentrations that we observed exceed the mics of the usual respiratory tract pathogens. objectives: acute necrotizing pancreatitis is still related to a high mortality rate, based on local infectious complications, particularly due to bacterial infections of the necrotic pancreatic and parapancreatic areas. limited penetration of antimicrobial drugs in these areas/compartments is considered to be a major cause of failure of therapy of severe local pancreatic infections. however, fluoroquinolones (e.g. ciprofloxacin, levofloxacin) have been shown to penetrate sufficiently into pancreatic tissue. on that score, the value of new quinolones such as moxifloxacin (mxf) has not been investigated yet. using a rat model of acute necrotizing pancreatitis mxf has been demonstrated to penetrate rapidly and efficiently into pancreatic tissue, also into the inflamed and necrotic pancreas. methods: addressing the penetration capability of mxf following intravenous (iv) or oral (po) administration with respect to the human pancreas, a prospective clinical trial was designed using a single iv ( patients) or po dose ( patients) of mg mxf for antimicrobial prophylaxis in patients undergoing pancreas resection. samples were taken from blood and from resection area of pancreatic tissue at two time points after application ( st sample at the beginning, nd sample at the end of resection). concentrations of mxf were determined by hplc/uv using ofloxacin as internal standard. results: mean plasma concentrations of mxf iv and po at first sampling time ( - . h) were . ± . and . ± . lg/ml, respectively. at the end of resection ( . - . h) . ± . and . ± . lg/ml were measured. corresponding mean concentrations of mxf in pancreatic tissue were to times higher ( . ± . and . ± . lg/g st sample; . ± . and . ± . lg/g nd sample). the mxf concentrations in pancreatic tissue exceeded the mic s of the relevant pathogens encompassed by mxf (e.g. e. coli . - . lg/ml, klebsiella spp. . lg/ml, and s. aureus . - . lg/ml) for at least five hours at the dosing interval. conclusion: mxf has been demonstrated to penetrate efficiently into human pancreatic tissue following iv as well as po administration. in this study mxf concentrations after po administration were found to be slightly lower than those observed in healthy subjects probably due to diminished or delayed intestinal absorption prior and during surgery. objective: in clinical practice on icu wards moxifloxacin (mfx) may be occasionally administered through a nasogastric (ng) feeding tube. in absence of an oral liquid formulation and since the divalent cations contained in enteral feeds may potentially impair absorption of mfx given via this way, we wanted to study the effect of concomitant enteral feeding on the pharmacokinetics and tolerability of mfx passed as a crushed tablet through a ng tube, firstly in healthy volunteers. methods: in a randomized -way crossover study the oral bioavailability of mfx was investigated in healthy volunteers ( f, m). each subject received separately an intact mg mfx tablet (a), a crushed tablet as a suspension through an ng tube with water (b), and while receiving enteral feeding (c). the concentrations of mfx in serum were measured by a validated hplc. auc and cmax were analysed statistically assuming log normally distributed data using anova. results: all mfx treatments were well tolerated. the auc was slightly, but not significantly decreased in treatments b and c [geo means ( . thus, rate of absorption was not affected by ng tube administration. this route of ingestion seems to be associated only with a slight loss of bioavailabilityindependent of the carrier medium used (water vs. enteral feed). no clinically relevant interaction with the multivalent cations contained in the enteral feed was observed indicating that mfx and enteral nutrition can be administered concomitantly. conclusion: there was no clinically relevant effect of enteral feeding on the pharmacokinetics of oral mfx in healthy volunteers. this results has to be evaluated in patients, especially those from icu, who are characterised by severe infectious and/or concomitant diseases, which might influence absorption of mfx. in the healthy volunteer studies, compliance with dosing regimens was strictly enforced and participants had no significant underlying diseases or concomitant medications that could confound interpretation of safety data. therefore, for the purpose of this analysis, the healthy volunteers served as a control group for the patient population. in both groups, most subjects received pos mg/d in divided doses. results: treatment-related aes (traes) occurred in % ( / ) of healthy volunteers; the most common were headache ( %), dry mouth ( %), and dizziness ( %). in patients, many aes were consistent with underlying diseases. traes occurred in % of patients ( / ); the most common were nausea ( %), vomiting ( %), headache ( %), abdominal pain ( %), and diarrhoea ( %). notably, gastrointestinal (gi) traes occurred in % of healthy volunteers and % of patients. treatmentrelated abnormal liver function test results were observed in % of healthy volunteers and up to % of patients. there were no clinically significant differences in mean qtc interval change from baseline in either population. serious aes (sae) considered possibly or probably related to pos occurred in ( %) patients and healthy volunteer. the most common saes in patients were altered drug level, increased hepatic enzymes, nausea, rash, and vomiting ( % each). no significant trends related to age, sex, or race were observed in either group. additionally, no unique traes were identified in patients during long-term exposure (> months) compared with those identified during shorter-duration therapy. conclusion: the safety profile of pos in patients was similar to that observed in a controlled population of healthy volunteers and is likely indicative of what will be observed in the clinical setting. headache and gi events (nausea, vomiting, abdominal pain, diarrhoea) were the most common traes observed in patients. given its favorable safety profile, pos should provide an additional treatment option for severely ill patients with ifis. objective: we evaluated the drug interaction (di) potential of posaconazole (pos), an extended-spectrum triazole antifungal agent, in phase studies. pos is an inhibitor of cytochrome p (cyp) a activity, but it does not affect the activity of other cyp enzymes. these studies were conducted to evaluate potential changes in the pharmacokinetics of pos and the coadministered medication, when given in combination. methods: seven open-label di studies were conducted in which subjects received pos ( - mg) for to consecutive days, alone or in combination with single or multiple doses of cimetidine, cyclosporine, glipizide, phenytoin, rifabutin, tacrolimus, or antiretroviral medications (zidovudine, lamivudine, ritonavir, and indinavir). in the cyclosporine and antiretroviral studies, patients received established doses (ed) of these drugs, and plasma concentrations were presumed to be at steady state (ss) before pos administration. bioavailability, based on logtransformed auc values, was expressed as the ratio of the combination treatment to pos or concomitant drug given alone. the effects of coadministration on the auc values of pos and each concomitant medication are summarized in the table. conclusion: no dose adjustments are required for glipizide, zidovudine, lamivudine, indinavir, or ritonavir when coadministered with pos, as the small differences in exposure are not considered clinically significant. however, when pos was given with glipizide, glucose concentrations decreased in some healthy volunteers more so than after glipizide administered alone. monitoring of cyclosporine and tacrolimus blood levels is warranted with pos coadministration, and dose adjustments of cyclosporine and tacrolimus should be made accordingly. concomitant use of pos with rifabutin, phenytoin, or cimetidine should be avoided unless the benefits outweigh the risks, due to the decrease in pos concentrations. objectives: the pharmacokinetics (pk) of many medications may be altered in the elderly population. because alterations in pk can potentially affect clinical outcomes, the pk parameters of posaconazole (pos), an extended-spectrum investigational triazole antifungal agent, were examined in healthy elderly persons ( ‡ years), and the clinical implications of age-related pk differences were evaluated. methods: we conducted a meta-analysis of pos steady-state pk data from clinical studies in healthy volunteers. pk data from elderly healthy volunteers were compared with those of elderly patients with invasive fungal infections ( objective: to develop a bioassay for measurement of csp blood levels using the hypersusceptible c. albicans cell wall sensor mutant delta-mid . methods: the c. albicans mutant delta-mid (mic for csp: . mg/l) was constructed by targeted deletion of the gene encoding for a cell wall sensor putatively involved in the maintenance of cell wall integrity. the bioassay was validated according to international guidelines (shah et al, ; fda, ) . standard curve included csp concentrations over the range . to mg/l. four quality controls were used ( . , , , mg/l) to study: i) stability of csp concentrations over time at different pre-analytical storage conditions, ii) accuracy (measured/nominal value x , validation range - %), iii) precision (coefficient of variation: sd/mean of measured values x , validation range %). the validation procedure included intra-run and inter-run measurements. results: the limit of detection and quantification was . and . mg/l (corresponding to and ng of csp in a sample volume of ml), respectively. reproducible standard curves were obtained over the clinically relevant concentration range ( . to mg/l) (r ‡ . ). analytical time was h. csp concentrations with a deviation from nominal values < % were measured: i) over days in plasma, and days in whole blood, at c, ii) over and days, respectively, at c, iii) over months in plasma at - c, iv) after freeze/thaw cycles. intra-and inter-run validation with the four quality controls (mean % value, ± sd) are shown in objective: to identify the pharmacokinetic and pharmacodynamic (pk-pd) parameters of antifungal therapy predictive for intra-and inter-run bioassay validation i intra (n = ) inter (n = ) accuracy . ± . . ± . precision . ± . . ± . invasive fungal infections in patients admitted to an intensive care unit (icu). the medical records of all ptt admitted to a tertiary hospital icu in a month period in were retrospectively reviewed, and the antifungal therapies were recorded and correlated to the findings of fungi from the ptt. the pk-pdparameters for each pt was estimated from the treatment given and the mic of the isolated candida, i.e. auc/mic-ratio (in h) for the azoles, and peak/mic ratio for the free fraction of amphotericinb. candida isolated from various infectious foci, or from the surveillance samples taken routinely three times a week, were susceptibility tested using e-tests for fluconazole, itraconazole and amphotericinb. results: in total ptt were included.among the ptt with invasive fungal infections, ptt had bloodstream infections at admission. one of the remaining ( %) received systemic prophylactic therapy before getting colonized, and ptt ( %) were prophylactic treated when colonized, ptt with fluconazole (auc/mic: - ), and one pt with liposomal ampho-tericinb (peak/mic = objective: to predict the pharmacokinetic properties of bal a new azole antifungal, in humans.methods: bal (the water-soluble pro-drug of bal ) was incubated at lg/ml in pooled heparinized rat, cynomolgus monkey and human plasma for minutes at °c. bal was incubated at , and lg/ml equivalent bal with rat, cynomolgus monkey and human liver microsomes for min at °c in the presence of mg/ml proteins. the extent of plasma protein binding of bal in rat, cynomolgus and human plasma was measured using the red blood cell partitioning method and c-bal . rats received a single oral dose of mg/kg equivalent bal and a single intravenous dose of mg/kg equivalent bal as bal . cynomolgus monkeys received single oral and intravenous doses of mg/kg as bal . serial blood samples were obtained. plasma concentrations of bal and bal were quantified using an hplc/fluorescence method or a lc-ms/ms assay. results: in plasma, bal is converted within minutes to bal in all species investigated. in the presence of rat, cynomolgus monkey and human liver microsomes, less than % of bal is metabolized during a min period. bal is bound to plasma proteins with a free fraction of . %, . % and . % in rat, cynomolgus monkey and human, respectively. after intravenous administration, the pro-drug bal is converted to bal within minutes. bal has a large volume of distribution with values greater than the total body water: l/kg and l/kg in rat and cynomolgus monkey, respectively. bal is slowly eliminated with halflives of h in rat and h in cynomolgus monkey. after oral administration of the pro-drug, bal reaches cmax-values between . to . hours. we observed high bioavailability of bal : % in rat and % in cynomolgus monkey. conclusions: based on these in vitro results and animal data, we predict that the pro-drug bal is very rapidly converted to bal in man. due to the protein binding and in vitro metabolic stability, bal is expected to behave as a low intrinsic clearance drug in human (clearance < % of liver blood flow). this combined with a large volume of distribution explains the long half-life > hours observed in man. animal data suggest a good oral bioavailability as is confirmed in humans. pharmacokinetics and fungicidal activity of aminocandin (hmr ), a novel echinocandin in healthy volunteers b. sandage, g. cooper, n. najarian, j. lowther (lexington, usa; romainville, f) objective: aminocandin (ac), an echinocandin, is being developed for treatment of systemic fungal infections. a study was carried out to determine the pharmacokinetic (pk) and fungicidal activity of ac in plasma samples against strains of c. albicans and strain of a. fumigatus following single iv doses. methods: single iv doses of - mg of ac were administered to healthy, male volunteers in a single-blind, randomized, placebo-controlled trial. individual plasma samples were collected from active and placebo (pbo) treated volunteers, serially diluted in % control plasma/ % growth medium, and the fungicidal titre determined on c. albicans strains and inhibitory titre against a. fumigatus strain. titres and pk results are the mean values (n = ). due to the dilution factor in test medium, the limit of detection was a titre of the germicidal effect of ceiling-and wall mounted ultraviolet c (uvc) light at nm in isolation units with negative pressure () pascal) was examined and compared with disinfection with chloramine during end-disinfection after patient stay. microbial samples were taken from surfaces before and after disinfection with uvc ( - min) and chloramine ( %, h exposure) using standard contact plates ( cm ). the uvc-distribution in the isolation units was monitored at positions. the output on the floor varied between . and . w/m , with an average ( ± sd) of . ± . w/m in the patient room, . ± . w/m in the sluice and . ± . w/m in the bath/ decontamination room. on other places, the values varied from . to . w/ m . the units were uvc-disinfected for - min, corresponding to doses ranging from j/m in shadowed area to j/m at the highest exposed site. according to published uvc-dosimetry, the survival of bacteria and bacterial spores are reduced by % with doses ranging from - j/m and - j/m , respectively. thus, uvc doses used in this study should be high enough to inactivate most bacterial organisms, including spores, even on surfaces not directly exposed to uvc. uvc-disinfection significantly reduced the bacteria on surfaces directly or indirectly exposed to uvc to a very low number (from ca to - cfu/plate), as did % chloramine disinfection (fom ca to - cfu per plate) alone; p < . ,and p < . , respectively. since cleaning before disinfection may be a risk for the staff in isolation units, disinfection with uvc-or chemicals should always be performed first. the presence of completely shadowed areas in the isolation unit (the bed rail, lockers, matresses etc.) still needs disinfection by chemicals before cleaning. therefore, uvc may not be used alone, but is a good additive to chemical disinfection, to lower the biological burden of infectious agents in isolation units for high risk infectious patients. disinfection of medical equipment, using dry mist of hydrogen peroxide objective: to compare the antimicrobial efficacy before and after disinfection of pine core wood surfaces with surfaces made of polyethylene and synthetic laminate against organisms typically causing hospital-acquired infections. methods: s. aureus (mrsa), e. faecium, e. coli, p. aeruginosa. c.albicans, m. terrae and p camembertii were uses as test organisms. after inoculation of the test organisms on surfaces made of pine core wood, polypropylene and synthetic laminate, samples were collected on rodac plates before and after disinfection with commonly used hospital germicides (alcohol, aldehyde, glucoprotamine and a quaternary ammonium compound). bacterial colonies were counted after min, min, h, results: substantially lower colony counts were measured on pine core wood surfaces before application of the disinfectant compared with counts on polypropylene and synthetic laminate surfaces. after bacterial contamination of the surfaces, significant colony counts were not observed on the surfaces after disinfection with alcohol, aldehyde and glucoprotamine. however, after disinfection with a quaternary ammonium compound, significant elevations in colony counts were found on the pine core wood surfaces in comparison to the polypropylene and synthetic laminate surfaces. discussion: the poor efficacy of quaternary ammonium compound on wooden material might be explained by the interaction between wood and the anionic polyphenols and cationic surfactants contained in the disinfectant. the study findings corroborate the antimicrobial effect of pine core wood against organisms typically causing nosocomial infections. with the exception of a quaternary compound, all the germicides displayed good disinfecting properties. from a hygienic point of view, pine core wood is suitable for use in hospitals. survey of the microbiological quality of drinking water supply in hospitals. drinking water cooler vs tap water fed dispenser vs bottled water objectives: hygienic, economic and ecologic comparison of drinking water coolers versus tap (mains) water-fed dispensers versus bottled drinking water. methods: samples were taken from each system without preflow or cleaning of the nozzle. these comprised samples from drinking water coolers each over months, sample per week from the tap water dispenser over months, and samples from different bottles of drinking water. total colony counts were analysed at °c and °c, respectively. total p. aeruginosa, e. coli, and coliforms, as well as sulphite-reducing clostridia were examined according to european norms (en iso) for compliance with the german drinking water ordinance (limit value for total colony counts at ± °c: cfu/ml, at ± °c: cfu/ml and cfu/ml for bottled water). costs per litre were calculated and compared based on the economic data supplied by the manufacturers. the ecological impact of the different systems was evaluated based on expert opinion. results: all the samples from the water cooler system exceeded german drinking water ordinance threshold values. all the samples taken from the tap water dispenser, and, with one exception, all the samples from the bottled water complied with the german drinking water ordinance. if more than l of water are consumed per month, the tap water dispensing system and bottled water are more economical than the water cooler system ( . per l for water cooler, . for month tap water dispenser, . per l for bottled water). in economic terms, the water cooler system can only be recommended for consumption levels below l per month. from an ecological point of view, the tap water dispenser is most favourable (expressed in co emissions per year for . l at freiburg university hospital), followed by the water cooler system and finally the bottled water. conclusion: for reasons of hygiene, the use of water cooler systems cannot be recommended in hospitals. furthermore, drinking water cooling systems are only economic for low consumption rates. tap (mains) water-fed dispensers feature the best hygienic, economic and ecologic properties. objectives: the application of antimicrobial finishes to textiles can prevent bacterial growth and might reduce the risk of infection resulting from fabrics that are contaminated with pathogenic microorganisms in hospitals. the main aim of this study was the determination of the antibacterial activities of chemical treatments applied to textiles. comparison of testing methods assessing antibacterial efficiency was conducted. these studies were performed in order to select the right methods of evaluating the antibacterial and bacteriostatic activity of finishes. methods: fibers and fabrics made from cotton ( %) applied with quaternary ammonium salts were tested. finishes treated with bactericidal agent were compared with samples (not treated with the disinfectant). the european standards iso / dis / and aatcc / suitable for assessment of antibacterial activity were applied. the bacterial strains recommended by the above standards such as: klebsiella pneumoniae (atcc ), staphylococcus aureus (atcc ), escherichia coli (atcc ) were tested. due to its high resistance to quaternary ammonium salts, additionally p. aeruginosa (atcc ) was examined. bacterial suspension - , x cfu/ml was prepared according to the standardsÕ requirement. the samples impregnated with biocidal products were examined with agar diffusion plate test. the specimens were located transversely across the bacterial inoculum on the nutrient agar. the level of antibacterial activity was assessed by examining the extent of bacterial growth in the contact zone between agar and the specimens. additionally, the extent zone of inhibition (minimal mm) around the specimens was considerate. results: tested fibers did not show antibacterial activity. the fabrics showed antibacterial activity against k. pneumoniae and s. aureus (inhibition zone mm). the examined specimens showed no bacteriocidal activity against escherichia coli and pseudomonas aeruginosa. the results obtained in two applied methods were comparable in assessing antibacterial activity of finishes. the antymicrobial finishes did not fulfill requirements of the standards. conclusion: based on the obtained result the tested antimicrobial finishes may not be considered effective in preventing bacterial transfer at contaminated areas in hospitals. a new interactive approch to improve hand hygiene compliance j. holt, e. tvenstrup jensen, d. buhl (copenhagen, dk) introduction: the compliance of guidelines for hand hygiene is well below % despite several at-tempts to improve. when planning an educational outline to promote a behavioural change in the hand hygiene practice in the aim of lowering the incidence of nosoco-mial infections the mapping of critical factors and implications that affect this practice is of utmost importance. objectives: this study was an empirical investigation of the aspects of the hand hygienic practice as it takes place in the danish health care system today. the study attempted to answer the question:''how can the health staff's lack of compliance with hand hygiene be explained and understood as basis for planning an educational material to support a behavioural change.'' methods: literature studies of hand hygiene, a questionnaire based done survey ( per-sons/ respondents) and qualitative interviews (n = ) was performed in order to uncover the explanations for this low rate of compliance.the data was analysed and discussed on the basis of theories on action, experience, reflective thinking, control and rituals in order to aim for a broader view and under-standing of this field. results: both the questionnaire and interviews showed that hygiene still is a field that has great implications on the interactions between individuals. furthermore it seemed difficult for the staff to draw a professional and not a private line between ''the clean and the unclean'' and thereby perform hand hygiene without compromising each other. further the interviews showed that it is difficult for the staff to react on something they cannot see and that not gives an immediate result when staff does not act as pre-scribed. introduction: leishmaniasis has increased in importance in recent years as hiv-infected patients have emerged as a risk group for the disease. however, real prevalence in the general population is unknown. objetive: the objective is to know the antibody seroprevalence for infection by leishmania infantum in the general population of castilla-leon (spain). methods: a random sample from the general population ( sera) and from hiv-infected patients in the autonomous community of castilla-leon was collected in . seroprevalence of antibodies against l. infantum was determined by an indirect enzymoimmunoanalysis (eia) test designed in the laboratory. results: anti-leishmania infantum antibody seroprevalence in the general population was . %. there is a significant increase in seroprevalence with age (p = . ), from . % in the - years group up to . % for those over years old. there were no significant differences between women and men ( . % versus . %; p = . ). seroprevalence was significantly higher in people from rural areas than in those from cities ( . % versus . %; p = . ). hiv-infected patients had a seroprevalence against l. infantum of . %. no differences were observed between women and men, nor was there a prevalence increase with age. efficacy of intralesional pentavalent antimony in combination with oral azole for treatment of oldworld cutaneous leishmaniasis results: a -year-old man presented with a -month history of slowly growing round lesions on his right arm and leg. he had a history of travel in the mediterranean area (including north african and middle east countries) months prior to presentation. examination showed one nodular lesion ( · cm) with margin induration and depressed central ulceration on the right arm, a second nodular lesion ( . · . cm) on the right leg, and a red papule ( . · . cm) on the left arm. a -year-old man with an history of a -month stay in iraq until months prior to presentation, showed lesions, which had grown during the last three months: two nodular lesions ( . · cm, . · cm) with erythematous margins and a thick crust on the left arm, round lesion ( . · . cm) with depressed central ulceration, at the left arm, right arm, right leg and second finger of the right hand. a -year-old man with a -month stay in iraq until months prior to presentation, showed two painless confluent nodular lesions ( . · . cm) with depressed central ulceration at the right elbow, which had grown over months, two round lesions ( · . cm and . · . cm) with a thick crust on the left poplitea fossa and one nodular lesion ( . · cm) at right leg. all patients received alternate-days intralesional injections of meglumine antimoniate and weeks of oral fluconazole. all lesions healed completely at the end of therapy and at the -month follow-up. none of the patients complained of any adverse events during treatment or follow up. conclusion: treatment with intralesional pentavalent antimony in combination with azole is effective and absolutely safe, in that it reduces the total amount of antimonial exposure. entomological study of sandflies (dipthera: psychodidea) in three foci of endemic cutaneous leishmaniasis in iran human leishmaniasis is a globally widespread parasitic disease caused by members of the genus leishmania. currently, leishmaniasis is considered to be endemic in countries including iran. phlebotomine sandflies the vectors of leishmaniasis have received considerable attention in recent years due to the resurgence of leishmaniasis in some endemic areas of iran ,so extensive studies have been conducted on the ecology of sandflies in different parts of the country in recent years. a total sandflies were collected by using funnel traps of rodent burrows in rural district of the province of iran (shiraz, yazd, khozestan). these sandflies identified in maine croups: phlebotomus. papatasi( %) , p. sergenti ( %) , p. sergentomya ( %). these findings indicate that p. papatasi could be a vector of humans and the gerbils (merion libycus). the close contact between vectors and resevoirs have created a very efficient cycle for the transmission of the disease in these areas and the villages around these provinces. erysipelas like cutaneous leishmaniasis: a case report a. erdogan, d. balaban, e. dervis, g. sengoz, g. barut, a. karaoglu (istanbul, tr) cutaneous leishmaniasis is an infectious disorder of the skin caused by leishmania major, tropica, aethiopica and infantum which are transmitted by phlebotomine sandflies. we present here a patient who demonstrated a morphologically rare form of erysipelas like cutaneous leishmaniasis. a -year-old woman with a four-month history of erythema and edema on her nasal and left malar area was referred to our clinic for further evaluation. her dermatological examination revealed an erythematous, edematous and fine desquamative plaque on her nose and left malar region resembling erysipelas. other physical and laboratory findings were normal. punch biopsy taken from the lesion and dyed with h.e revealed dense lymphocytic infiltration between the layers of just beneath the epidermis and deep dermis. in addition, a huge number of amastigots were found in macrophages and histiocytes that formed granulomas. in light of these findings the patient was diagnosed with cutaneous leishmaniasis and commenced a regimen of meglumin antimonat mg b.i.d. after two weeks of therapy the lesion was gradually disappeared without any scar formation. in conclusion, cases with cutaneous leishmaniasis are still observed in our country not always with usual clinical appearance, but also like the present case, it may clinically resemble erysipelas and provides difficulties in the differential diagnosis. study of human infection of cutaneous leishmaniasis in a focus of the disease, southern iran ) with scar and cases ( %) with ulcers. the mean of acute lesions and scars per infected cases was . and . , respectively. totally cases were observed with sores and scars, and out of them were infected in the area. the highest rate of the acute lesion was observed in - years old age group ( . %); meanwhile the highest rate of scars was in - years old age group ( . %). sores were located on hands ( . %), foot ( . %) face ( . %) and other parts of body ( . %). in the study of schools, students ( . % boys, . % girls) were visited. the infection rate to acute lesion was . % and . % of students had scar. there is no significant difference between males and females based on the acute infection (p > . ), but this difference was significant based on scar (x = . , p < . ). the highest infection rate was observed in tashkooieh village ( . %). two injected mice were developed the acute lesion and the agent of the disease was identified as leishmania major by pcr test. conclusion: the infection rate of the sores and scars shows that the disease is located in the studied area in recent years. the disease had one peak during - and has been increased from up to now. this is the first time that the parasite isolated from human in the area. therefore, this focus must be added to the zoonotic cutaneous leishmaniasis foci of iran. the disease is endemic in the area because more than % of cases were infected locally. giardia and cryptosporidium in the netherlands objectives: • the studies were designed to get an insight into the incidence of protozoal-, bacterial-, and viral infections in patients with diarrheal complaints in different groups: patients consulting their general practioner and the dutch population. here we focus on giardia and cryptosporidium • to decrease diagnostic deficit • study the risk factors methods: three studies were designed and conducted : • ÔhaarlemÕstudy: - , general practitioners, haarlem region • nivel: case-control study in sentinel general practitioners practices ( general practitioners practices ( - • sensor: prospective population based cohort study with a nested case-control study in the dutch population. ( )the studies differ in inclusion criteria and the diagnostic laboratory techniques used, esp. virological stool examinations results: incidence of gastroenteritis in the nivel (gp) study (after correction) was . per , personyears. this means that . - . persons will consult a gp annually for gastroenteritis. incidence of gastroenteritis in the populationbased study was per personyears. giardia was detected in . % of the cases in haarlem, in . % of the cases in the nivel study and . % of the controls. for cryptosporidium this was resp . %, . % and . %. the diagnostic deficit decrease substantialy by testing for viral pathogens like nlv. detection of pathogens was influenced by age, season and duration of symptoms. we were able to construct an algoritm for diagnostic workup in gi patients. statistical surveys have been made for the effect of the climate on the epidemic diseases in tropical area, but not so much has been clarified on the relation between the variations of meteorological elements and of the number of patients. in this paper, we apply eof analysis method to time series of diarrhoea patients and meteorological elements in bangladesh, to understand effects of the meteorological variation to the prevalence of the diarrhoea disease. the eof analysis of the time series of patients and meteorological elements averaged every two weeks for years from to shows that in the dominating component, the anomaly of the number of the diarrhoea patients has different signs for the periods before and after june, corresponding to the two seasonal peaks of the number of the patients. higher maximum temperature and more humidity in the pre-monsoon period are found to have a tendency to enhance the first peak of the diarrheal occurrence. we will also report the result for the different types of diarrhoea as v. cholera, rota and etec. serologic evidence for babesiosis in the northern and eastern tyrol (austria) and the southern tyrol (italy) methods: leptospirosis was diagnosed by leptospiral igm enzyme linked immunosorbent assay (elisa) and the serovar was confirmed by the microscopic agglutination test (mat) using a battery of live leptospiral strains as antigens. results: most of them were outdoors manual workers ( %), housewives ( %), indoors non-manual workers ( %) and unknown ( %). mean duration of symptoms was . ± . days. majority of the patients presented with fever ( %), jaundice ( %), chills and rigors, vomiting ( % each), cough ( %), abdominal pain ( %), diarrhoea, and altered sensorium ( % each), purpura/bleeding ( %). salient laboratory abnormalities included anaemia ( %), leucocytosis ( %), thrombocytopaenia ( %), elevated erythrocyte sedimentation rate (esr) ( %), hyponatremia ( %), hypokalemia ( %). acute renal failure occurred in %. hepatic function derangement occurred in %. three patients had pulmonary infiltrates and sputum revealed haemosiderin laden macropahges in them. two others manifested exudative pleural effusion which was bilateral and sequential in one of them. the common serovars encountered included l. autumnalis ( %), l. hebdomadis ( %), l. grippotyphosa ( %), l. icterohaemorrhagiae and l. javanica ( % each), l. australis ( %). all patients were treated with parenteral crystalline penicillin, oral doxycycline and were managed conservatively. haemodialysis was required in four patients. two patients died, both of whom developed multiorgan system failure. conclusions: leptospirosis is an important cause of acute febrile illness with renal, hepatic dysfunction and bleeding abnormalities. a high index of clinical suspicion is required confirm the diagnosis early as the condition responds well to conservative management. determination of fr , a promising antimalarial agent, in human serum by capillary electrophoresis for pharmacokinetic studies the acetyl derivative of fosmidomycin, fr , was demonstrated to be twice as active against plasmodium falciparum in vitro and in the plasmodium vinckei mouse model. fr , as fosmidomycin, is an inhibitor of -deoxy-d-xylulose- -phosphate (doxp) reductoisomerase, an essential enzyme of the non-mevalonate pathway of isoprenoids biosynthesis. the biosynthesis of isoprenoids in plasmodium is depending on this doxp pathway, as found in eubacteria, algae and plants, but not in human. in plasmodium, the doxp pathway is localised inside a plastid like organelle, the so-called apicoplast. here, we report on the development of a high-performance capillary electrophoresis (hpce) technique for the determination of fr in serum. various instrumental setting for migration (voltage, capillary temperature) were tested and the buffer properties (ph values, component molarities) were optimized. the assay was submitted to standard quality control procedures: within-, between-days reproducibility, accuracy, limits of detection (lod) and quantification (loq), linearity, short and long term stability. finally, the working buffer used was mm kh po / mm k hpo , % methanol, and . mm hexadecyltrimethylammonium bromide (htab). the ph was adjusted to . . the electroosmotic flow was modified by the cationic ion pairing reagent, htab. the assays were linear over the large concentration range tested, from . to lg/ml. the recovery of the sample pre-treatments was higher than %. good precision was obtained, with betweendays reproducibilities resulting in coefficients of variation (cv%) of . , . and . %, and within-days reproducibilities of . , . , and . % (cv%), for , , and lg/ml, respectively. the lod was . lg/ml, and the loq was . lg/ml. the studies of short and long-term stability of fr in serum showed a good stability of the molecule, at room temperature, + °c or ) °c. moreover, fr was resistant to numerous cycles of freezing and thawing. indeed, after four freeze-thaw cycles ( days), . %, . %, and . % of fr were recovered from the , , and lg/ml samples, respectively. in conclusion, we have developed a convenient ce technique for the determination of fr in serum which offers advantages of speed, sensitivity, and accuracy. at present, the procedure is applied for pharmacokinetic studies in an animal model of gö ttingen mini-pig. present and future of malaria in kahnouj endemic area, southern iran objective: kahnouj district is associated with one of the malaria regions in southeast of iran. the anopheleine fauna does not appear to have changed much over several decades. methods: entomological studies and mosquito collection were performed every days from indoor and out door shelters. malaria surveillance was carried out by health centers, ministry of health. microscopy was performed on out patients with fever or suspected malaria. malaria cases detection were mostly performed passive and rarely actively. the positive cases were treated with chorolquien/primaquen. results: in the present study five vectors species of malaria were found in this study which had been previously recorded decades ago. this district like other malaria endemic areas in iran has been under pressure of anti malaria programmes including case finding and residual spraying insecticide as well as larviciding against the vectors since . annual incidence of malaria declined from . to . during ten years. the most transmission occurred in october to december when the temperature was suitable for vector activity in kahnouj area. electricity was recently supplied into the rural area where most malaria cases were found. therefore, most houses equipped with air conditioner and the resident keep windows close, so they are secured from mosquitoes bites during hot season but not in beginning of spring and autumn when temperature is mild enough, in order to save the electricity cost, the residents do not use air conditioners whereas they leave windows open and no other protection against mosquito bites is provided. residual spraying insecticide of indoor shelters have been stopped for five years and the most activity of antimalaria programme set up base on case finding. conclusion; in order to control malaria, indoor residual spraying of insecticide would not assist effectively, so given knowledge to people to use bed net particularly in season people are more expose to mosquitoes bite may be considered as an effective measure in controlling malaria in khanouj district. also development in this area is effectively pushing back malaria in near future. entomological studies and mosquitoes collection were performed every days from indoor and outdoor shelters as well as breeding places with the aid of suction tube and dipper. results: entomological researches were found that five vectors species of malaria in this study had been previously recorded decades ago. anopheles stephensi was recognized as the main vector of malaria in this area with two peaks, one in may and the other in december. the most malaria transmission occurred in june and december. the larval habitats includes draying river bed with pools, rocky river pools, stagnant streams, slow foothill streams, temporary pools, slow moving water with or without vegetation. conclusion: operational of insecticides for adult and larval control, as well as surveillance of malaria cases, would not assist effectively to control of malaria, so given another malaria control methods as impregnation of bed nets as well as repellent particularly in seasons that people are more expose to mosquitoes bite, may be considered as an effective measure in controlling malaria in this area . case report: a year old boy presented with fever and a generalized exanthema at the local dermatology clinic. the mixed appearance of pustules and umbilicated non purulent vesicles led to the suspicion of an orthopoxvirus infection. the patient reported that little red dots had occurred weeks earlier at the extremities after contact with a sick pet rat. - weeks later a generalized pustular exanthema appeared, which was associated with high fever (> °c) and a pronounced feeling of sickness. histologically no viral inclusion bodies were found in pustule material but poxvirus particles were detected by elmi in swabs from pustule ground. following the anamnestic suspicion of transmission by an infected rat, which unfortunately had perished soon afterwards, and the experience made at the recent monkeypox outbreak in the usa initiated by imported rats, one of the major aims was the exclusion of monkeypoxvirus. this was accomplished by pcr typing, which like elmi had been established on occasion of the implementation of the austrian plan for poxvirus preparedness (Ôpocken-alarmplanÕ) and despite the diagnostic means for detection of variola vera also included the ability to discriminate between animal orthopoxviruses. additionally serological diagnosis was performed and presumably due to the protracted course of infection cowpox specific igg antibodies were already present at admission with a titre of : . interestingly the humoral immune response initially seemed to be rather cowpox specific as there were no antibodies crossreacting with vaccinia virus detectable in indirect immunofluorescence. as an exclusive immune reaction with one species alone would be a very rare situation observed with orthopoxviruses we did more extensive testing by western blot analysis. although not exclusively directed against cowpoxvirus antigens a very restricted reactivity of the patient serum was observed in the early course of infection using different recombinant and virus-derived orthopoxvirus antigens. this would suggest that also serological methods might be able to give a hint towards rapid determination of orthopoxvirus species, which certainly is also the case with immunological antigen detection methods. crimean-congo haemorrhagic fever in results: forty veterinerians from endemic region (tokat), and from non-endemic region (aydin) were included. demographic characteristics in two groups were similar, whereas professional activities of veterinarians in non-endemic region were more intense (p = . ). the cchf igg positivity ( . % vs %), brucella agglutination titre of > / ( . vs . %) were more common in endemic region than non-endemic region. three veterinarians in tokat had malaise, myalgia, and fever. in multivariate analysis to detect the risk factors for serum tube agglutination of > , the veterinarians living in endemic area were found to have . times higher risk of brucella infection than the ones living in non-endemic area (or; . , confidence interval; . - . , p = . ). the prevalence of coxiella burnetii serology was equal in both regions as %, and none of the seropositives had complaints. the history of tick bite was significantly more common in the endemic region than the nonendemic region ( % versus %, p = . ). conclusions: cchf and brucellosis compose infection risks for veterinarians in endemic region, despite the veterinarians in endemic region perform less riskfull professional activities. veterinarians in cchf endemic regions should be warned to protect themselves against tick bites according to universal precautions. the use of masks should be employed to prevent inhalational transmission of brucellosis in endemic regions. changes in temperature and the crimean congo haemorrhagic fever outbreak in turkey o. ergonul, s. akgunduz, i. kocaman, z. vatansever, v. korten (ankara, istanbul, tr) objective: to investigate the role of the climatic factors that could effect the activation of hyalomma marginatum marginatum population, and consequently the emergence of crimean-congo hemorrhagic fever (cchf) epidemic. methods: the meteorological data were obtained from three meteorology stations (tokat, sivas, and yozgat), where the majority of the cchf cases were reported in the last years. these provinces are located at the northern parts of eastern anatolia and southern parts of black sea region. temperature data have been observed and recorded by the turkish state meteorological service (tsms), and were available for years in sivas, for years in tokat, and for years in yozgat. meteorology stations are located at the city centres, not at the airports. temperature variations and trends for turkey were analysed by using a data set including monthly averages of daily mean, and minimum temperatures. annual rainfall, and the number of days in april with the temperature of > °c were also included in the analysis. in order to detect homogeneity in mean annual series, first a homogeneity analysis was performed by using the non-parametric kruskal-wallis (k-w) test. the non-parametric mann-kendall (m-k) rank correlation test ( ) the interest in the coordination properties of acrylate acid and its homologues was generated by the facile synthesis of the Ômetal-containing monomersÕ (mcm) materials. these compounds can be polymerised with a lot of organic monomers leading to various metal-containing polymers. polymeric transformations of mcm led to a new research field of current interest due to the practical importance of the obtained products which exhibit a number of unique features: high catalytic activity, unusual magnetic, electro-physical and biological properties.these polymers are especially appropriate for biological applications (tissue engineering, implantation of medical devices, dentistry, bone repair etc.) because of their molecular weight, compositions and architectures which can be regulated through controlled reactions. we report here the antibacterial and antifungal activity of new complexes of type m(phen)(c h o ) (h o)y (( ) m: mn, y = ; ( ) m: ni, y = ;( ) m: cu, y = ;( ) m: zn, y = ; phen = phenantroline and c h o is acrylate anion), representing the first step products in the synthesis of polymeric materials. the in vitro antimicrobial testings were performed by broth microdilution method, in order to establish the minimal inhibitory concentration (mic), against gram-positive (bacillus subtilis, listeria monocytogenes, staphylococcus aureus), gram-negative (psedomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, both concerning the microbial spectrum and the mic value. the mics values widely ranged between mg/ml and mcg/ml. all the tested compunds were highly active against salmonella and listeria (mic = mcg/ml objectives: c. glabrata is innately less susceptible to azole than most other species of candida and it acquires azole resistance after short-term exposure to fluconazole, as recently noted in oropharyngeal isolates. since c. glabrata has emerged as a significant cause of candidemia, we examined the change in azole mic and karyotype in sequential isolates of c. glabrata during the course of fungaemia, and its relationship to antifungal therapy. methods: serial bloodstream isolates of c. glabrata were obtained from patients with fungaemia over periods of up to days. forty-seven c. glabrata isolates from patients ( patients who received antifungal therapy and patients who did not receive antifungal therapy) were analysed using electrophoretic karyotyping (ek) and tested for antifungal susceptibility to fluconazole, voriconazole, and itraconazole. results: the overall rates of resistance to fluconazole (mic ‡ lg/ml) and itraconazole (mic ‡ lg/ml) for all isolates were and %, respectively. for most patients, the sequential strains from each patient exhibited the same or similar azole susceptibilities. however, sequential isolates from two patients showed three-or four-fold increases in the mics of all three azole antifungals, while they retained the same karyotypes. azole-resistant strains were isolated from both patients after fluconazole therapy was discontinued and the intervals after the first blood isolation were and days, respectively. the isolates from one of these patients exhibited increased expression of the cgcdr efflux pump. the sequential strains from each patient had identical karyotypes in ( %) patients, but two or four different karyotypes in ( %) patients. the sequential isolates from these five patients exhibited the same or similar antifungal susceptibilities, showed only one or two chromosome band differences, and had no association with previous antifungal therapy. conclusion: this study showed that sequential bloodstream isolates of c. glabrata were able to acquire azole resistance in association with fluconazole therapy, and that they developed two or four different karyotypes in some patients, during the course of fungaemia. results: the next table shows the mics obtained for the tested drugs. from the three isolates with voriconazole mic > mg/l, two of them had a fluconazole mic ‡ mg/l. six isolates with voriconazole mic = mg/l were found: the fluconazole mic for one of them was mg/l, for another four was mg/l and for the last one was mg/l. conclusions: a. there wasn't found any fluconazole or voriconazole ÔresistantÕ isolate in the haart era. b. the percentage ( . %) of isolates with voriconazole mic ‡ mg/l is higher than it has been described in other works. c. the % of the voriconazole ÔresistantÕ isolates were fluconazole ÔresistantÕ too, so a cross resistance mechanism could be implicated. in vitro susceptibiility testing of micafungina (fk- ) and anidulafungin (ly ) against candida spp. objectives: despite antifungal therapy, mortality in disseminated zygomycosis is still too high. we analysed the in vitro activity of posaconazole (pos), voriconazole (vrc) and caspofungin (cas) against strains of the genera rhizopus, mucor, absidia, and cunninghamella in different media compositions. methods: the following five media compositions were compared: rpmi ± % glucose, am ± % glucose and hr-medium. mics were determined by microdilution method following nccls guidelines with minor modifications. each well was read visually, the growth in each well was compared with the growth control. two endpoints were evaluated for each drug: an inhibition of growth > % war recorded as mic and an inhibition of growth > % was recorded as mic . the final concentrations of the antifungal agents were . - mg/l for pos and vrc, and . - mg/l for cas. results: pos was significantly more active than cas and vor, both in r + g and in hr media (p < . when comparing the mic of pos in hr medium to that of vrc; p < . for all other comparisons). growth in rpmi and am media supplemented with glucose was more robust than in the corresponding media lacking glucose. glucose had little influence on mic values. the agreement when comparing the mic evaluated in am ± glucose was > %, while the use of mic endpoint yielded % agreement for all genera. when comparing the data obtained in rpmi ± glucose to that in hr, the agreement was good. the percentage agreements using the mic and mic endpoints were % and > %, in contrast, the agreement between am ± glucose and the other media was generally poor. moreover, the average mics obtained in am medium were lower than those obtained in either hr or rpmi which was due to a difference among genera. conclusions: our results suggest the following: a) pos is active in vitro against zygomycetes at clinically relevant concentrations. b) within zygomycetes, there are differences between genera in terms of their antifungal susceptibilies. c) growth medium is an important variable for mic determination in zygomycetes, and the more relevant medium appears to be rpmi supplemented with % glucose. objectives: here we describe first clinical case of a caspofungin resistant c. glabrata infection. the patient was a year old male with aml. he received a matched unrelated hsct, months prior to his death. he had a complicated hospital course which included c. glabrata sepsis. c. glabrata was cultured from his stool, from the time of transplant to death. initial blood culture isolates were azole resistant and amphotericin b was started. this was stopped due to renal insufficiency and caspofungin was started (mic . lg/ml). he had a prolonged duration of therapy which comprised of alternating courses of iv voriconazole and caspofungin. four months after initial fungaemia c. glabrata was cultured which was both caspofungin resistant (mic > lg/ml) and azole resistant. despite the addition of amphotericin b the patient died weeks later. c. glabrata was isolated from the bone marrow at autopsy. methods: the series of patient isolates, from time of transplant to death, had susceptibilities performed as per nccls document m -a. the isolates were typed using cg probe and mlst. results: he susceptibilities demonstrated caspofungin resistance in the blood isolate ( > lg/ml) and azole resistance in all but a few of the initial stool isolates. all the isolates were shown to be identical by cg and were determined to be mlst group , which has previously been shown to be the most prevalent clade of c. glabrata worldwide. discussion: this describes the first caspofungin resistant clinical isolate of c. glabrata. it has been recently reported in c. albicans. this data details an isolate that has developed resistance in the presence of therapy. resistance to caspofungin is not common and is thought to be due to mutations in the beta- - -glucan synthase (fks) gene. there are fks genes in c. glabrata. preliminary sequencing data of one of these genes has so far revealed no non-conservative mutations. the remaining genes are yet to be sequenced and the presence of other mechanisms cannot be ruled out. objectives: the aim of this study was to investigate the production of slime factor among candida species which were isolated from hospitalized patients. another aim of this study was to see in vitro activities of antifungal agents and to compare these results with slime production. methods: total candida spp ( c. albicans and nonalbicans candida spp) isolated from various specimens were included to the study. fluconazole, itraconazole, amphotericin b and caspofungin susceptibilities of these strains were determined by broth microdilution method according to nccls m -a standards. biofilm production of candida spp. was determined by microplate method on polystyrene microtiter plates using brain heart infusion broth supplemented with . % glucose as a growth medium. results: caspofungin and amphotericin b was the most active agents which mic values mg/ml and . mg/ml respectively. fluconazole resistance (mics > ) was obtained from of the isolates ( %). biofilm formation was detected in of the total strains ( %). statistically important difference (p < . ) was determined between the biofilm production of c. albicans ( . %) and nonalbicans species (% . ). significant correlation between biofilm production and amphotericin b mic values was established (p < . ) conclusion: candida species are one of the most important etiologic agents of catheter related infections especially biolfilm producing strains. this study showed us biofilm production rates are too high particularly in non-albicans strains. this will explain the rising incidences of non albicans strains especially c. parapsilosis in serious infections. our study also implicated that the mic values amphotericin b which was one of the most active agent against candida infections had a significant correlation with slime production. this will be a problem in treatment of slime producing candida infections with this drug. conclusions: (i) scedosporium spp. carry on with high mics of antifungal agents, even for new antifungal agent as pos. (ii) s. prolificans is a multi-resistant organism. (iii) s. apiospermum is resistant to itc and tbf, but % of strains are susceptible in vitro to amb (mic < mg/l), % to vrc (mic < mg/l), and % to pos (mic < mg/l). (iv) these findings reinforced the need of continued surveillance programmes that analyze antifungal susceptibility profiles of medically important fungal isolates. assessing susceptibility of fungi isolated from hospital environment to disinfectants objectives: the past decade has witnessed a worldwide increase in serve invasive gas infections. these rapidly progressive infections are associated with high mortality rates despite prompt antimicrobial therapy. the aim of this study was to characterize gas isolates causing severe invasive disease in different regions of poland by emm-typing, multilocus sequence typing (mlst), pfge, virulence genes distribution and their susceptibility to antimicrobial agents. methods: a total of gas isolates from blood ( . %), pus ( . %), sputum ( . %), peritoneum fluid ( . %) and other sources were examined. susceptibility to penicillin, erythromycin, clindamycin, telithromycin, tetracycline, levofloxacin, chloramphenicol, quinupristin/dalfopristin and linezolid was determined by the microdilution method according to the nccls guidelines. clonality of all isolates was studied by emm typing, pfge of sma i-restricted bacterial dna as well as mlst. the strains were also tested for the presence of spea, speb, spec, spef and ssa genes by pcr. results: resistance to erythromycin was found in four isolates ( . %), two of them exhibited the imlsb and two the cmlsb phenotype. twenty-one ( %) and five ( . %) were resistant to tetracycline and chloramphenicol, respectively. all tested isolates were fully susceptible to penicillin, levofloxacin, quinupristin/ dalfopristin and linezolid. twenty different emm types were detected, of which emm ( . %) and emm ( . %) were most common, followed by emm , emm and emm . all emm types and emm types corresponded to the st and st , respectively. altogether, different pfge patterns, designated a-y were discerned among the isolates, with two predominant profiles a (n = ) and b (n = ). our study showed that all isolates possessed speb and spef genes, while the frequencies of spec and spea were . % and %, respectively. conclusion: two clones predominated among gas strains causing severe invasive disease in poland; these clones were of emm type and . we present actinomyces spp isolations, their identification to a species level and their clinical sources. in addition, we perform susceptibility testing of of those strains to drugs. the identification of actinomyces spp was done taking into account their cultural features, growthÕs atmosphere and biochemical and enzimatical tests, according to schemes proposed by sarkonen n., funke g., moncla b.,hillier s., and bernard k. we studied too, the clinical sources of actinomyces spp, if they were isolated lonely or in association with mucosa's normal flora. the susceptibility testing was performed by the agar dilution method, with mueller hinton agar supplemented with % sheep blood. the reading of minimum inhibitory concentration (mics) were done after hours incubation at °c in at atmosphere enriched with co . all strains were grown at °c on sheep blood agar plates with co added. a. radingae, a. europaeus, and a. odontolyticus were the most frequent isolated species ( each one) followed by a. israelii, a. graevenitzii, a. turicensis and a. viscosus. in isolations, actinomyces spp were recovered as sole microorganism, and in the remainder, in association with mucosa's normal flora. there were not relations between actinomyces species and the clinical sources of the samples. mics for penicilin, ampicilin and cefotaxime were from < . to . lg/ml. there was a bimodal behaviour with macrolides : erythromycin, azithromycin and clarithromycin (mics from . to lg/ml) and the same was observed with quinolones: levofloxacin, ciprofloxacin and moxifloxacin (mics from . to lg/ml). all isolations presented mics for vancomycin < . lg/ml. the identification of the actinomyces genus presents diagnostic difficulties due to its growth requirements. some species are not so infrequent, and the sources from which they are recovered suggest that they may be of clinical relevance, for they are often isolated as sole pathogen. isotretinoin versus tetracycline: a comparative study with regard to efficiency in the treatment of acne vulgaris c. oprica, l. emtestam, c.e. nord (stockholm, s) tetracyclines are most commonly used for treatment of moderate and severe inflammatory acne, and systemically administered isotretinoin has proved to be the most efficient treatment, used in patients with moderate or severe acne that fails to respond to other therapies. objectives: a randomized clinical trial was conducted to compare the clinical efficacy and the antimicrobial susceptibility of propionibacterium acnes strains isolated before, during and after treatment with either tetracycline or isotretinoin in patients with acne vulgaris. methods: male and female patients, - years of age, with moderate or severe acne, were randomized into two groups of patients each. they received oral tetracycline hydrochloride gram/day together with topical retinoid (differine gel . %) or isotretinoin (roaccutane) mg/kg/day. the therapy was given for a -month period. clinical evaluation (leeds acne grading system and lesions counting) and bacterial samples were taken before the treatment started, during the treatment and months after the treatment had stopped. dermatology life quality index questionnaire was completed by patients before and after treatment, in order to determine impairment of life quality. results: acne severity was significantly reduced by both regimens during therapy, and patients in the isotretinoin group continuously improved the acne scores after the treatment had stopped. after months of treatment, isotretinoin produced greater lesion reductions than tetracycline. the mean per cent reduction in the different lesion counts was as follows: % versus % for non-inflammatory lesions (p < . ) and % versus % for inflammatory lesions (p < . ) in isotretinoin or tetracycline group, respectively. in the drug-free period, the group of patients treated with isotretinoin presented significantly less inflammatory lesions compared to the group treated with tetracycline (p < . ). both treatments had improved the life quality (p < . ), independent of acne severity. resistant p. acnes strains were isolated after treatment in both the tetracycline group ( %) and isotretinoin group ( %). conclusions: both treatments were effective during treatment period. more resistant strains were recovered from the tetracycline group. psoas abscesses. differences between pyogenic and tuberculous abscess objectives: to compare the demographic characteristics, clinical features, laboratory, microbiologic and imaging data, therapeutic options and outcome of pyogenic and tuberculous psoas abscesses. patients and methods: retrospective descriptive study of the medical records of the patients diagnosed of psoas abscess in our institution, in the period between january/ and october/ . results: in this period patients were diagnosed of psoas abscess, all of them secondary to a adjacent source of infection. nine patients had pyogenic abscess, and m. tuberculosis was the causal microorganism in patients. in patients there were bilateral involvement, and all three were tuberculous abscess. an underlying disease was present in % patients of pyogenic abscess, and only in % of tuberculous abscess. these one were malignancy, intravenous drug use, cirrhosis, use of steroids, and with inflammatory bowel disease. patients with tuberculous abscess were younger ( vs years), and they presented with a longer duration of symptoms from presentation to diagnosis ( vs days) than patients with pyogenic abscess. abdominal pain was the most frequent symptom at diagnosis in pyogenic abscess ( %), whereas lumbar pain ( %) was in tuberculous abscess. other clinical manifestations were similar in both groups. the source of infection in pyogenic abscess was gastrointestinal in patients ( polymicrobial), and in patient infection of an aortobifemoral bypass and sacroilitis (both caused by s. aureus). all tuberculous abscess were secondary to spondilitis. there were no differences between both groups in analysis or imaging alterations. culture of the abscess was positive in all cases practised. drainage was performed in patients ( percutaneous), without differences in both groups. clinical improvement was more delayed in patients with tuberculous abscess than in patients with pyogenic abscess ( vs days). there were no deaths in any group. conclusions: tuberculous psoas abscesses presents in younger patients with lesser underlying diseases and a more prolonged clinical presentation than pyogenic abscesses. these tuberculous abscesses are secondary to spondilitis, usually with bilateral involvement. prevalence of micro-organisms isolated from wounds of inpatients versus outpatients in a spanish teaching hospital objectives: hidradenitis suppurativa (hd) is a chronic disorder characterized from dilatation of sweat glands and recurrent bacterial infections. vitamin e was administered in several patients as an antioxidant in an attempt to relieve tissue function probably altered by the locally increased oxidant status. methods: twenty nine patients with hd, male and female, were enrolled over a period of twelve months. all have presented with more than three episodes of bacterial exacerbations for at least two years. a detailed medical history was taken upon first evaluation and patients were examined for areas affected by the disease. they were asked to self-evaluate the severity of their condition on a scale of to ( representing intact skin and maximum severity). patients were divided into three groups of treatment: a (n = ), controls; b (n = ), vitamin e orally mg bid; and c (n = ), vitamin e mg tid orally. patients were withhold from any antimicrobial regime. patients were followed-up at three-month intervals; they were asked to re-evaluate their condition, and to provide details regarding frequency of relapses before and after the initiation of treatment. results: mean ± se duration of the disease was . ± . ) years and of involved areas . ± , with axillas and groin being involved in the majority of cases. mean interval between exacerbations before initiation of therapy with vitamin e was . ± . days and after initiation of therapy . ± . days (p: . ). for group b, mean ± se time interval between exacerbations before initiation of treatment was . ± . days and after initiation of treatment . ± . days. for group c, respective values were . ± . days and ± days. mean ± se of self-evaluation scores before therapy with vitamin e was . ± . and . ± . for patients of groups b and c respectively. they were . ± . and . ± . respectively after twelve months of follow-up. the latter changes constituted a significant improvement (p: . ). conclusion: vitamin e seems to improve the overall clinical condition of patients with hd substantially. further placebocontrolled studies are necessary to confirm these results. clonal diversity and toxin genes of staphylococcus aureus causing infections in a clinical ward over a -year period ( - ) s. pollini, g. zanelli, a. sansoni, s. cresti, c. cellesi, g.m. rossolini (siena, i) objectives: s. aureus remains one of the leading bacterial pathogens worldwide, representing a major challenge for antimicrobial chemotherapy. the aim of this study was to evaluate the molecular epidemiology of s. aureus infections observed in an infectious disease ward during the past years. clinical microbiology and infection, volume , supplement , methods: nonreplicate s. aureus isolates were collected from patients with staphylococcal infections (including food-borne infections, osteomyelitis, skin and soft tissue infections [ssti], pneumonia, meningitis and bacteraemia/endocarditis; mostly community-acquired) at the infectious diseases clinic, university of siena, during the period st feb - st march . all isolates were analysed for antimicrobial resistance, clonal diversity and toxin genes (sea-e, eta-d, tst, luks-pv and lukf-pv). susceptibility testing was performed according to the nccls guidelines. clonal diversity was determined by pfge and by analysis of the coagulase (coa) and protein a (spa) genes polymorphism. toxin genes were analysed by pcr and restriction mapping. results: most isolates ( , %) were methicillin-susceptible (mssa), while were methicillin-resistant (mrsa, mecapositive) and borderline. genotyping revealed a single mrsa clone and remarkable clonal diversity among the mssa isolates (at least distinct clonal lineages). several clones (including mssa and the mrsa clone) were detected over prolonged times ( - years). the most prevalent clone was detected over a -year period and was exclusively associated with ssti (mostly bullous impetigo). toxin genes were overall detected in % of isolates, the most frequent being sea ( %), tst ( %) and eta and/or etb genes ( %). six isolates (all mssa) harboured the luks-pv and lukf-pv genes. conclusions: mrsa were rare and appeared only since . significant clonal diversity was observed within mssa. a significant relationship was observed between toxin production and some infections (e.g. ssti). severe group a streptococcal infections in romania. a surveillance within the strep-euro project b. luca, m. straut, v. ungureanu, c. schalen, a. jasir on behalf of the strep-euro study group objectives: in the last years, severe invasive streptococcal infections, often afflicting otherwise healthy subjects and yielding high mortality rates, have been increasingly recorded in europe and other areas. our main objective was to investigate the situation in romania lacking earlier data in the field. methods: the strains used in this study were clinical isolates from nine regions in romania. the strains were mainly blood isolates, but some were from other sterile sites. the in vitro susceptibility to antibiotics was tested by disk diffusion on pdm agar following the standard instruction. t-typing was performed by slide agglutination using sera from sevapharma, prague. spe gene detection and emm sequence typing was performed according to provious publications. results: during eighteen months cases were reported. most prevalent t-type was , followed by types and . more than fifteen different emm types were recognised, most of them unusual types; only four were type . half of the reported cases were from children below years. out of all strains, % harboured spec gene, and harboured spea. erythromycin resistance was uncommon (one isolate), whereas an overall high rate of tetracycline resistance was found ( %). conclusion: this is the first report on severe invasive streptococcal infections since no surveillance has been previously done in romania. the population covered was from eight out of provinces. the emm type distribution might be of concern since many unusual types, not previously associated with severe disease, were found. this may be partly related to the dissemination of new types in populations immune to classical types. since tetracycline is not used in the treatment of streptococcal infections the level of tetracycline resistance among clinical isolates, appeared comparatively high. in contrast to several european countries, macrolide resistance seems not to be common among invasive group a streptococci in romania. acknowledgment: the strep-euro project is funded by the european commission. severe soft tissue infection and secondary bacteraemia due to community-acquired mrsa in a traveller returning from the congo r. padmanabhan, r. shrestha, g. hall, s. gordon, l. saravolatz (cleveland, detroit, usa) objectives: community acquired mrsa is increasingly becoming a global problem. these isolates possess the staphylococcal cassette chromosome (scc) mec type iv in their genome and a different pattern of antibiotic susceptibilities than hospital acquired strains. they are frequently virulent and cause predominantly skin and soft tissue infections by virtue of a panton valentine leukocidin (pvl) toxin gene. a returning traveler, who had spent the preceding four weeks in congo, presented with a staphylococcal bacteraemia and a large cutaneous ulcer of the right lower extremity that progressed to extensive soft tissue necrosis. he first experienced symptoms in kinshasa international airport, while awaiting his return flight to the united states, approximately hours prior to presentation at our facility. methods: mrsa that was isolated from blood was susceptible to vancomycin, clindamycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and gentamicin. pulse field gel electrophoresis (pfge) analysis was performed using sma on the patient's isolate along with another strain from our hospital and compared to other strains isolated from the midwestern united states. pcr amplification of the genes encoding panton-valentin leukocidin (pvl) was performed using primers described elsewhere. results: pfge demonstrated that the congo strain was different from any of the other midwestern united states strains. scc mec typing of this isolate revealed that this strain was type iv. pcr analysis did not detect sequences specific for the pvl gene. conclusions: the differential diagnosis of an ulcer in a returning traveler is large and includes bacterial, fungal, helminthic, protozoal, viral and arthropod borne causes. mrsa should be added to this list. the widespread occurrence of ca-mrsa in many parts of the world would support the finding of this organism on the african continent. to our knowledge, this represents the first ca-mrsa with the scc type iv mec gene reported from congo. ten-year of community-acquired methicillinresistant staphylococcus aureus st -iv clone in denmark objectives: to determine the epidemiology in denmark of the pandemic european ca-mrsa clone st -iv with respect to subtypes, dissemination, acquisition and types of infection. methods: all methicillin resistant staphylococcus aureus (mrsa) isolated in denmark between - were referred to and stored at the staphylococcus laboratory, statens serum institut (n = ). the isolates were characterized by macro-restriction (smai) analysis using pfge, sccmec typing, dru sequence typing, antibiotic susceptibility testing and pcr amplification of the panton valentine leukocidin gene (pvl). clinical and epidemiological information from all patients were obtained from discharge summaries and registered. results: between and , the mrsa st -iv clone accounted for % ( / ) of the total number of mrsa in denmark and % ( / ) of infections due to st -iv had primarily community onset ( / ) and thereby st -iv accounted for % of all community onset mrsa in the period. more than % of the st -iv infections were skin and soft tissue infections. by comparing the antibiogram of st -iv (streptomycin-, kanamycin-, tetra-cycline-and fusidic acidresistant and gentamicin sensitive) with the isolates stored in our s. aureus bacteraemia collection, we traced the st -iv clone back to . before one to five st -iv isolates were encountered pr. year, which increased to , , , and isolates in the years after. by pfge, the st -iv isolates exhibited nine different patterns (a - ), of which % were type a . nine type a isolates, isolated - were subjected to dru typing, which has earlier enabled separation of pfge clones into subtypes. this was however not possible for the st -iv isolates. conclusions: st -iv isolates cause a large proportion of the mrsa infections in denmark and in particular among the infections with a community onset. the success of st -iv as an infective clone outside the hospital environment may in part be due to its small sccmec type iv and its ability to cause skin and soft tissue infections probably associated with the expression of pvl. the dru sequence typing could not subdivide the major type of st -iv found in denmark pointing out the strict clonality of this clone, which may indicate that it is a very well adapted pathogen. epidemiology and management of pressure ulcers in an acute care hospital objectives: the aims of the study were to determine pressure ulcer prevalence in an acute care hospital, to identify risk factors, to evaluate the medical and non medical management of pressure ulcers and to study the microbiological contamination of pressure ulcers. methods: for each patient, a standardized questionnaire was completed. the questionnaire included demographic data (age, sex, previous hospitalizations…) and the risk factors of the braden scale. detection of pressure ulcer was performed by skin examination of patients by two experts in skin care. management of ulcer pressure was evaluated by reviewing the clinical charts of each patient with pressure ulcer. each pressure ulcer was swabbed and inoculated on selective media. all the data were entered and analysed with epiinfo . d (cdc, atlanta, ga). results: a total of patients ( ± years old) were included : ulcer pressures were observed in patients (prevalence = . %). heel anckle was the most frequent localization ( %), followed by sacrum ( %), elbow ( %), spinous processes ( %) and ischial tuberosities ( %). pressure ulcers were stage i ( %), stage ii ( %), stage iii ( %) and stage iv ( %). eighty percents of pressure ulcers were acquired within the hospital. using univariate analysis, risk factors significantly associated with pressure ulcer were : braden cutoff < (or = . , p < . ), neurological disorders (or = . , p = . ), previous ulcer pressures (or = . , p < . ), and hospitalization in an intensive care unit (p = . ). among the criteria used for braden scale, humidity, activity, motility, nutrition friction and shear were significantly associated with ulcer pressures (p < . ). among the pressure ulcers, . % were diagnosed only by the experts in skin care and . % were treated. treatment was considered inappropriate in . % (mostly in stage i and iii) according to the french guidelines. microbiological results showed that . % of ulcer pressures, mostly from stage iii and iv were colonized with multiple resistant bacteria (i.e. methicillin resistant staphylococcus aureus, extended spectrum beta-lactamase enterobacteriaceae). conclusion: this prospective prevalence study led to a better awareness of patients at risk for pressure ulcer. this surveillance also contributed to a better knowledge of the mobile unit of geriatrics recently created within the hospital. background: following surgery for a musculoskeletal infection (mi), a positive suction drainage culture (sdc) is consistent with persistent sepsis and an unfavorable evolution in the majority of cases. the objective of this study was to determine the effect of a negative sdc obtained in a subsequent operation or repeated operations on the outcome of mi. methods: one hundred patients treated surgically for mi utilizing suction drainage for - hours postoperative and appropriate antimicrobial therapy were enrolled in this prospective study. surgical treatment consisted of the routine practice developed for the treatment of mi, consisting of drainage of purulent material, débridement, and prosthetic exchange or implant removal. the accumulated drainage fluid in the reservoir was cultured. sdc was considered negative if all bottles resulted in negative cultures. patients were placed into one of three possible mi treatment groups according to the sdc results identified after the first surgical procedure, as follows: (i) patients with a negative sdc and no new operation was performed; (ii) patients with a positive sdc and a new operation(s) was performed until the sdc was negative; and (iii) patients with a positive sdc and there was no new operation. the duration of antibiotic therapy for those with osteomyelitis ranged from to weeks, while others with mi (soft tissue infection) were treated for - days. results: the groups were similar with regards to gender, age, underlying conditions, mi, bacterial organism, surgery, and antibiotic therapy. the majority of patients ( %) had osteomyelitis in the presence of an implant. at final review, a cure was obtained in % of patients ( of ) in group i, % of patients ( of ) in group , and % of patients ( of ) in group . conclusion: a negative sdc following mi surgery is a strong indication as to the eventual outcome of the infectious process. microbial aetiology of osteomyelitis of the foot in diabetic patients related hospitalization and lower-extremity amputation.acute infections in pts who have not recently received antibiotic therapy are predominantly caused by aerobic gram-positive cocci, often as a monomicrobial infection. although chronic wounds tend to develop polymicrobial flora. objective: to know the microbial etiology of osteomyelitis of the foot in diabetic pts, to determine the best choice empirical antibiotics combination. methods: diabetic pts with osteomyelitis of the foot admitted in our unit between and were included. the infection was documented with intraoperative samples and osteomyelitis was histolopathologically prouved. clinical and biological data were collected. results: diabetics pts ( males, females) with a median age years( - ) were included.the co-morbidities were chronic renal failure (n = ), arteriopathy (n = ), obesity (n = ), alcoholism (n = ), immunodepression (n = ). the clinical data were fever ( %), shock ( %), local signs as erythema ( %), fistula ( %), necrosis ( %) and foul odor ( %). the median rates of neutrophils polynuclear, c-reactive protein and erythrocyte sedimentation were respectively . g/l, mg/l, mm. pts ( %) had a polymicrobial infection.the intra-operative samples (n = ) yieled a majority of gram positive cocci [mssa:n = ;mrsa:n = ; cns:n = ; streptococcus sp:n = ; enterococcus sp: n = ], following with gram negative bacteria [ pseudomonas sp: n = ; proteus sp: n = ; morganella sp: n = ; e. coli: n = ; klebsiella sp: n = ; e. cloacae: n = ; citrobacter sp: n = ; serratia sp: n = , others: n = ] and anaerobes (n = ). pts had positive blood samples. s. aureus is the most frequent pathogen isolated especially in monomicrobial infections and infections with positive blood samples. pseudomonas spp. seems to be correlated with chronic renal failure and immunodepression. no clinical data was significantly associated with a special bacteria , except necrosis and foul odor with gram negative bacteria or anaerobes. conclusion: our results are according with clinical studies in the literature. the major difficulty to treat osteomyelitis of diabetics foot is to well documented the infection. selecting an appropriate antibiotic to treat is particularly important because of the prolonged duration of therapy required and potential resistance of pathogens. the duration of hospitalisation (length of stay) in patients hospitalised with complicated skin and skin structure infections: identifying clinical and microbiologic risk factors in a comparison of tigecycline with vancomycin/aztreonam % of patients had polymicrobial infections. about % had a gram-negative causative pathogen, mostly escherichia coli. gram-negative causative pathogen prevalence was greatest in asia ( %). conclusions: this analysis showed significant global variations in csssi subdiagnoses, etiologies, comorbidities and causative pathogens. consistent with findings of the sentry antimicrobial surveillance program, s. aureus was identified as the predominant pathogen in csssi, especially in the us. gramnegative pathogens were also identified as causative in a substantial proportion of complicated skin infections. these findings may offer guidance in selecting effective empiric therapy, especially regarding newer agents with expanded broad-spectrum activity. a novel approach for evaluating the microbiological efficacy of tigecycline in patients with complicated skin and skin-structure infections a. meagher, p. ambrose, j. passarell, b. cirincione, t. babinchak, e.j. ellis-grosse (buffalo, albany, collegeville, usa) objectives: tigecycline (t) is a glycylcycline in development for the treatment of patients (pts) with serious infections, including complicated skin and skin-structure infections (csssi). while csssi can be caused by a mixture of grampositive and -negative bacteria, staphylococcus aureus and streptococci are the predominant pathogens. previous analyses combining all pathogens have failed to identify an exposureresponse (er) relationship. a method was developed to create more homogenous pt populations for the microbiological (m) er analysis of t in the treatment of csssi. methods: pts from csssi clinical trials (one phase & two phase ) with t pharmacokinetic data and classified as both clinically and m evaluable, were pooled for analysis. pts received mg loading dose (ld)/ mg q h ( / ) or mg ld/ mg q h ( / ). at the test of cure visit, m (eradication or persistence) response was evaluated. indeterminate responses were excluded. non pathogenic baseline isolates were excluded. five homogeneous pt cohorts (c) were created based on baseline pathogens: s. aureus only (c ); s. aureus or streptococci (c ); gram-positive pathogens (c ); polymicrobial (c ); other monomicrobial infections (c ). prospective step-wise procedures for combining c to increase sample size were used. logistic regression was used to evaluate steady-state hr area under the concentration-time curve (auc) to mic ratio (auc/mic) to predict response. results: the dataset included pts with observations. c (n = ) and c (n = ) could not be evaluated due to small sample size. analysis began with pooled c + c . continuous auc/mic ratio was marginally significant (p = . ); a pt was . % more likely to have successful response for every oneunit increase in auc/mic. adding c , including pathogens with mic values up to mcg/ml, decreased auc/mic, added cures to the lower end of the distribution, and added significant noise to the analysis. adding c increased sample size and further decreased the ability to detect a relationship. conclusion: analysis of all pathogens combined could not identify an er relationship. polymicrobial infections with gramnegative and anaerobic pathogens, associated with high mic values, added noise to the analysis and decreased the predictive capability of the model. the approach of creating homogenous populations based on two key pathogens in csssi, s. aureus and streptococci, was critical for identifying significant er relationships. exposure-response analysis of the efficacy of tigecycline in patients with complicated skin and skin-structure infections a. meagher, j. passarell, b. cirincione, s. van wart, k. liolios, t. babinchak, e.j. ellis-grosse, p. ambrose (buffalo, collegeville, albany, usa) objectives: tigecycline (t), the first glycylcycline to reach clinical trials, is in development for the treatment of patients (pts) with serious infections, including complicated skin and skin-structure infections (csssi). pharmacokinetic-pharmacodynamic (pk-pd) relationships, including pt covariates, for microbiological (m) & clinical (c) efficacy of t were evaluated in pts with csssi. methods: pts from csssi clinical trials (one phase & two phase ), with pk data and classified as both c & m evaluable, were pooled for analysis. only those pts with infections due to staphylococcus aureus and/or streptococci, the predominant pathogens in csssi, were prospectively evaluated. pts received mg loading dose (ld)/ mg q h ( / ) or mg ld/ mg q h ( / ). at the test of cure visit, m (eradication or persistence) & c (cure or failure) outcomes were assessed. indeterminate responses were excluded. steady-state hr area under the concentration-time curve (auc) and auc/mic ratio were evaluated as predictors of response. pt covariates included: age, weight, country, baseline pseudomonas aeruginosa or anaerobes, & comorbidities (diabetes, peripheral vascular disease). classification and regression tree (cart) analyses determined auc/mic breakpoints (bp). logistic regression (one observation/pt) was performed to determine predictors of efficacy. results: the dataset included pts with s. aureus and/or streptococcal baseline pathogens. mic values ranged from . to . mcg/ml. clinical cure was achieved in ( . %) pts and ( . %) pathogens were successfully eradicated. the median auc/mic ratio was . and for the and mg dose groups, respectively. covariates were not significant predictors of efficacy. cart identified a significant auc/mic bp of . (p = . for m and . for c). the continuous auc/mic ratio was marginally significant based on sample size (p = . for m & . for c) and was deemed the most informative model. for each unit increase in auc/mic, within the observed range, pts were . % more likely to have a successful c response and . % more likely to have a successful m response. conclusion: pts with auc/mic ratios ‡ . were . times more likely to have successful m response. at the median auc/ mic ratio of . & for the / & / dose groups, the model-predicted probability of c success was . & . , respectively. t is likely to be an important treatment option for csssi. introduction: vertebral osteomyelitis and/or sacroiliitis due to streptococcus pneumoniae are extremely rare. although it is one of the most common pathogen isolated in blood cultures, there are only about cases reported in the literature. in the main series of vertebral osteomyelitis (vo) spsi represents less than % of cases ( / ). material and methods: we report cases of spinal infection due to s. pneumoniae ( vo and si with psoas abscess) seen between and at different acute care hospitals. results: the cases reported were part of a total of episodes of pneumococcal bacteraemia ( . %).mean age of the patients was . years (range - ); there were men. alcohol abuse / , immunosupression / and smoking / were the most common comorbilities. blood cultures were positive in all cases, epidural pus and vertebral biopsy were also positive in / . all strains were susceptible to penicillin. diagnoses were made by mri in and ct scanning . all patients recived betalactam agents during a mean of seven weeks ( . - . ) in monotherapy (cefotaxime / , ampicillin / ) or in combination (with vancomicin+gentamicin in case). one case needed surgery, because of progression of a psoas abscess. other foci were found in three cases (psoas and paravertebral abscesses and acromio-clavicular arthritis). conclusions: spsi is a rare presentation of invasive pneumococcal disease. underlying disease is comnmon and other territories are frequently involved. infective endocarditis must be ruled out. its course appears to be benign. beta-lactam antibiotics are a good option, and surgery is occasionally indicated for progressive disease despite appropriate antimicrobial therapy. lumbar spine spondylitis due to staphylococcus schleiferi following coronary angioplasty: isolation of the pathogen in peripheral blood cultures a. karakolios, c. karagiannidou, t. kallinikidis, f. markou, v. kontos, g. tsinopoulos (serres, gr) a -year old man presented with acute, severe lower back pain weeks after having been subjected to coronary angioplasty for ischaemic heart disease. he was treated with nsaids but not antibiotics without any improvement. ct scan of the lumbar spine showed changes in l -s vertebrae that were consistent with spondylitis. mri imaging, as well as confirming spondylitis at l -s , it also revealed the presence of a left paravertebral abscess. on admission to hospital, low grade fever was documented. clinical examination of the lower limbs was normal. staphylococcus schleiferi, sensitive to several antibiotics, was isolated from sets of blood cultures taken from both arms. initially, ciprofloxacin mg bd iv was administered for weeks. despite continuous clinical improvement, mri imaging weeks after the commencement of antibiotic treatment showed no improvement of the radiological findings. thus, levofloxacin mg bd p.o. rifampicin mg bd p.o. were given for further weeks. at the end of the treatment, the patient was symptom-free, resumed full activity and mri imaging showed considerable improvement. multiple blood cultures taken from both arms at different time points can help isolate pathogens causing spondylitis averting thus the need for difficult, interventional sampling of the focus of infection. diabetes and use of topical ocular antibiotics: a population-based case-control study objectives: we conducted this population-based case-control study to examine whether patients with diabetes mellitus have an increased relative risk of being treated with topical ocular antibiotics, as compared with population controls. methods: incident cases were defined as , individuals who redeemed a prescription for a topical ocular antibiotic in in north jutland county, denmark. ten gender-and agematched population controls per case were selected, using a unique personal identifier. diabetes prior to the topical ocular antibiotic prescription was determined by record-linkage with the county prescription database and hospital discharge registry. we did conditional logistic regression to estimate odds ratios (ors) for ocular antibiotic use among diabetic individuals and population controls, with adjustment for a range of comorbid diseases. results: among individuals treated with topical ocular antibiotics, . % had diabetes as compared with . % among control subjects. the overall adjusted or for use of ocular antibiotics in diabetic individuals was . ( % confidence interval [ci]: . - . ). stratified analyses showed that children between and years with diabetes had a % increased relative risk for ocular antibiotic use, whereas the effect of diabetes as a risk factor was low in individuals over years and in those with presence of other diseases. conclusions: these results suggest that diabetes is a risk factor for the use of topical ocular antibiotics, especially in younger individuals. objectives: determination of bacterial species and their susceptibility to antibiotics in infectious conjunctivitis in general practice. methods: patients suspected of having infectious conjunctivitis were included from general care centres in the netherlands. from both eyes a swab was taken and pathogens found in the bacterial cultures were tested for their susceptibility using the etest for chloramphenicol, fusidic acid, trimethoprim, ciprofloxacin, and gentamicin (mics according to dutch national criteria). results: / affected eyes were culture positive. streptococcus pneumoniae (n = ) was the most predominant pathogen, followed by haemophilus influenzae (n = ), and staphylococcus aureus (n = ). only for chloramphenicol and ciprofloxacin the mic s were in the susceptible or intermediate susceptible range for all three pathogens, whereas this was not the case for the other antibiotics tested, of which at least one mic was in the resistant range. conclusion: in only one thirdth of the patients suspected of infectious conjunctivis in general practice a bacterial pathogen was found. although the prescription of antibiotics can be debated on the basis of our results, chloramphenicol and ciprofloxacin seems to be superior above fusidic acid, trimethoprim, and gentamicin. investigation of the aetiology of a trachoma-like condition in a rural population in guangxi province, prc p. cho, m.v. boost, w. ng (hong kong, hk) objectives: to determine the presence of trachoma in primary school children of the zhuang and yiao ethnic minority groups in du¡an county in guangxi, china. method: primary school children were examined using a slit lamp and several were noted to display follicular/papillary conjunctivitis suggestive of trachoma. to confirm the etiology of the condition, children were examined on a follow-up visit. for each subject, after examination with the slit lamp, the upper lid was everted after the application of a topical anesthetic (novesin), and a dacron swab was passed along the upper tarsal lengthwise four times. strict aseptic precautions were employed to prevent cross-contamination during sample collection. swabs were placed in dna-free tubes, transported to hong kong, and tested using the roche amplicor kits for detection of chlamydia trachomatis following the manufacturer¡s instructions. twenty children displayed significant conjunctival signs and photographs of the everted upper lids were taken to determine if these visual signs correlated with presence of trachoma as confirmed by pcr. results: sixty-five of primary school students were willing to be tested, and % of these were positive for trachoma by pcr. comparison of photographs and pcr result indicated that not all children who showed signs of conjunctivitis were positive for trachoma. in addition, some children whose eyelids displayed milder form for conjunctivitis were positive by pcr. no obvious corneal involvement was observed for these children. conclusions: it was confirmed that hyper-epidemic levels of trachoma can be found among ethnic minority children in du¡an county. this may be related to the poor hygiene standards and the scarcity of water in this limestone region. pcr was not positive for all children showing signs of follicular/papillary conjunctivitis, which may indicate that some cases were in a dormant stage. it is also possible that failure to detect the organism was due to sampling error or delay in pcr analysis. nevertheless, the high level of infection is a cause for concern as trachoma remains one of the leading causes of blindness, and further investigation and treatment are warranted. clinical and epidemiological considerations about anthrax in constanta county, romania s. rugina, i.m. dumitru, c.n. rugina, e. dumea, e. basca (constanta, ro) introduction: anthrax is by primarily a disease of herbivores but humans can become infected as they come into contact with infected animals or their products. objective: to study the cases of human anthrax diagnosed in the last twelve years and their characteristics: epidemiology, clinical forms and evolution. material and method: it is a retrospective study on a period of twelve years performed in infectious diseases clinical hospital. the diagnosis was based by smear, and culture of vesicular fluid or csf. results: during this period fifteen cases of anthrax in humans were identified. the repartition of cases according to sex groups revealed a high predominance of male cases ( cases). the disease was most prevalent in - age group and after years old ( cases), although anthrax can be observed between years and years. in all cases anthrax was occupational disease and the provenience of patients was rural area. the annual incidence of anthrax was characterized by a low value and irregular frequency of the disease. the most cases were in ( cases) and ( cases). according to the seasonal repartition, anthrax was most prevalent in the summer months ( cases). the most frequent clinical form of anthrax was the cutaneous anthrax ( cases), and only one patient achieved anthrax meningitis. under specific treatment with penicillin g, all the patients with cutaneous anthrax recovery complete, only the patient with anthrax meningitis died. conclusions: in constantza county, anthrax remained sporadic in the last years. after a free period ( ) ( ) ( ) cases of coutaneous anthrax were registered in . a good cooperation between veterinary and physicians and individual awareness of the materials is the key to prevention of anthrax. hemolytic activity. all of the strains tested have hemopeptic activity, which is indirect evidence of their virulence. the strains have no phosphatase enzymes or lecithinase. the strains we studied ferment glucose, sucrose, glycerin, and rhamnose, and they form acid without gas. the strains all grew in synthetic "a" medium, although growth was weak. when l-tyrosine, ltryptophan, l-threonine, and l-methionine were added to the synthetic "a" medium, the pathogen grows in the typical manner. two anthrax strains are moderately resistant to rifampicin (mic . - ), and three are not sensitive. the anthrax strains are highly sensitive to tetracycline, benzylpenicillin, gentamycin, and oxacillin. conclusions: regardless of their source of isolation and time of storage, the anthrax strains isolated during outbreaks are virulent, spore-forming, and capsule-forming cultures. this study confirms that virulent forms of b. anthracis are still circulating in kazakhstan. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency under the project «kb - -al- » and administered by u.s. crdf. the importance of laboratory methods in diagnosing anthrax in humans l. lukhnova, a. aikimbayev, t. meka-mechenko, g. temiraliyeva, s. zakaryan, m. sagatova (almaty, kaz) objectives: isolation of anthrax bacillus using bacteriological methods is slow and frequently misleading. the literature suggests anthrax bacillus is isolated in % to % of patients infected with bacillus anthracis. the pathogen quickly perishes or transforms into atypical forms. the number of bacilli in a clinical specimen is often low or below the sensitivity of culture. this suggests other methods would be useful in establishing the diagnosis of anthrax, specifically; serological methods would be beneficial in diagnosing infection with bacillus anthracis. methods: we conducted studies to assess various methods for diagnosis of bacillus anthracis infection in patients. we reviewed archival data and our own results on pathological material from patients infected with bacillus anthracis in the southern and eastern kazakhstan oblasts. results: the most informative test for detection of anthrax in patients was the delayed type hypersensitivity (dth) or dermatoallergic test with anthraxin. eighty one per cent of patients with signs and symptoms of anthrax gave a positive reaction with anthraxin. the ascoli reaction complete with scab was positive in . % of patients, and the indirect hemeagglutination (iha) test was positive in . % of patients. the clinicoepidemiological diagnosis of anthrax was confirmed with culture in % of cases. serum samples from patients diagnosed with anthrax in the eastern kazakhstan oblast were evaluated using iha in - . high titres of specific antibodies ( : - : ) were detected in patients with dermatologic anthrax lesions from which anthrax was isolated. conclusion: our data suggest the anthraxin dth test and iha are good methods for establishing a diagnosis of anthrax. cultures for anthrax bacillus were the least efficient method for establishing the diagnosis. when results of laboratory tests are negative or inconclusive, one still has to rely on the clinicoepidemiological diagnosis. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency (dtra) under the project «kb - -al- » and administered by u.s. civilian research and development foundation (crdf). objectives: the fluoroquinolones are bactericidal agents frequently used in the treatment of respiratory tract infections. the viridans streptococci (vgs), although associated with disease, generally exist as part of the normal human oral flora. as such, they may exposed to antibiotics with greater frequency than oppurtunistic pathogens. preliminary data suggest that the vgs may act as a reservoir for fluoroquinolone resistance in s.pneumoniae through horizontal transfer. here, we compared the killing kinetics of moxifloxacin (mxf) and levofloxacin (lev), against vgs at simulated saliva concentrations. methods: eight strains of vgs, s. mitis ( with parc mutation) and s.oralis, ( with parc mutation) were challenged with mxf and lev in a pharmacodynamic model utilizing mueller-hinton broth supplemented with % lysed horse blood. cultures were inoculated at a density of · cfu/ml, incubated at ordm;c, and examined for viable growth at , , , , , , and hrs after exposure to the antibiotics at concentrations simulating salivary levels. salivary concentrations of mxf ( mg), and lev ( mg) were . and . lg/ml respectively. protein binding of mfx and lev were taken to be % and %. adjusted salivary levels were . (mxf) and . (lev) ug/ml. pcr was used to amplify parc and gyra, and mutations detected by dna sequencing. results: both mxf and lev were highly bactericidal against susceptible strains of vgs at salivary concentrations. mfx typically eradicated the vgs by hours and lev by - hours. for parc mutants, both mxf and lev were consistently bactericidal resulting in eradication after hours for mxf and hours for lev. regrowth was observed in both strains of s.mitis (parc) exposed to lev ( of experiments). the mic of these strains to lev increased fold and dna sequencing revealed the selection of a gyra mutation in each strain. no regrowth was detected in the s. oralis strains. conclusions: both mxf and lev are extremely active against susceptible vgs at concentrations of drug found in saliva, however, superior killing kinetics were consistently observed with mxf. mxf consistently eradicated strains with a parc mutation, however, regrowth was observed on occasions with lev suggesting that mxf may be less likely to drive resistance among vgs. again, this may have implications due to the ability of vgs to exchange its genetic determinants with s. pneumoniae. moxifloxacin and ciprofloxacin uptake by human neutrophils and their influence on viability, phagocytosis, oxidative burst, and chemotaxis a.m. berg, j. altrichter, b. drewelow, r.g. mundkowski (rostock, d) objectives: the influence of antibiotics on the immune defence is of increasing interest. the purpose of this study was to set up a cellular model for correlating the intracellular concentrations of the fluorochinolones moxifloxacin and ciprofloxacin with selected cell functions of early immune response. a human promyelotic cell line which is used for sepsis treatment by the extracorporeal immune support system (eissÒ, teraklin gmbh) was chosen to establish the model. the second stage involved native human polymorphonuclear neutrophiles (pmns) as target cells. methods: the promyelotic model cell line was differentiated towards neutrophils with all-trans retinoic acid, human pmns were isolated using dextran sulfate sedimentation and density gradient centrifugation. the cells were incubated with antibiotic concentrations within the therapeutic range and above ( . - cmax) for time periods up to hours. the extra-and intracellular concentrations were determined by hplc-uv. the viability of the cells was controlled using the trypanblue-exclusion assay. phagocytosis and oxidative burst were assessed by a combined flow cytometric assay after stimulation of the cells with serum-opsonised fluorescein-isothiocyanate-conjugated e. coli in the presence of dihydroethidium as an indicator of superoxid production. a two-compartement chamber system was used to study the influence of either fluorochinolone on the chemotaxis. results: with either cell type, moxifloxacin accumulated rapidly with peak concentrations within min whereas intracellular maximum concentrations of ciprofloxacin were achieved after h. the accumulation rate of both antibiotics inside the native pmns was more than twice as high as in differentiated eiss Ò cells. neither moxifloxacin nor ciprofloxacin showed a significant influence on viability and the immune response parameters phagocytosis and oxidative burst of native pmns; chemotaxis ability was reduced to a minor degree.viability and phagocytosis of the diffentiated eiss Ò -cells remain unaffected whereas the oxidative burst appeared decreased. chemotactic acticity of eiss Ò -cells could not be stimulated. conclusions: a significant accumulation of moxifloxacin and ciprofloxacin was determined in both cell types. however, no relevant influence on viability, phagocytosis, oxidative burst, and chemotaxis was observed. hence no hints were found suggesting a restricted use of moxifloxacin and ciprofloxacin for immunocompromised patients. prospective, multicentre in vitro study to determine resistance rates and comparative activity of moxifloxacin vs. clinical bacterial isolates from patients with respiratory tract infections (moxiaktiv study) a. dalhoff, g. korfmann, e. jacobs (kiel, leverkusen, dresden, d) objective: to determine the prevalence of resistance in current clinical isolates of s. pneumoniae, h. influenzae, m. catarrhalis, s. aureus and k. pneumoniae as well as the in vitro susceptibility to moxifloxacin and other iv antibiotics in germany. methods: up to isolates of each of the above-mentioned species were cultured on standard media and tested for in vitro susceptibility using etestÒ in each of the study centres in germany. for validation, an atcc control strain was included for each species. drugs tested were moxifloxacin, levofloxacin, amoxicillin/clavulanate, cefuroxime, clarithromycin and penicillin g. the latter two were not tested for klebsiellae. s. aureus was additionally tested for oxacillin resistance and k. pneumoniae was tested for production of extended spectrum beta-lactamases (esbl). din breakpoints were used where applicable. results: overall, pathogens of , hospitalised patients with respiratory tract infections were analysed. results for moxifloxacin are summarized in table . of s. pneumoniae isolates . % were susceptible to penicillin g and . % to clarithromycin. with an mic of mg/l, the in vitro activity of levofloxacin was markedly lower than that of moxifloxacin. h. influenzae showed almost % susceptibility to fluoroquinolones, but only . % to clarithromycin (mic mg/l). as beta-lactamase production is common among h. influenzae isolates in germany, amoxicillin/clavulanate showed far higher susceptibility rates than penicillin g ( . % vs. . %). due to a high rate of beta-lactamase production only . % of moraxella isolates were susceptible to penicillin g, compared with . % to amoxicillin/clavulanate. moraxella isolates were fully susceptible to the fluoroquinolones. overall, . % of klebsiella isolates produced an esbl. interestingly, esbl prevalence was . % in eastern germany, but below % in western germany. esbl producers of klebsiella were less susceptible to antibiotics other than cefotaxime or ceftazidime than non-esbl producers. conclusion: for all species tested, the fluoroquinolones achieved the highest overall susceptibility rate ( . %) compared to the other antibiotics (penicillin g: . %, clarithromycin: . %, ampicillin/clavulanate: . %, cefuroxime: . %). moxifloxacin showed a high activity against current respiratory pathogens in germany and was the most active fluoroquinolone, in particular against gram-positive pathogens. objectives: drug combinations have become the best choice in treatment of serious infections with resistant microorganisms including p. aeruginosa (pa). we tested ciprofloxacin (cip) in double and triple combinations with ceftazidime (caz), imipenem (imp), piperacillin (pip), and amikacin (amk) against mdr clinical isolates of pa. methods: the mic for each drug alone was determined by broth microdilution technique described by nccls. double and triple combinations of cip with other drugs were tested by using -well plates and by time-kill assay. rows a to g were activity of moxifloxacin on s. pneumoniae (sp), h. influenzae (hi) and m. catarrhalis (mc) clinical isolates collected from the respiratory tract by determination of the mic's using the etest and to compare these results against other antimicrobial agents.these results were also compared for sp versus those of the previous study (winter periods - ) for interpretive criteria s -i -r (according the nccls -breakpoints). methods: belgian university and non-university medical microbiology laboratories participated during the past winter period in this multicentre in-vitro evaluation of moxifloxacin. each laboratory included strains of sp and strains of hi and mc. the total number of evaluable strains (with the exclusion of duplicate isolates) was . testing conditions: method and antibiotics: susceptibility testing was performed in the participating centres by using the e-test. the antibiotics tested were penicillin, ampicillin, amoxi/clav., doxycycline, clarithromycin, cefuroxime, ceftriaxone, ciprofloxacin, levofloxacin and moxifloxacin.medium : sp and mc : mueller-hinton agar with % blood; hi : htm agar. inoculum : direct suspension and . mc farland standard.incubation : °c for hours in % co . quality control was performed using atcc reference strains (s. pneumoniae atcc ; h. influenzae atcc ). results: see graphs and . conclusions: this follow-up belgian multicentre study confirms the excellent in-vitro potency of moxifloxacin against clinical isolates of sp, hi and mc and it demonstrates that it is the most potent fluoroquinolone available in belgium for the treatment of lower respiratory tract infections. according the nccls -breakpoints, there are no major s -i -r differences for sp between these results ( ) ( ) and the results of the previous study ( ) ( ) ( ) . this means that the use of new fluoroquinolones like moxifloxacin and levofloxacin has not led, to date, to an increase of resistance in sp. however, repeated follow-up surveys will continue to be needed in order to assess the activity of fluoroquinolones and to monitor the ecological impact of the recently increased fluoroquinolone usage for respiratory tract infections in belgium in the forthcoming years. objectives: the objective of several experiments was to evaluate the in vitro activity of moxifloxacin against porphyromonas gingivalis (p.g.). methods: mics of moxifloxacin against strains of p.g. were determined by e-test (ab biodisk, solna, sweden). furthermore the spontaneous mutation rate and the induction of resistant strains by the . -fold mic of the antibiotic were determined. to find the target of resistance fragments of gyra and gyrb were sequenced. finally the efficacy of moxifloxacin against p. gingivalis atcc considering special conditions such as effects on the strain in a biofilm or within epithelial cells (kb cells) was evaluated. results: moxifloxacin had very low mic values (ranging from . - . lg/ml), but subinhibitory concentrations of the fluoroquinolone induced very fast mutations. the spontaneous mutation rate was up to · ) after the two-fold mic and . · ) after the eight-fold mic. often the mutants exhibited a high resistance mics > mg/l. all these mutants bore ser- ->phe substitution in gyra. the -fold mic eliminated p.g. atcc within biofilms after h, and the -fold mic was able to kill all intracellular p. gingivalis within kb cells. conclusions: moxifloxacin showed a very good activity against planctonic p. gingivalis and p.g. within a biofilm. this antibiotic in a higher concentration was also efficient to intracellular bacteria. but a rapid development of resistance was observed in laboratory. moxifloxacin might be an alternative in the antibiotic treatment of p. gingivalis associated periodontitis, nevertheless clinical studies should focus not only on improvement of clinical parameters but also on occurrence of resistant strains. levofloxacin activity on respiratory isolates of streptococcus pneumoniae with decreased susceptibility to ciprofloxacin in spain j. garcía-de-lomas, j.l. juan-bañ ó n, c. garcía-rey, r. dal-ré on behalf of the spanish surveillance group for respiratory pathogens objectives: to test the activity of levofloxacin against recent respiratory isolates of s. pneumoniae with reduced susceptibility to ciprofloxacin (mic ‡ mg/l; n = ) belonging to the national surveillance sauce- (n = ; nov -oct ) and to identify chromosomal mutations in isolates with a levofloxacin mic ‡ mg/l. methods: the activity of levofloxacin was tested by broth microdilution following nccls recommendations. genotypic sequenciation of the qrdr (gyra, gyrb, parc and pare) was done on all the strains with a levofloxacin mic ‡ mg/l. results: levofloxacin mic distribution, mic and mic (in bold) are given in the table for the whole sample and broken into ciprofloxacin mic categories. mutations in gyra (e k) were found in ( . %) strains. no gyrb mutations were found. mutation in parc occurred in % these strains ( s f; s y; s i; s r; d ; d n). finally, mutation of pare was found in ( . %) strains and all of them had the change i v. one single parc mutation occurred in ( . %) strains, ( ) ( ) ( ) ( ) ; > . % were at £ . mg/l conclusions: gemi continues to be a highly potent fq when tested against the two most commonly isolated gram-negative carti pathogens; h. influenzae and m. catarrhalis. this potency and spectrum was consistent across six years and three continents. norfloxacin as a marker of decreased susceptibility to fluoroquinolones in enterobacteria using the vitek system l. ló pez, j. alcala, f. torres, e.j. perea, a. pascual (seville, e) objective: the detection of nalidixic acid resistance acquires importance in enterobacteria since already resistant strains develop more frequently resistance to fluorquinolones than susceptible strains. many clinical laboratories have incorporated the vitek system because their benefit in routine, but nalidixic testing is not included for enterobacteria cards. the aim of this study was the use of norfloxacin vitek mics value as alternative marker to detect nalidixic acid resistant enterobacteria strains. material and methods: a total of isolates of enterobacteriaceae were analyzed and routinely inoculated into the vitek id-gnb and ast-n cards (biomérieux) and then loaded into the vitek system, as recommended by the manufacturer. ninety-six strains with a higher norfloxacin vitek mic value ( - mg/l), but still within the susceptibility range established by the nccls breakpoints, and strains with a norfloxacin vitek mic £ . mg/l were selected. they were further tested with a microg-nalidixic acid-disk (nal) (oxoid) according to nccls reference. results: the norfloxacin - mg/l and susceptible to ciprofloxacin phenotype represented the % of total isolates loaded in the vitek system during the study, similar to the prevalence observed in previous analysis in spain. both selected groups, one with the highest and the other with the lowest norfloxacin vitek mic value, included % and % e. coli isolates, % and % s. enterica isolates, respectively. among the norfloxacin vitek mic - mg/l strains isolates ( %) were nal resistant, and ( %) were ciprofloxacin susceptible. the agreement between norfloxacin vitek mic £ . mg/l and nal susceptibility was foun in ( %) strains. two isolates (one e. cloacae and one k. pneumoniae) with norfloxacin vitek mic - mg/l were nal-s and isolates (two e. coli and one p. mirabilis) with norfloxacin vitek mic £ . mg/l were nal-r. conclusion: when using vitek system for enterobacteria, the consideration of mic of norfloxacin > . mg/l could be an alternative marker of nalidixic acid resistance (and decreased susceptibility to fluoroquinolones). antibacterial susceptibility studies -i objectives: p. aeruginosa is an important nosocomial pathogen, which causes serious and often lethal infections in immunocompromised hosts. this pathogen is intrinsically resistant to many antibiotics and can easily develop resistance towards many currently available agents. natural resistance can be attributed to the low permeability of the p.aeruginosa outer membrane to a variety of antibiotics including glycopeptides (glys). since glys are powerful antibiotics against grampositive bacteria and resistance is very rarely develop, it seemed interesting to evaluate the effect of combining these antimicrobial agents with antibiotics that might disorganize the structure of the outer membrane allowing the entrance of glicopeptides into the gram-negative cells. in order to verify this hypothesis, ceftazidime (caz) has been tested in association with vancomycin (van) or teicoplanin (tei). the same experiments have been carried out also in the presence of azithromycin (azi), which is normally a non anti-pseudomonal agents but has been shown to interfere with some virulence factors. methods: a bacterial suspension of about cfu/ml was seeded on plates containing a fixed concentration of glys ( mg/l) and increasing doses ( x, x, x, x) of caz. survivors were counted after hr at °c. results were interpreted as synergism ( %), additivity ( %), and indifference ( %) of the cfu/ml reduction found in the drugs combination in comparison to the drug alone.the same experiments have been repeated adding azi ( mg/l) and using glys at concentrations ranging from to mg/l. results: caz in combination with glys reacted synergically in out of cases, additivity was found in / interactions and indifference was noted in / tests. preliminary results ( tests performed) indicated that the addition of azi increased the incidence of synergisms and additivities even when using glys concentration of mg/l. conclusions: caz combined with glys gave additive or synergistic results in the great majority of experiments, while the simultaneous combination of azi, caz and a gly always produce an additive or synergistic effect against p. aeruginosa. these data, given the high concentration of glys employed, could be of particular interest in clinical situations where the drugs could be topically administered. a multicentre correlation study of vitek with reference methods for telithromycin, mupirocin, and daptomycin against staphylococcus spp. k.e. aldridge, a. dizney, k. thomson, g. procop (new orleans, omaha, cleveland, usa) objective: clinical studies have shown that appropriate antimicrobial therapy based on in vitro susceptibility data correlates with better patient outcome. vitek is a fully automated susceptibility testing methodology providing rapid( - h) results for a variety of aerobic and facultatively anaerobic gram-positive and -negative pathogens which have been shown to correlate to established reference methods. the objective of this study was to correlate the susceptibility results of vitek testing of telithromycin, mupirocin, and daptomycin against staphylococcus spp. with those from reference methodology. methods: three geographically distinct clinical microbiology laboratories participated in the testing with each site testing consecutive clinical islates and blinded challenge isolates of staphylococci sent to each site.vitek testing was performed according to the manufacturer's recommendations. the challenge isolates were also tested using a manual preparation of the inoculum. nccls recommended reference agar and broth microdilution methods were used to test telithromycin, mupirocin, and daptomycin. mic results were collated to determine the per cent (%) of isolates susceptible ( in vitro efficacy of tigecycline tested against community-acquired respiratory tract infection and nosocomial pneumonia pathogens conclusions: among r subsets of commonly occurring pathogens, % were inhibited by £ mg/l of tig and > % were inhibited by £ mg/l (the current nccls breakpoint for tet). tig is highly stable to most tet-r determinants, including protected ribosomes and efflux mechanisms, and may represent a superior choice among parenteral agents for broad-spectrum coverage, including the most commonly occurring-and problematic-r phenotypes. determination of the minimum inhibitory and mutant prevention concentration of telithromycin against clinical isolates of streptococcus pneumoniae s. borsos, c. hesje, l. blondeau, j. blondeau saskatoon, can objective: telithromycin (tl) is a novel ketolide antimicrobial agent that has recently been approved for use in north america for respiratory tract infections of which sp is a principal pathogen. the global escalation of penicillin (p) and macrolide (m) resistant sp has compromised the use of beta-lactam and macrolide compounds. tl was reported to be active against p and m resistant sp. objectives: garenoxacin (grn) is an investigational des-f( )quinolone with enhanced in vitro activity against gram-positive cocci. the global escalation in antimicrobial resistance amongst respiratory tract pathogens -particularly sp-has highlighted the importance of quinolone compounds for treating sp infections. the mutant prevention concentration (mpc) is a novel in vitro measurement that defines the drug concentration threshold that would require an organism to simultaneously acquire two or more resistance mutations for growth in the presence of the drug. we measured the mics and mpcs for grn against clinical isolates of sp, including multidrug resistant strains and those with elevated mpcs to levofloxacingrn. methods: microbroth dilution in accordance with nccls guidelines was followed for mic testing using todd-hewitt broth and two-fold concentration drug increments. for mpc testing, ‡ billion organisms were applied to agar plates containing grn, incubated at - °c and % co and screened for colony growth at and hours. the mpc was the lowest drug concentration that completely prevented growth. select organisms with grn mpc values > lg/ml were screened for amino acid substitution in the quinolone resistance determining region. great concern nowadays because the therapeutical alternatives are limited. fusidic acid inhibits protein synthesis. it has only a few side effects and is usually well-tolerated by patients as an oral drug. fusidic acid can be considered as an alternative drug for the treatment of infections due to both methicillin susceptible and resistant s. aureus strains. evaluation of synergy between glycopeptides, levofloxacin and beta-lactams against methicillinresistant staphylococcus aureus (imi) + va/tp, lvx + piperacillin/tazobactam (ptz) + va/tp] were considered. synergy was evaluated by means of checkerboard assay against (double combinations) and (triple combination) mrsa strains isolated from respiratory tract infections and by means of time kill curves ( strains). in checkerboard assay synergy was defined as fractional inhibitory concentration index (fici) of < . , additivity as fici > . and > , indifference as fici > and antagonism as fici > , while in time kill studies synergy was defined as a > log decrease in bacterial count of combinations in respect to the most active single drug. moreover, mutational rates of single and combined drugs at antimicrobial concentrations equal to the resistance breakpoints were calculated in strains results: synergy and additivity were the prevalent effects, while no antagonism was observed in checkerboard assay. in particular, lvx + va/tp and ctx + tp/va gave synergy in / , / , / and / strains, respectively. combinations of lvx + va + caz/cpm/ptz and of lvx + tp + caz/cpm/ptz yielded synergy or additivity in all the tested strains, with combinations of lvx + tp + caz / ptz giving synergy in / strains. time kill curves evidenced synergy for lvx/ctx + va/tp after and h of incubation. synergistic triple combinations occurred more frequently with lvx + tp than with lvx + va after and h. for single antibiotics, mutational frequencies ranged between - and < - for lvx, ctx, amk and imi, and < - for va and tp. when tested in double and triple combinations, mutational frequencies fell below - for all the combinations. conclusion: the study provide in vitro evidence of synergy between glycopeptides with fluoroquinolones, cephalosporins and beta-lactams and of limitations of occurrence of mutations in mrsa strains responsible of respiratory infections, thus underlining a potential role in therapy of such infections. comparative activity (mic and mbc) of daptomycin, quinupristin/dalfopristin, linezolid, vancomycin and teicoplanin against a large worldwide collection of visa/hvisa a. bolmströ m, a. engelhardt, Å . karlsson, e. edling, p. ho (solna, s) objective: the worldwide emergence and possible dissemination of vancomycin (va) resistance in staphylococci involving vana (vrs), visa and hetero-visa phenotypes comprise formidable clinical threats. comparisons of the in vitro activity of newer gram positive agents using larger worldwide collections of visa strains become increasingly important both for individual therapy guidance and for epidemiologic purposes. method: a country collection of staphylococci ( mrsa, mssa), of which were visa (e-test macromethod and pap-auc, walsh et al, jcm ) was assessed. mic of dp, qd, lz, va and tp were determined using the nccls broth microdilution (bmd) and e-test. mbc for all agents for a subset of visa and non-visa strains were determined using an etest mbc procedure and bmd. results: inter-method agreement (n = ) for all antibiotics were ± % ± dilution for mic and ± % for mbc results. the continuous gradient format in e-test allowed detection of subtle upward shifts in mic and mbc distributions of visa, as well as the detection of persister colonies in mbc assays seen with the less bactericidal compounds. conclusion: mic distributions showed that daptomycin and linezolid maintained potency against the large collection of visa while quinupristin/dalfopristin and glycopeptides showed a shift towards reduced activity. mbc results showed daptomycin to be superior to linezolid (bacteriostatic) and the other agents. the potential usefulness of newer agents for visa should be continually assessed with respect to both their static and cidal activities in order to detect subtle shifts in susceptibility as early as possible. performance of a unique e-test daptomycin gradient + calcium gradient on isosensitest compared to nccls broth microdilution in mueller hinton objective: daptomycin (dp) requires physiologic levels of free calcium ions (ca + ) for expression of adequate activity. susceptibility testing methods can be significantly influenced by ca + variations in agar and broth. nccls broth microdilution (bmd) uses a final physiologic level of lg/ml calcium for dp testing. e-test dp is a unique gradient which incorporates a constant level of calcium. e-test dp has been shown to be equivalent to bmd when tested with commercially available mueller hinton (mh) agar. since isosensitest (iso) is used in some parts of europe, and has an inherently low level of calcium (approximately - lg/ml), it is important to evaluate the performance of e-test dp on iso in comparison to bmd. method: a lab study compared e-test dp on iso to nccls bmd. a total of clinical and stock strains comprising clindamycin (cc), ciprofloxacin (c) and tetracycline (t) and also to determine whether there are any differences in the antimicrobial susceptibility patters among the difference species of the vgs and s. bovis. methods: the vgs and s. bovis were isolated from blood and identified at species level by id -strep system ( s. mitis, s. anginosus, s. sanguis, s. salivarius and s. mutans). mics were determined by the agar dilution method and the percentages of resistance determined according to nccls criteria for streptococci other than s. pneumoniae. results: overall, . % of the streptococci were resistant at least to one of the antibiotic tested and of these, % was resistant to or more. the most susceptible species were s. anginosus and s. salivarius ( % susceptibility to all antibiotics tested). the only s. mutans strain was susceptible to all the antibiotics tested. the resistance phenotypes for the different species are shown in the table. conclusions: as expected cc resistance was always associated with e resistance. overall, the phenotype most frequently founded was ecct in strains ( . %) and it was present in the % of s. bovis and % of s. anginosus. the pattern eccpt was the second more common phenotype ( . %) and the most frequent in s. sanguis and s. mitis with % and . % respectively. s. mitis was the specie that showed more number of phenotypes ( ). heterogeneity in the susceptibility patterns in the species of vgs indicates the need for accurate identification. in vitro antibacterial activity of temocillin against extended spectrum beta-lactamases enterobacteriaceae producing clinical isolates objectives: temocillin is a methoxy-derivative of ticarcillin, active on enterobacteriaceae and stable against b-lactamases, including ampc and extended spectrum b-lactamases (esbl). however data concerning its activity on esbl producing strains are limited. the aim of this study was describe the range of minimal inhibitory concentration (mic) of temocillin in different species of esbl producing enterobacteriaceae from a single tertiary care centre in belgium . methods: esbl producing enterobacteriaceae ( e. aerogenes, e. coli, e. cloacae, k. pneumoniae and other ) strains from patients hospitalised in our institution during the period of january to december . esbl production was screened by double-disk test and confirmed by oxoid combined disk method. characterisation of enzymes was performed by multiplex pcr for bla tem, bla shv and bla ctx-m gene families detection and by dna sequencing and/or iso-electric focusing. mics (mg/l) were determined by agar dilution method according to nccls guidelines. susceptibility was determined according to breakpoints provided by fuchs et al: susceptible mic £ mg/l. (eur j clin microbiol ) results: isolates originated from screening rectal swabs ( %), urinary tract ( %), respiratory tract ( %), wound ( %), blood ( %), gastrointestinal tract ( %), or other sites ( %) from male and female patients with a mean age of (range, - ) years. the antimicrobial activity of temocillin against esbl producing enterobacteriaceae is summarised in the table. conclusions: this study confirmed the good in vitro activity of temocillin against multiresistant esbl-producing clinical isolates of enterobacteriaceae. its activity included enterobacter ampc depressed mutant strains that co-produced multiple esbl. these data suggest that temocillin could be a valuable drug to be considered in the treatment of infections by some esbl-producing enterobacteriaceae. in vitro activity of ertapenem against cefpodoxime-resistant gram-negative bacilli from urine introduction: resistance to commonly used antibiotics for urinary tract infection (uti) is of growing concern. this is due to the emergence of extended-spectrum beta-lactamase (esbl) producing bacteria in the community with reports from different parts of the world. the appearance and dissemination of these resistant bacteria cause serious concerns for the treatment of urinary tract infections (utis). objectives: ertapenem is the latest member of the carbapenem class of antimicrobials. the aim of this study was to assess the activity of ertapenem against a collection of cefpodoximeresistant gram-negative bacilli isolated from urine samples of community and hospital-based patients. materials and methods: between june and october , our laboratory received , urine samples. of these gave a positive culture, of which from hospitalised patients and from the community. of these were gram negative bacilli resistant to cefpodoxime mg/l. the identity of the isolates was confirmed using api e or api ne (biomerieux, france). esbl production was sought using the mastdd and the identity of the resistant determinant(s) was carried out using a combination of pcr and nucleotide sequence analysis tech- different antibiotics in order to evaluate alternatives for intrapartum chemoprophylaxis to women allergic to penicillin and colonized by a gbs strain resistant to macrolides and/or lincosamides; ( ) characterize the mechanisms of resistance to macrolides and lincosamide; ( ) evaluate the clonal relationship between these strains. methods: a total of strains collected in a multicentre study: isolated between and from newborns diagnosed of early-onset gbs disease and strains collected in from vagina or rectum of pregnant women. microdilution method was used to study the antimicrobial susceptibility and disk diffusion to define the macrolide-lincosamide resistance phenotype. pcr was performed to determine the presence of ermb, ermtr and mefa resistance genes. to evaluate the clonal relationship, pfge of total dna was done using smai. results: all the strains were susceptible to penicillin, ampicillin, vancomycin, quinupristin/dalfopristin, levofloxacin and teicoplanin. the . % were resistant to erythromycin and azithromycin, . % to josamycin, . % to clindamycin, . % to telithromycin and . % to fosfomycin. among the macrolide resistant strains, the . % presented a constitutive mlsb phenotype (cmlsb), . % an inducible mlsb phenotype (imlsb) and the remaining . % a m phenotype. three strains were susceptible to macrolides and resistant to clindamycin. the ermb, ermtr and mefa genes were presented in . %, . % and . % of the macrolide resistant strains. two strains did not amplified none of the studied genes. the % of the strains harbouring the ermb gene showed a constitutive phenotype. all the strains showing an inducible phenotype harboured the ermtr gene. the telithromycin resistant strains presented a cmlsb phenotype; of them posses the ermb gene and one the ermtr. conclusion: in spain, there is a high rate of resistance to macrolides and lincosamides that makes mandatory to perform susceptibility testing to gbs strains isolated from pregnant women allergic to penicillin. in our region ermb methilase is the main cause of macrolide resistance, followed by the ermtr and the macrolide eflux pump in a low proportion. there is a wide clonal diversity among resistant strains to macrolides, lincosamides and telithromycin. detection of the ermx determinant of macrolide resistance in clinical strains of corynebacterium urealyticum a. ortiz, j.i. garcía-cía, r. fernández-roblas, j. esteban (madrid, e) objectives: to detect the presence of the ermx macrolide resistance gene in clinical isolates of c. urealyticum. methods: c. urealyticum strains were isolated from clinical samples in the fundació n jiménez díaz hospital. the strains were maintained frozen until the studies to evaluate macrolide resistance were performed. antibiotic susceptibility testing was performed by agar dilution assay in cases and by disc diffusion assay in cases. detection of ermx gene was performed by using primers cerm- and cerm- according to the protocol described by rosato et al. dna was extracted by boiling a suspension of bacteria in distilled water. amplification products were analysed by agarose gel electrophoresis and molecular weight of the amplicons were calculated by using the photocapt software (biogene, usa). results: strains were resistant to erythromycin and clindamycin, and were susceptible to both antimicrobials. we detected the gen ermx in cases (all of them resistant). macrolide-resistant strains gave negative results for ermx detection. the susceptible strains did not show any amplification product. the ermx gene is detected in . % of macrolideresistant corynebacterium urealyticum strains. however, there were . % macrolide-resistant strains in which ermx gene was not detected. other resistance determinants must be involved in macrolide resistance among corynebacterium urealyticum. characterisation of phenotype and genotype of macrolide-resistant streptococcus pyogenes objectives: the resistance of streptococcus pyogenes to macrolide is a world wide problem. the aim of present study was to determine the prevalence of antibiotic resistance rate of erythromycin (em) resistant isolates from korea, and evaluate their genetic mechanism, phenotype distribution and clonal relationship. methods: total em resistant s. pyogenes from clinical specimens from to were studied; agar dilution method to determine minimal inhibitory concentrations (mics) to antimicrobial agents was performed. resistance phenotypes were determined by triple-disc test and resistance induction experiment with sub-inhibitory concentration of erythromycin ( . lg/ml). their genetic mechanisms of resistance were determined by pcr. the genetic relatedness of them was also investigated by means of emm genotyping and random amplified polymorphic dna (rapd) analysis. results: an average em resistance rate was % and their cross-resistance rate to clarithromycin, azithromycin, and spiramycin were %, %, and %, respectively. the most common resistance phenotype was inducible mls (imls; %), followed by constitutive mls (cmls; %), and m type ( %). of imls isolates ( % ) showed novel imlsd phenotype, which had small ( - mm) inhibition zones around all three discs on triple-disc test and high level resistance to clinamycin and spiramycin after erythromycin induction. all isolates of the cmlsa and imlsd harboured ermb gene, while imlsc and m isolates harboured erma and mefa gene, respectively. all imlsd isolates were emm except one while all multidrug resistance cmlsa isolates were emm genotype. imlsd isolates made a tight cluster on phylogenetic tree and % of them showed a common pattern by rapd analysis. conclusion: imlsd was most common macrolide resistance phenotype in korea and emm gene and rapd analysis was suggestive of its clonal relationship. detection of inducible clindamycin resistance in cutaneous staphylococci clinical isolates by phenotypic and genotypic method , and ( . %) isolates with constitutive clindamycin resistance ( s. aureus and coagulase-negative staphylococci). the mrsa gene was present in all the clindamycin susceptible isolates except for cns. among the isolates with clindamycin inducible resistance, isolates possess either erma or ermc while cns isolate possess both ermc and mrsa. one cns and s. aureus isolates were negative for all the genes tested. all isolates with constitutive clindamycin resistance amplified to any of the genes tested. among the s. aureus isolates, have the erma gene, the ermc and both ermc and mrsa genes. all the cns isolates possess the ermc but of them possess also the mrsa gene (table ) . the disk-diffusion test detected all the clindamycin resistant strains, and none of the clindamycin inducible resistant strains was detected by the microscan. the ermc gen was the most prevalent among the clindamycin resistant, in both cns and s. aureus isolates. ribosomal protein l mutations in bacillus anthracis associated with cross-resistance between macrolide, lincosamide, streptogramin and ketolide antibiotics rob chemother ; : ) . the aim of this study was to determine ribosomal mutations associated with this resistance. methods: primers were designed and used to amplify and sequence the l , l riboprotein genes and the copies of the s rrna gene using b. anthracis str. ames complete genome (genbank accession no. nc_ ). results: (mics in mg/l) two significant mutations associated with resistance were found. a amino acid (aa) insertion (a tandem duplication of mgra ) into the l gene at position was found in the st- strain with a quinupristin/dalfopristin (q/d) mic of selected after passages in q/d. this strain was cross-resistant to telithromycin, erythromycin, clarithromycin and clindamycin with mics of , , and respectively. an amino acid insertion (mgramgra) into the l gene (also at position ) was found in the sterne strain with a clarithromycin mic of after passages in clarithromycin. this strain was cross-resistant to telithromycin, q/d, erythromycin and clindamycin with mics of , , and respectively. conclusions: tandem duplications of l mgra were found to be important in mlsk resistance in separate strains of b. anthracis demonstrating that this locus is important for resistance development under selective pressure of q/d and clarithromycin. importantly, these mutations were also associated with cross-resistance to other mlsk antibiotics. horizontal transfer of the mlsb resistance gene erm(b) between the human pathogen clostridium difficile and the ruminal anaerobe butyrivibrio fibrisolvens p. mastrantonio, f. barbanti, p. spigaglia (rome, i) objectives: the aim of this study was to investigate the possibility of in vitro transfer of the mlsb resistance gene, erm(b) between the human pathogen clostridium difficile and the ruminal commensal butyrivibrio fibrisolvens. methods: the erm(b)-positive c. difficile strain cd was used in filter matings as donor, whereasthe b. fibrisolvens strain . , tetracycline resistant, and the b. fibrisolvens strain r, rifampicin resistant, were used as recipients. the strains were cultured in m gsc broth, with % of sterile rumen, in anaerobic conditions and the filter matings were performed on sheep blood agar plates, supplemented with hemin ( . %) and vitamin k ( . %). the same medium, supplemented with erythromycin ( mcg/ml) and tetracycline ( mcg/ml), were used to select the transconjugants. mic values were assessed by e-tests. the erm(b) transfer was confirmed by pcr and by hybridisation assays, using an erm(b) probe on transconjugant genomes digested with pvuii and with smai (pfge). results: the transfer average frequency was · ) . mic values for erythromycin were > mg/l both in donor and transconjugants. an internal fragment of the erm(b) gene ( bp) was amplified in all the transconjugants and a specific band was visualised in hybridisation assays on transconjugant genomes digested with both pvuii and smai. the re-transfer of the erm(b) gene from b. fibrisolvens transconjugants to c. difficile , an erythromycin susceptible strain, was also obtained. conclusion: the in vitro transfer of the mlsb resistance gene erm(b) from c. difficile to b. fibrisolvens and vice-versa, is an important result supporting the possibility that horizontal transfer of resistant genes between human pathogenic bacteria and animal commensal microorganisms could occur, under natural conditions, more easily than expected. candida spp. attach on polymers, create a biofilm protecting the yeast and pose a reservoir for entering the bloodstream. candida spp. in biofilm are less susceptible to the antifungal drugs currently in use. we investigated the in vitro antifungal activity of micafungin (mcf) against biofilms of c. albicans, c. parapsilosis, c. dubliniensis, c. tropicalis, c. glabrata and c. kefyr. methods: biofilm formation was performed in vitro on polystyrene -well plates and on hickman catheter discs with minor modifications to kuhn ( ). mcf was tested at six concentrations ( . - lg/ml). measurements were done by xtt reduction assay at nm, % inhibition was calculated in comparison to the growth control. the biphasic structure of biofilms were imaged by reverse light-through microscopy. results: on polystyrene plates and on hickman catheters all candida spp. produced intermediate biofilm after - h incubation. at all concentrations on polystyrene plates micafungin reached at maximum % inhibition in biofilm against all candida spp.: c. albicans ( % at lg/ml), c. parapsilosis ( % at lg/ml), c. dubliniensis ( % at lg/ml), c. tropicalis ( % at lg/ml), except for c. glabrata ( % at lg/ml) and c. kefyr ( % at lg/ml) methods: this multicentre, randomized, double-blind study compared mem with ipm (both mg iv every hours) in patients hospitalized with csssi. the primary efficacy endpoints were clinical and bacteriological responses at follow-up in the fully evaluable (fe) patient population (all patients meeting eligibility criteria and who had an identified pathogen prior to treatment) polymicrobial infections were seen in % of all documented infection s. agalactiae (n = ; % s to mem and ipm), and e. faecalis (n = ; % s to mem and % s to ipm) pseudomonas aeruginosa (n = ; % s to mem and % s to ipm), bacteroides spp. (n = ; % s to mem and % s to ipm), and peptostreptococcus spp % (mem) and . % (ipm) in patients with polymicrobial infections; bacteriological (documented or presumed eradication, colonization) response rates were we used a cox proportional hazard (cph) model adjusting for potential clinical and microbiologic risk factors, to evaluate los. results: among the modified intent-to-treat patients, the most common infection diagnoses at baseline were cellulitis ( . %) and major abscess ( . %). the most common underlying etiologies were spontaneous infection ( . %) and trauma ( . %). about . % of patients had diabetes; . % had peripheral vascular disease. staphylococcus aureus was the most common causative pathogen (about % of patients) among patients with confirmed microbiology and complete hospitalization data. however, about % of patients had a gramnegative causative pathogen among patients with confirmed microbiology, staphylococcus aureus was the most common causative pathogen but varied significantly in prevalence across regions (about % of patients overall, % in the us, to % in europe and asia, and % in south africa). about assigned for the drugs as follows: row a for cip alone, b for second drug, c for third drug, d for cip + second drug, e for cip + third drug, f for second drug + third drug and g for the three drugs together. row h was left for drug-free control. briefly, all wells were filled with ll of cation adjusted muller hinton broth. the drugs at x of the highest tested concentrations in ll of the medium (alone, in double or in triple combinations) were delivered to wells a -g . two-fold serial drugs. results: the best synergistic effects were observed in combination of amk with caz ( %) or pip ( %) and in combination of caz with pip ( %) or cip ( %) as quinolones are widely used for treating sp infections and quinolone resistance is a concern, we tested gm-a newly approved compound-by mic and mpc against ps and prsp. methods: mic testing was by microbroth dilution in accordance with nccls guidelines. for mpc testing, billion organisms were applied to agar plates containing drug and read at and hours following incubation. the lowest concentration preventing any growth was the mpc. organisms with high mpc values ( ‡ lg/ml) were screened for amino acid substitution in the quinolone resistance determining region. results: a total of clinical sp isolates were tested: ps, penicillin intermediate (pi) and pr. the following represents the mic / (lg/ml) and range (lg/ml) respectively against pr tr) objective: to determine the in-vitro activities of moxifloxacin and ciprofloxacin against methicillin-susceptible s. aureus (mssa) and methicillin-resistant s. aureus (mrsa) strains. methods: the study was conducted in a university hospital in turkey. a total of non-repeat isolates ( mssa and mrsa) from various clinical specimens were included in the study. all isolates were identified as s. aureus by phenotypic characteristics, gram staining, catalase and tube coagulase tests. mics of the drugs were determined by an agar dilution method, according to the nccls criteria. the drugs were obtained from their manufacturer (bayer inc). s. aureus atcc was used as quality control strain. results: twenty-seven ( %) of mssa strains and ( %) of mrsa strains were resistant to ciprofloxacin. mic and mic values of ciprofloxacin-resistant mssa strains for moxifloxacin were . mg/l and mg/l, respectively. mic and mic values of ciprofloxacin-susceptible mssa strains for moxifloxacin were £ . mg/l and . mg/l, respectively. moxifloxacin was -fold more active than ciprofloxacin against mssa strains. there was not any difference between these two quinolones against mrsa strains (mic s: ‡ mg/l). conclusion: mrsa strains are major causes of nosocomial infections. despite advances in antibacterial therapy, treatment of infections caused by mrsa is still troublesome. new quinolone-derived compounds such as moxifloxacin are reported to have improved activities against s. aureus including mrsa. in this first study on in-vitro activity of moxifloxacin against s. aureus from turkey, the moxifloxacin mics were higher than those reported from different countries objectives: a multicentric study during the winter period of - was performed in belgium to assess the in-vitro two mutations in ( . %) ( parc+gyra and parc+pare), and mutations in parc+pare+gyra in ( . %) strains. conclusions: ) levofloxacin mic and mic for pneumococcal strains with decreased susceptibility to ciprofloxacin were . and mg/l, respectively. resistance to levofloxacin (mic ‡ ) occurred in isolates ( . % of the sauce- collection). ) activity of levofloxacin was negatively affected only among pneumococcal isolates with a very high mic of ciprofloxacin ( ‡ mg/l). in this case, levofloxacin mic and mic were and , respectively, and . % of these strains were resistant to levofloxacin. ) all sequenced strains with a levofloxacin mic ‡ mg/l had a mutation in the parc gene (single in % strains), but none of them in the gyrb. the most common mutations identified were parc/s f, pare/i v, parc/s y and gyra/e k. ) one single parc mutation was organism/year (no. tested) gemi cipro levo gati moxi / (na) methods: the organisms numbered , , a total of , from np and , from carti. the most frequent carti pathogens were: s. pneumoniae (spn; , ), h. influenzae (hi; , ) and m. catarrhalis (mcat; ) usa) objective: to evaluate the activity and potency of tigecycline (tig) when tested against a large international collection of common bacterial pathogens displaying increasing and worrisome resistance (r) profiles. tig is a novel glycylcycline derivative of minocycline that has demonstrated activity against a variety of gram-positive and -negative pathogens mic / (mg/l) strains) were collected from to in > medical centres participating in the global tig surveillance program results: tig results for the r organism subsets are in the table: tig was highly active (mic and , £ . and £ . mg/l, respectively) against all resistant sa, cons, esp, spn and vgs with > % of strains inhibited by £ mg/l. while potency of tig against r subsets of ent was less (mic and , £ . and £ mg/l, respectively), the vast majority of tet-r isolates remained s to tig (> % of isolates were inhibited by £ mg/l). no geographic differences in tig potency among esbl-producing, cip-r or tet-r ent strains were noted. grn drug concentrations in serum would be expected to remain above the mpc value for > hours. conclusion: grn exhibits potent activity against sp, as measured by both mic and mpc, and is active against strains with high mic/mpc values for other quinolones. grn is less likely to select for quinolone resistant sp isolates s. agalactiae ( ) and s. dysagalactiae ( ) were used. interlaboratory reproducibility was evaluated using bias/precision strains tested at all sites and nccls qc strains were used to compare e-test dp on iso values to nccls mh values. the various methods were used according to standard recommendations. nccls susceptible breakpoints ‡ lg/ml for staphylococci and streptococci and ‡ lg/ml for enterococci were used as is and as adjusted upwards by dilution. results: percentage essential agreement (ea), category agreement methods: e test strips were used to establish the fici of the antibiotic combination pairs. combination testing was carried out on strains of pseudomonas aeruginosa (pa), strains of burkholderia cepacia (bc) and strains of stenotrophomonas maltophilia (sm) from cf patients. , and combination pairs were tested between and times against pa, bc and sm respectively. the fici results were categorised as synergistic (fici £ . ), additive (fici = . to £ . ), indifferent (fici = . to £ . ) and antagonistic (fici > . ). results: combinations were tested. synergy was found in . % of pa, . % of bc and . % of sm. . %, . % and . % of pa, bc and sm respectively demonstrated an additive effect. . % of pa, . % of bc and . % of sm demonstrated an indifferent effect. antagonism was found in . %, . % and . % of pa, bc and sm respectively results: of the isolates resistant to cefpodoxime, were positive for esbl production and the enzymes produced were: tem- , shv- , shv- , ctx-m- , ctx-m and ctx-m singly or in combination. ertapenem proved active against all enterobacteriaceae regardless of production of esbls (mic range . - . mg/l). for acinetobacter sp giamarellou on behalf of the hellenic study group for the susceptibility of streptococcus pneumoniae results: of the strains studied, were ery s and were ery r. all strains but two (mic and mg/l) were susceptible to tel. in particular, results for telithromycin for the ery s strains, showed an mic range for tel between . - . , with mic of . and mic of . . for ery r in total, mic and mic were . and . (range . - ). with respect to macrolide resistance phenotype, the distribution per type was: m . % (ery mic range - ), and cmlsb and imlsb (ery mic range to ( ) % and . % respectively. the two strains resistant to tel displayed the cmlsb phenotype (ery mic ( ). for each different macrolide resistance phenotype no isolates demonstrated an inducible mlsb phenotype by this method. forty-nine of these carried the ermam gene ( . %) and three carried both ermam and mefe genes ( . %). eighty-seven isolates ( . %) had the m phenotype (macrolide resistance) and all of these contained the mefe gene. conclusions: our findings are in contrast to european studies and to previous greek studies, where the erm gene prevailed or at least equaled the prevalence of mef gene. this is the first report of isolates carrying both ermam and mef genes in greece. knowledge of the resistant mechanisms to macrolides is crucial for the empiric antibiotic treatment of community acquired infections. p antibiotic susceptibility, mechanisms of macrolide resistance and clonal relationship in group b streptococci microbiological characteristics of anthrax strains l. lukhnova, g. temiraliyeva, t. meka-mechenko, y. pazylov, s. zakaryan, j. denissov (almaty, kaz)objectives: the kazakh science center for quarantine and zoonotic diseases (kscqzd) has a collection of pathogens of especially dangerous infections that have been isolated in kazakhstan. by kazakh government resolution, the kscqzd's collection is the official collection of pathogenic bacteria for kazakhstan. kscqzd has strains of b. anthracis that were isolated in various years . methods: we studied the biological properties of anthrax strains from our collection: from humans, from soil, from sheepskins, from sheep organs, and from the external environment. we tested the anthrax strains using standard identification tests. results: the growth of the bacillus anthracis strains on the hottinger's agar was typical (r -form), and no distinctions based on the origin of the strains were noted. all of the strains form spores and are immobile. when stained with specific antibody directed toward anthrax antigens, all strains stained positive. three of the b. anthracis strains did not have capsules when originally studied, but after cultures on the hottinger's agar, the strains' ability to form capsules in a co atmosphere was restored. the anthrax strains are sensitive to anthrax bacteriophage and penicillin. the strains have lipolytic and background: we studied the application of pk/pd modeling techniques as an alternative method to define pharmacodynamic breakpoints complementary to conventional microbiological breakpoints for fluoroquinolones (fqs). methods: staphylococcus aureus strains with mics ranging from . - mg/l were exposed to fluctuating concentrations of levo-(lfx) gati-(gfx) and moxifloxacin (mfx) mimicking their total serum concentration profiles following oral doses of mg mfx, mg gfx, and mg lfx over h using the sedimentation model. strains were cultivated in brain heart infusion broth at °c. viable counts were quantitated at sampling points. as pd-measure the aubkcnorm (i.e. area under the bacterial kill curve normalized to the initial inoculum [h] ; aubkcnorm < indicates a bactericidal activity, > bacterial growth) was determined for the time kill experiment and plotted as a function of mic for each treatment. results: the aubkcnorm vs mic function was similar for all treatments following a bimodal profile. while the slope of all fqs studied was shallow for mics below mg/l with au-bkcnorm < h, aubkcnorm started increasing steeply beyond this point. it intercepted the cut off line at concentrations of . mg/l for lfx mg, mg/l for gfx, . mg/l for mfx and . mg/l for lfx mg. this indicates that s. aureus with susceptibilites beyond these mics would no longer be eradicated by the corresponding treatments. thus, according to our investigations a breakpoint for fqs integrating both microbiological and clinical pharmacokinetic relation-ships results in a value of mg/l for lfx mg, and mg/l for mfx, gfx, and lfx mg. conclusion: pharmacodynamic breakpoints determined by pk/pd m+s which are taking into respect the fluctuating concentration time profiles encountered in patients are suitable surrogates complementing the data obtained in microbiological experiments. a multicentre agar and broth dilution susceptibility testing study of fluoroquinolones against the bacteroides fragilis group k.e. aldridge, d. snydman, d. hecht, c. edlund, j. herrington (new orleans, boston, chicago, usa; stockholm, s; west haven, usa)objectives: the development of fluoroquinolones (fq) antimicrobials has resulted in effective therapy for a variety of infections due to aerobic and facultatively anaerobic grampositive and -negative pathogens. fq activity against anaerobes has not been as promising with only trovafloxacin receiving fda approval for anaerobic infections. moreover, more recent reports of increased resistance to fq among the b. fragilis group has prompted interested to determine if these increases are due to susceptibility testing methodology problems. this study was designed to compare the susceptibility results from five laboratories that tested the same isolates against fq using agar dilution(ad) and broth microdilution (bmd) testing. methods: seventy clinical isolates of the b. fragilis group identified by only a sequential number were distributed to each of the five participating laboratories. four of the laboratories performed ad and one laboratory performed bmd susceptibility testing using nccls recommendations including breakpoints where appropriate. serial two-fold dilutions of moxifloxacin (mox), gatifloxacin (gate), trovafloxacin (trov), and metronidzaole (metro) were prepared in the test medium within a range of . to mg/l. plates were inoculated with · cfu per spot or well, incubated anaerobically for hours and the mic read. qc was performed using nccls-recommended atcc strains. results: mic values for each antimicrobial were collated as mic and per cent (%) of isolates inhibited at £ and £ mg/l: these data indicate that inter-laboratory testing of the same test isolates gave very similar results for each antimicrobial. bmd gave slightly lower mic results than ad but was not significantly different. we conclude that susceptibility methodology is not responsible for unusually high resistance rates. global evaluation of gemifloxacin activity tested against community-acquired respiratory tract pathogens, haemophilus influenzae and moraxella catarrhalis ( ) ( ) ( ) ( ) ( ) ( ) fluoroquinolones are a therapeutic option in treatment of infections caused by non-fermentative gram-negative bacteria.however, resistance to these antibiotics rapidly increase in clinical isolates. the efflux mechanism is the most common cause of multidrug resistance of strains p. aeruginosa, a. baumanii and s. maltophilia. these mdr pumps belonging to the rnd family are inhibited by phe-arg-beta-naphthylamide [mc , ] . however, different results of reserpine effect on the activity of rnd pumps was established. in our study, we searched for effect of the efflux pump inhibitors (mc , , reserpine and rescinnamine) on the mic of quinolones for mdr clinical isolates of p. aeruginosa, a. baumanii and s. maltophilia. we looked for a interdependence between structure of quinolones and ability of tested inhibitors to inhibit efflux of these antibiotics. results: the mdr strains were resistant to all tested quinolones (nalidixic acid-nal, pipemidic acid-kp, norfloxacin-nor, ciprofloxacin-cip and ofloxacin-of). among studied inhibitors only mc , affected the susceptibility of all tested strains to quinolones, first of all to nal. effect of mc , on the mic depended on its concentration, but did not depend on the temperature. for majority of p. aeruginosa and a. baumanii strains the mic of all quinolones decreased in the presence of mc , . the highest decrease of the mic of quinolones was obtained for p. aeruginosa ( - -fold). for a. baumanii strains the mic of kp, nor, cip and of decreased only from -to -fold but the mic of nal decreased to -fold. moreover, the presence of this inhibitor increased the sensitivity of % of s. maltophilia only to nal ( -to -fold). reserpine and rescinnamine did not effect on the mic of quinolones for all studied mdr strains. however, reserpine alkaloides showed inhibitors activity against control strains of s. aureus (decrease mic of nor). conclusions: our data confirm that mc , inhibited efflux pumps not only in p. aeruginosa but also in a. baumanii and s. maltophilia strains. additionally, obtained results suggested possibility that for quinolones, depending on their structure, are different binding sites in efflux pumps. furthermore, probably also mc , binds to specific site of pumps depending on the efflux systems. so, the effect of this inhibitor on mic of quinolones depends on genus of bacteria, kind of pumps and structure of agents. in vitro activity of fucidic acid against staphylococcus aureus strains objectives: viridans group streptococci (vgs) resistant to penicillin, macrolides and other antimicrobial are increasingly found. linezolid and quinupristin-dalfopristin exert their mechanism of action by protein synthesis inhibition and are agents with activity against multidrug resistant gram-positive cocci. this study was aimed to determine, by time-kill curves, the activity of linezolid and quinupristin-dalfopristin against five vgs expressing various degrees of resistance to erythromycin and harbouring different erythromycin resistance determinants methods: the species, erythromycin mics (mg/l) and the erythromycin resistance genes present for each of the five isolates tested were: s. anginosus/ . /none, s. sanguis/ /mef(a), s. sanguis/ /erm(b), s. mitis/ /erm(b) +mef(a), s. anginosus/ /erm(b) +mef(a). quinupristin-dalfopristin and linezolid mics for all strains were and . mg/l respectively. strains were incubated in cation-adjusted mueller-hinton broth supplemented with % lysed horse blood. each of the five isolates were exposed to linezolid and quinupristin-dalfopristin for hours in a shacking water bath in presence of · mic. colony counts were performed at , , and hours. results: time-kill studies showed that for linezolid, the antibiotic concentration of · mic was bacteriostatic for each strain irrespectively of the resistance to erythromycin and the genes harboured, showing a % of killing at h. the in vitro activity of quinupristin-dalfopristin was predominantly bactericidal with a . % of killing at h. the exception were the strains with erm(b) + mef(a) determinants, in one of them, a s. milleri, the bactericidal activity was delayed to h, and in the other strain, a s. mitis, the activity was bactericidal at h but with regrowth at h. conclusions: when vgs are implicated in infections in neutropenic patients and in cases of endocarditis where bactericidal activity is usually considered mandatory, we have to considered that: linezolid was bacteriostatic, at the concentration tested, against all strains and the rapid killing of quinupristin-dalfopristin against vgs was lost when the strain presented the erm(b) and mef(a) genes together. comparative antimicrobial susceptibility patterns in different groups of species of viridans group streptococci and streptococcus bovis isolated fom blood cultures i. rodriguez-avial, c. rodriguez-avial, j.j picazo (madrid, e)objectives: viridans group streptococci (vgs) and s. bovis are part of the normal human microbiota and are implicated as a cause of endocarditis and sepsis in neutropenic patients. because of their resident nature are frequently exposed to the antimicrobial agents. this study was performed to study the resistance phenotypes to penicillin (p), erythromycin (e), key: cord- -i reanan authors: nan title: ecb : th european congess on biotechnology date: - - journal: j biotechnol doi: . /j.jbiotec. . . sha: doc_id: cord_uid: i reanan nan in the last years biotechnology has made tremendous progress in its different application fields: red biotechnology, the use of biological methods for medical purposes, is firmly established in the development of new drugs. the use of plant or green biotechnology is under controversial discussion in politics and public. nevertheless, genetically modified herbicide and insect resistant crops are cultivated to a large extent. industrial biotechnology, now often named white biotechnology, seems widely underestimated in the public perception. it includes all industrial processes for the production of chemical products and enzymes, which fully or partly rely on the biological toolbox of nature. white biotechnology processes are carried out in a contained environment, typically in a bioreactor in a dedicated industrial plant. well-known examples are the fermentative productions of antibiotics, amino acids, vitamins and enzymes, products related to medical, food and feed applications. many products like the amino acids glutamic acid, lysine, threonine and tryptophane are exclusively produced using microbes in large scale industrial processes. in other cases, like the water soluble vitamin b , biotechnological processes successfully replaced chemical productions, due to lower costs and improved ecoefficiency. in contrast to this, most industrial chemicals and polymers are produced by chemical synthesis based on oil and gas. however, there are some examples for bioproducts among industrial chemicals. the solvents acetone and butanol, for instance, were manufactured by fermentation for several decades in the last century. since the s these fermentations have been replaced by more efficient and cheaper chemical synthesis. recently, new pilot and production processes for biopolymers like pha or biomonomers like , -propanediol or lactic acid were announced by different companies. currently, ethanol is by far the largest white biotech product by volume. in brazil, where bioethanol is used as liquid transportation fuel, the annual production is in the range of mio m . bioethanol is of growing importance also in the united states. business consultants predict a tremendous growth of biotechnological products within the chemical industry. high prices for crude oil, dropping prices for renewable resources, and scientific progresses nourish the expectation that industrial biotechnology will replace many bulk chemicals. is this realistic? will we switch from a petrochemical industry to a biobased chemistry within the next years? based on economic considerations it can be stated that this is a long term goal. to achieve this it remains a scientific challenge to make renewable raw materials available for competitive bioproduction of bulk chemicals at low costs. conversion of lignocellulosic material to fermentation sugar may be a solution. also green biotechnology can contribute to the supply of cheap fermentation raw materials. innovative ideas for downstream processing or further chemical conversion of fermentation products are required to enter the chemical value chains. furthermore, the identification of new higher value bioproducts is a chance for short term successes in white biotechnology. enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. by continuously increasing the efficiency and throughput of dna sequencing we, together with colleagues, have sequenced the human genome and the genomes of all the major model organisms. the challenge now centers on understanding these vast instruction sets. our ability to read these instructions must be enhanced through collection of key additional data sets. one productive path for delineating the functional sequences and inferring their function is comparative sequence. the mouse genome sequence, for example, led to estimates that only % of the human genome is functional. sequencing of an extensive set of additional mammalian genomes promises to define these functional sequences with a resolution of less than base pairs. on a different course, we have sequenced the chimpanzee genome to learn what has changed in the evolution of humans. beyond providing for the first time a catalog of the differences between the two genomes, the comparison of the chimpanzee and human genomes reveals the patterns of neutral mutation and regions that deviate from that. the talk will summarize these and related findings. the sequence of additional primate genomes will help delineate what has changed specifically in humans and add power to the analysis. ultimately, capturing human sequence variation and correlating with phenotypic variation will be required to understand function. but learning what these functional elements do requires new sets of experimental data. for this, we have turned to the nematode c. elegans. in this simple system most of the ∼ k genes have been defined and experimentally confirmed. beyond the hundreds of genes with already known mutants, two centers are systematically producing gene knockouts or rnai can be used to inhibit any gene temporarily. sequences of three caenorhabditis species are already available, and two more are underway. expression data has been collected for all the genes under many conditions and time points through development. to enhance the resolution of expression data and to simplify phenotypic analysis of embryonic mutants, we are developing a system that will automatically trace the cell lineage and assign gene expression to precise cells with high temporal resolution. the latest results with the system will be described. in the longer term, this and similar datasets should provide an understanding of how the genome specifies the form and behavior of the worm. uhlen department of biotechnlogy, albanova university center, royal institute of technology (kth), stockholm, sweden here, we present a new protein atlas database (www.proteinatlas.org) showing the expression and localization of human protein in normal and cancer tissues. the atlas is based on the use of antibodies (agaton et al., ) to generate high-resolution immunohistochemistry images representing normal tissues and different cancer types (uhlen and ponten, ) . each antibody is used to generate more than individual images and each image has been annotated by a pathologist (kampf et al., in press) . the database has been created by the swedish human proteome resource (hpr) and the program has been set-up to allow the exploration of the human proteome with antibody-based proteomics (nilsson et al., in press) . the basic concept is to generate, in a systematic and high-throughput manner (uhlen and ponten, ) , specific antibodies to all human proteins, and subsequently used these for functional analysis of the corresponding proteins in a wide range of assay platforms, including (i) a protein atlas for tissue profiles (kampf et al., in press) , (ii) specific probes to evaluate the functional role of individual proteins, and (iii) affinity reagents for purification of the specific proteins and their associated complexes for structural and biochemical analyses. ments, most effective source of variation was perturbation in growth medium, followed by perturbation in growth rate. effect of gene deletion on data variation was found to be less apparent when compared to other perturbations. a significant similarity in variation of metabolome and mrna data was observed, which may be used as the key point for integration of these two sets of data in functional analysis of genes. projection to latent structures (partial least squares, pls) is used for integration of transcriptome and metabolome data. comparison of pca and pls shows that linear model constructed via pls to predict the metabolome data does not make use of all the variation in transcriptome data. thus, pls allows the discrimination between the portion of gene expression change that affects the metabolome profile and the portion that is not directly effective on metabolome. both pca and pls can be used to detect the open reading frames (orfs) which are the main sources of variation in transcriptome data and/or effective on metabolome profile. extracellular metabolomics to accelerate the discovery of key genes involved in fibre degradation silas g. villas-bôas, geoffrey lane, graeme attwood, adrian cookson agresearch limited, grasslands research centre, tennent drive, private bag , palmerston north, new zealand. e-mail: silas.villas-boas@agresearch.co.nz (s.g. villas-bôas) the genome of the hemicellulose-degrading microbe clostridium proteoclasticum is been sequenced and an array of candidate genes with diverse activity relevant to fibre degradation have been identified by automated gene annotation methods. c. proteoclasticum falls within the butyrivibrio-pseudobutyrivibrio assemblage of rumen bacteria which are though to play an important role in the degradation of plant hemicellulose-lignin complexes which limit fibre degradation in the rumen. for new zealand it makes strategic sense to invest in microbial genomics efforts applied to agriculture where the country holds a strong competitive advantage and where ruminants constitute the vast majority of farmed animals. in conjunction with dna sequencing, proteomics and transcriptomics (micro-array analysis) we are using metabolomics as an additional functional genomics tool for gene discovery. we have established a footprinting approach for microbial metabolome analysis focused mainly on metabolic intermediates of polysaccharide degradation to provide quantitative information on end products of fibre-degrading enzymes. a gc-ms method has been developed that is able to resolve complex biological mixtures containing mono-, di, and oligosaccharides, in addition to a series of organic acids. we are currently phenotyping a series of c. proteoclasticum mutants to validate our analytical methodology and we are going to fully characterize the fibrolytic ability of c. proteoclasticum to be compared with other fibre-degrading microbes. we believe that our metabolomics data will complement current proteomic analysis of fibre-degrading enzymes and micro-array analysis of gene expression from a series of mutants by providing direct evidence of the metabolic function of key genes involved in fibre-degradation processes. many gram-negative bacteria utilize cell-to-cell communication systems that rely on diffusible n-acyl homoserine lactone (ahl) signal molecules to monitor the size of the population in a process known as quorum sensing (qs). in human pathogens this form of gene regulation ensures that the cells remain invisible to the immune system of the host until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish the infection. the qs regulon of pseudomonas aeruginosa and burkholderia cepacia, two important pathogens for patients suffering from cystic fibrosis, has been studied by proteome analyses. comparative twodimensional gelelectrophoresis of pre-fractionated protein mixtures (extra-, surface-, and intracellular proteins) coupled to mass spectrometry analysis or n-terminal sequencing has been employed to recognize and identify qs-controlled proteins. our findings strongly support the importance of ahl-mediated cell-cell-communication as a global regulatory system and suggest that qs control also operates via post-translational mechanisms. as qs has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in opportunistic human pathogens it represents a highly attractive target for the development of novel anti-infective compounds. functional genomics technologies (transcriptomics and proteomics) have been exploited to validate the target specificity of natural and synthetic qs inhibitors, thus having a great potential as alternative therapeutics for the treatment of bacterial infections. modeling cell cycle complex formation from high-throughput data sets lars juhl jensen european molecular biology laboratory, meyerhofstrasse , heidelberg, germany. e-mail: jensen@embl. de to analyze the dynamics of protein complexes during the mitotic cell cycle, we integrated data on protein interactions and gene expression. the resulting time-dependent interaction network for the first time places both periodically and constitutively expressed proteins in a temporal cell cycle context, thereby revealing novel components and modules. we discover that most complexes consist of both periodically and constitutively expressed subunits, suggesting that the former control complex activity by a mechanism of just-in-time assembly. consistent with this, we show that additional regulation through targeted degradation and phosphorylation by cdk (cdc p) specifically affects the periodically expressed proteins. alessandra luchini, andrea callegaro, silvio bicciato department of chemical engineering processes, university of padova, padova, italy. e-mail: alessandra.luchini@unipd.it (a. luchini) since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. however, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. in addition, common data analysis tools, like anova, require replicates and disregard correlation structure among times. we present a method for the identification of differentially expressed genes in un-replicated time-course experiments. the procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. the identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. specifically, (i) data are structured so that measurements are correlated in time, within the same biological condition; (ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; (iii) time point t represents the system before the perturbation. therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t , and given an empirical null distribution constructed using permutations. statistical significance is assessed by the q-value. the method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: (i) skeletal muscle cells treated with a histone deacetylase inhibitor (iezzi et al., ) and (ii) immature mouse dendritic cells (dc) exposed to larval and egg stages of s. mansoni (trottein et al., ) . differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from anova model and sam paired test. the biological significance and soundness of selected transcripts was also verified using global functional profiling by means of ontotools. results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches. carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. the calcium-dependent antibiotic (cda) is a lipopeptide synthesised non-ribosomally and produced by streptomyces coelicolor a ( ). cda contains several non-proteinogenic amino acid residues. hydroxyphenylglycine ( -hpg) is one of the unusual amino acids in the structure of the cda and vancomycin groups of antibiotics. for the members of the vancomycin group of antibiotics, the -hpg residue plays crucial roles in the structure and function of the final glycopeptide antibiotic. to reveal the putative biosynthetic pathway of this amino acid in cda, a standard "double crossover replacement strategy" was used to delete -hydroxymandelic acid synthase ( -hmas, encoded by hpd) from s. coelicolor mt and , using the delivery plasmid pzmh . there was no cda production in the disrupted strains. plates containing a gradient of hydroxymandelic acid were used to restore cda production in both s. coelicolor mt hpd and hpd. exogenous supply of -hydroxyl phenylglyoxylate and -hydroxyphenylglycine reestablished cda production by the hpd mutant. feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). a cxcl tandem repeat promoter polymorphism is associated with susceptibility to severe sepsis in the spanish population n. maca-meyer , c. flores , l. pérez-méndez , r. sangüesa , e. espinosa , j. villar : research institute, hospital universitario n.s. de candelaria, s/c tenerife , spain; department of anesthesiology, hospital universitario n. s. de candelaria, s/c tenerife , spain. e-mail: nmacame@ull.es (n. maca-meyer) sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria, and remains an important cause of mortality in the intensive care unit. cxcl chemokine (or mip- ) exhibits a pivotal role in the immune response, and several functional studies in animal models of sepsis have catalogued cxcl as a candidate gene for the development of sepsis. we have performed a case-control association study of cxcl gene variants and susceptibility to severe sepsis in hospitalised patients and healthy individuals. after the examination of linkage disequilibrium in the region, we analysed whether two promoter polymorphisms (snp rs and a newly described polymorphic short tandem repeat d s ) were associated with the syndrome. we found a significant association of common variants at d s with the development of severe sepsis (heterozygote carriers or . ; % ci . - . , and homozygote carriers or . ; % ci . - . ; mantel-haenszel χ test for linear trend p = . ). the risks remained significant even after a genomic control adjustment, based on additional genotyped polymorphisms not linked to the candidate gene. these preliminary results suggest that cxcl gene variants may contribute to the development of severe sepsis. kasper møller , ana paula oliveira , jens nielsen , mark johnston : center for microbial biotechnology, biocentrum, technical university of denmark, denmark; department of genetics, school of medicine, washington university, st. louis, usa glucose is the preferred carbon and energy source for most cells. in saccharomyces cerevisiae, a complex regulatory network ensures that s. cerevisiae ferments glucose to ethanol even in the presence of oxygen. to obtain a better understanding of this crabtree effect and the logic of the glucose signalling network in s. cerevisiae, we are analyzing glucose sensing and signalling in the related species saccharomyces kluyveri, which exhibits much less of a crabtree effect (it prefers not to ferment glucose when oxygen is available). we show that there are only two major glucose transporters in s. kluyveri, and that these are regulated in response to the availability of glucose via a glucose sensor and a signalling pathway similar to the glucose induction (rgt /snf -rgt ) pathway in s. cerevisiae. we have used dna-microarrays for s. kluyveri to find targets of the s. kluyveri glucose induction pathway, as well as to evaluate the global response to a change in environment from growth on ethanol to growth on glucose. this study identifies a number of differences in the regulation of glucose uptake and global responses to glucose between s. kluyveri and s. cerevisiae, which may contribute to their different glucose metabolism. detection and analysis of microrna using lna probes nana jacobsen, christian lomholt, peter mouritzen, peter stein nielsen, mikkel noerholm exiqon a/s, bygstubben , dk- vedbaek, denmark. e-mail: mouritzen@exiqon.com (p. mouritzen) micrornas are a class of short endogenous rnas that act as post-transcriptional modulators of gene expression. growing evidence suggest that micrornas exhibit a wide variety of regulatory functions and exert significant effects on cell growth, development, and differentiation. recent studies have shown that human microrna genes are frequently located in cancer associated genomic regions and perturbed microrna expression patterns have been observed in many malignant tumors. we have exploited the significantly improved hybridization properties of lna oligonucleotides against rna targets to design lna-modified dna probes for detection of different micrornas in animal and plants by northern blot analysis, microarray hybridization and in situ hybridization. we will describe the results obtained from detection and analysis of different micrornas in c. elegans, zebrafish, mouse, and plants. in addition, we will describe a novel lna-based method for expression profiling of mature micrornas by quantitative rt-pcr. expression profile of the sty and paa genes in pseudomonas sp. y by means of dna microarrays david bartolomé-martín , david juck , m a teresa del peso-santos , charles w. greer , julián perera : departamento de bioquímica y biología molecular i, facultad de ciencias biológicas, universidad complutense de madrid, madrid, spain; environmental microbiology group, biotechnology research institute, national research council canada, montréal, que., canada h p r . e-mail: perera@bio.ucm.es (j. perera) dna microarrays are a new and powerful tool to study gene expression in very diverse systems. environmental biotechnology and biodegradation are some of the fields of research where this technology may be very promising. pseudomonas sp. y is a bacterium able to grow in minimal medium plus either styrene (sty) or phenylacetic acid (paa) as the sole carbon and energy sources. this bacterium is the only organism where the genes that code for both the upper (sty genes) and the lower (paa genes) catabolic pathways for the styrene degradation have been described till now. it is unique in having two active copies of the genes encoding the lower pathway (paa and paa gene clusters). we have designed a dna microarray with the sty and paa genes in order to analyse their expression in the wild type pseudomonas sp. y , in p. sp. y t (a paa deletion mutant) and in p. sp. y c (a crc gene mutant). this analysis has been performed on bacterial cultures grown in media with different carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. dynamics in induced repression of phosphomannose isomerase pmi gene of saccharomyces cerevisiae anssi törmä , , juha-pekka pitkänen , , laura huopaniemi , risto renkonen : medicel ltd., haartmaninkatu , helsinki, finland; rational drug design program, department of bacteriology and immunology, haartman institute and biomedicum, university of helsinki, p.o. box , helsinki, finland. e-mail: juhapekka.pitkanen@medicel.com (j.-p. pitkänen) gdp-mannose is the precursor of cell wall biosynthesis in s. cerevisiae. to understand the system level role of gdp-mannose, we studied a conditional knock-out strain of the key enzyme in its synthesis; pmi . the experimental procedure allowed us to study the order of mechanisms the cells launch in order to adjust to a sudden malfunction in the metabolic machinery. we collected samples from continuous cultivations over h and measured genome-wide gene expression levels, enzyme activities, and concentrations of intracellular metabolites. for sampling we have built a sample robot, which automatically takes and preserves the samples. in order to carry out this magnitude of experimentations and generated data, we have constructed a proprietary software platform to handle all the phases from project management in wet-lab to workflow and pathway management in in silico. after normalization and clustering, significantly changed genes and metabolites were searched for enrichment in biological processes and molecular complexes. further, gene expression levels, metabolite concentrations, and enzyme activities were searched against each other for causality over time. overall, we focused on thorough analysis of our own data and known database data in order to reward our efforts with knowledge. at the transcriptome level, repression of pmi led to two major types of activation profiles, one peaking at the time when pmi p activity and gdp-mannose were depleted and the other later during recovery from the perturbation. the primary response was most enriched with genes known to play roles in mating and filamentous growth and associated with the transcription factors ste p, tec p, dig p, and mcm p, whereas the secondary response consisted of genes involved in carbon metabolism and associated with the general stress response regulators msn p and msn p. skn p, a high-level transcription factor was associated with both the primary and the secondary response, consistent with its suggested role of coordinating environmental responses and developmental processes. transcriptome of pig ovarian cells: discriminant genes involved in follicular development bonnet a., le cao k.a., low-so g., san cristobal m., tosser-klopp g., hatey f. laboratoire de génétique cellulaire, centre inra de toulouse, castanet-tolosan , france in order to identify genes and gene networks involved in pig ovarian follicular development, we built subtractive suppressive hybridization libraries (ssh) from granulosa cells of healthy follicles (small, medium or large). the rna isolated from these cells was used to hybridize cdna nylon micro-arrays. data analysis using a gaussian linear mixed model showed that % of the variability is due to the genes. two hundred fifty one regulated genes (from the expressed) were identified and clustered into three groups according to the follicle size. moreover, we found previously identified genes such as aromatase, igfbp which supported the validity of our experimental model. ramdom forest analysis put forward the most discriminant genes between the three follicle classes. this study put forward gene sets such as those involved in cell modeling, regulation of transcription, apoptosis during follicle growth. the next step will be to describe more precisely the spatio-temporal expression patterns at the mrna levels of the genes identified by these experiments. microalgae constitute a significant source of valuable natural products, e.g. sulfated polysaccharides, polyunsaturated fatty acids, and phycobiliproteins that find applications in wide range of industries, including food, pharmaceutical, agricultural and cosmetics. however, genomic and molecular genetic studies of microalgae lag far behind those of higher plants. in order to accelerate red microalgal genomic studies by taking advantage of current genomics and post-genomic technologies, we have generated expressed sequence tag (est) databases of two red microalgae porphyridium sp. and dixoniella grisea grown under various physiological conditions. to date we have sequenced and ests of porphyridium sp. and d. grisea, respectively. the sequence assembly resulted into, ca. non-redundant unigenes for each microalga, only % of which were identified by similarity to sequences in the public databases. porphyridium sp. and d. grisea unigenes were compared with the whole-genome predicted proteomes of three microalgae and those of representative eukaryotic and prokaryotic organisms. both microalgae have highest similarity to the red microalga cyanidioschyzon merolae. the order of sequence similarity to other organisms examined was arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, thalassiosira pseudonana (diatom), saccharomyces cerevisiae, caenorhabditis elegans, archaea and cyanobacteria. although red microalgae are considered as phylogenetic bridge between prokaryotes and eukaryotes, our data show that the red microalgae have strong similarity to eukaryotes and only distant similarity to prokaryotes. gene expression profiles were studied by analyzing cdna and subtraction libraries constructed from algae grown under various physiological conditions. we observed that top three most abundant ests in the stationary phase of porphyridium sp. were adp ribosylation factor like- , flavohemoglobin and adp ribosylation factor- . in addition, we have identified several genes which were specific to nitrate-and sulfate starvation. the sarco(endo)plasmic reticulum ca + -atpase (serca, "the calcium pump"), is responsible for pumping the ca + released into the cytoplasm during muscle contraction back into the sarcoplasmic reticulum store while proton are pumped the opposite way as counter-cations. these transport processes go against the concentration gradients and are therefore energy consuming. the energy is derived from atp hydrolysis via formation and break-down of a phospho-enzyme intermediate. over the last year a number of new crystal structures have been published which have added to our understanding of how this task is accomplished, which provides an impressive insight to the mechanism of a molecular pump. rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. this work was supported principally by embo and the mrc. structure and target-specificity of thioredoxin h kenji maeda , anette henriksen , per hägglund , christine finnie , birte svensson : biochemistry and nutrition group, biocentrum-dtu, technical university of denmark, dk- kgs. lyngby, denmark; biostructure group, carlsberg laboratory, dk- valby, denmark. e-mail: kenji@biocentrum.dtu.dk (k. maeda) thioredoxins are ubiquitous small proteins with protein disulphide reductase activity. thioredoxins can alter the structures and the activities of various target proteins by reducing their disulphide bonds. seeds of several plants are abundant in cytosolic thioredoxins referred as h-type. in barley, two thioredoxin h isoforms, hvtrxh and hvtrxh that share % sequence identity but differ in temporal and spatial distributions were previously identified and characterised. in the present study, the relationship between structures and targetspecificities of h-type thioredoxins are analysed. the d-structures of hvtrxh and hvtrxh are determined by x-ray crystallography as the first crystal structures of thioredoxin h. comparison of the structures shows that the majority of solvent exposed residues near the active sites are conserved between the two isoforms. this is in agreement with previously observed similarity in target-specificity of the two isoforms. thioredoxins from organisms distantly related to barley, such as e. coli, have highly similar folds but different surface charge distributions compared to barley thioredoxins. a comparison of the target-specificities of hvtrxh , hvtrxh , e. coli thioredoxin and several thioredoxin mutants will be attempted to reveal the structural features that influence specificity of barley thioredoxin h isoforms. enbrel is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human kda (p ) tumor necrosis factor receptor (tnfr) linked to the fc portion of human igg . the cho-expressed molecule contains both n-and o-linked oligosaccharides with a total carbohydrate content of % by mass. the o-linked oligosaccharides were released by hydrazinolysis and their structure determined by exoglycosidase sequencing and maldi-tof mass spectrometry. to locate precisely the o-linked sites, the glycosylation heterogeneity of tnfr:fc was simplified by treatment with n-acetyl neuraminidase and n-glycanase. the remaining molecule, which only carries core o-linked glycan structures, was cleaved by trypsin and analyzed by lc-ms. precise localization of o-glycosylation sites was determined based on the concept of a specific modification of the o-glycosylated serine into aminopropenoic acid and o-glycosylated threonine into -amino- butenoic acid. the deficit in mass resulting from this transformation was the marker used to localize the modified residues on the peptides by tandem mass spectrometry sequencing (ms-ms). ms-ms spectrum of enbrel glycopeptides were interpreted based on the presence of -aminopropenoic acid and -amino- -butenoic acid, resulting in a complete map of o-linked glycans precisely located at different sites. anu mursula, beatrix fahnert, sari krapu, eija-riitta hämäläinen, ritva isomäki, peter neubauer bioprocess engineering laboratory and biocenter oulu, university of oulu, oulu, finland. e-mail: anu.mursula@oulu.fi (a. mursula) wnt proteins form a highly conserved family of secreted glycoproteins important in cell-cell signaling events during embryogenesis and adult tissue maintenance. impairments within this complex signaling pathway can lead for example to developmental defects in embryos, degenerative diseases and cancer. respectively, wnt proteins can be used as tools in basic research concerning wnt function, developmental biology, screening for interacting compounds, and for medical applications (e.g. therapeutics, stem cells). hence, recombinant wnts provide a valuable basis for these purposes. however, production of recombinant wnt proteins is challenging, because they contain multiple disulfide bonds making the folding very difficult. a process for production of murine wnt- in e. coli has been developed in our laboratory. the knowledge obtained from this research has also been applied to the expression of other wnts, namely wnt- and wnt- , and can be used to approach other cysteine-rich proteins as well. since the expression level of wnt proteins is rather low so far, tools for monitoring and optimizing the production process have been established. by means of this sandwich hybridization method the level of target (wnt) mrna can be measured. the technique has already been applied to analyzing wnt- mrna levels. probes also for wnt- and wnt- have been generated. thus, transcription of wnt genes in all kind of cells (e.g. tissue, recombinant hosts) in general as well as kinetics of transcription can be studied using these tools. in growth factor signaling, stimulation of cell-surface receptors first triggers activation of the receptor itself and then of a large number of intracellular effector molecules. the stimulus is integrated with a host of other cellular processes, leading to cytoskeletal changes, activating transcriptional programs in the nucleus and ultimately resulting in cell proliferation, differentiation or motility. classical signaling pathways and networks depict potential protein-protein interactions only in a static form. in the cell, these interactions are dynamic and occur in an ordered fashion. here, we apply a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance in order to study the global dynamics of signaling events. briefly, three cell populations are metabolically labeled with either normal arginine or a c substituted form, or a c n variant (stable isotope labeling by amino acids in cell culture, silac). each population was then stimulated with egf for a different time period and tyrosine phosphorylated proteins were affinity purified with anti-phosphotyrosine antibodies. the proteins from the precipitated complexes were quantitatively analyzed and identified using lc-ms/ms. arginine containing peptides occurred in three forms, directly indicating protein activation at the corre-sponding time point. combination of two experiments sharing one common time point of activation then generated five-point dynamic profiles. from the proteins quantified, we identified signaling proteins, including virtually all known egfr substrates and novel effectors, and the time course of their activation upon egf stimulation. discriminating proteins involved in the signaling network from unspecific binders was straightforward as these presented an activation profile. we have now further extended this study by directly measuring in vivo phosphorylation sites in response to growth factor stimulation and monitoring the time evolution of the phosphorylation events. finally, we determined and quantitatively compared the global egf and pdgf tyrosine phosphoproteomes in human mesenchymal stem cells and revealed a control point in their differentiation into bone-forming cells. such global activation profiles provide a novel perspective in cell signaling and will be crucial to model the highly dynamic signaling networks in a systems biology approach. klaus schneider , dave g smith , steven skaper , alastair d. reith : discovery research, glaxosmithkline, coldharbour road, harlow, essex, uk; neurology & gastrointestinal cedd, glaxo-smithkline, coldharbour road, harlow, essex, uk over the last years, progress in signal transduction research has revealed an astonishing degree of complexity in cell signalling which is manifested in positive and negative regulations and feedback loops within signalling pathways and by cross-talks between pathways, all of which are highly cell-type dependent. it has become evident that protein phosphorylation by protein kinases plays a major role in this complex regulation of cell signalling (hunter, ) . due to the importance of signal transduction in disease processes, many protein kinases may constitute key targets for disease intervention. yet, the lack of a full understanding of the regulation, the activation and, importantly, of downstream substrates of particular protein kinases requires often more detailed studies before initiation of resource-intensive efforts to find disease-modifying molecules. technologies for the study of protein kinase signalling include p labelling, mutational and knock-out studies and more recently rna interference. these tools are complemented by approaches that are based on proteomic technologies developed over the course of the last years. in this presentation, the scope of proteomics technologies to contribute to an understanding of kinase signalling will be discussed. an overview of available technologies will be given and results will be presented from a proteomic study of glycogen-synthase kinase (gsk ) signalling (coghlan et al., ) . novel findings will be presented on a study of gsk inhibition in a primary neuronal cell line by differential d gel electrophoresis, which resulted in the identification of more than proteins that were significantly regulated. proteome analysis is typically done by nanospray lc/ms in order to achieve higher sensitivity and thus a greater number of protein identifications. however, nano-scale lc systems can be more challenging to use and maintain. to obtain the best chromatographic performance, connections must be made carefully to minimize band broadening. improved chromatographic performance can enhance the mass spectrometric results by tandem ms as a greater number of peptides can be detected. a microfluidic chip-based system has been developed (yin et al., ) that minimizes the number of connections and the delay volumes. this work evaluates the performance of this device against the traditional nanospray approach. a yeast extract sample was separated by sds-page and bands were excised from the gel for further analysis. after in-gel digestion, the sample was analyzed by both traditional nanospray and the microfluidicbased chip device. after protein database searching, the identified proteins and the protein sequence coverage's were compared for the two approaches. the microfluidic device was demonstrated to be equivalent or better compared to the traditional approach. yin, h., killeen, k., brennen, r., et al., . anal. chem. , - . plant cytochromes p (p s) play key roles in the biosynthesis of most bioactive compounds with agronomic and therapeutic applications. a collection of about plant p s was expressed in yeast. the cdnas were isolated from the model plant with a sequenced genome arabidopsis thaliana, some others from wheat, helianthus tuberosus or vicia sativa. they were expressed under the control of a galactose-inducible promoter in an engineered strain of saccharomyces cerevisiae in which the gene of the native p reductase was replaced with the gene of a p reductase from a. thaliana under the control of the same galactose-inducible promoter, in order to provide an optimal context for plant p expression and activity (pompon et al., ) . an original procedure was designed for the high-throughput functional screening of this enzyme collection. it is based on the detection of oxygen consumed during the catalytic reaction by a fluorochrome embedded in the bottom of the microwell plates. this method was validated using several recombinant p s of known activity. it also allows for a very efficient screening for enzyme inhibitors. the advantages and limits of the method will be discussed. this work was carried out with the support of génoplante programme (no ) . reference pompon, d., louerat, b., bronine, a., urban, p., . methods enzymol , - . folding of a bacterial membrane protein studied by protein engineering daniel e.otzen, pankaj sehgal, peter a. christensen department of life sciences, aalborg university, sohngaardsholmsvej , dk - aalborg. e-mail: dao@bio.aau.dk (d.e. otzen) we have carried out a kinetic analysis of the folding of the -helix transmembrane protein dsbb in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate and dodecyl maltoside. this analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. the analysis also takes into account the composition of the mixed micelles, which is different from the bulk detergent composition. refolding and unfolding are consistent with a three-state folding scheme involving the sdsdenatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of sds. the temperature-dependence of the folding reaction displays an unusual decrease in heat capacity accompanying unfolding, which probably reflects the amphiphilic environment of the membrane protein. destabilization of dsbb by different short-chain alcohols correlates very well with the alcohols' respective hydrophobicities. data from a series of ala-scanning mutants tentatively identify a nucleus for folding, which is relatively diffuse and involves all four helices. we are currently complementing this work with studies of the association of peptides corresponding to individual transmembrane segments of dsbb. the reca protein of e. coli plays a crucial role in homologous recombination and dna repair. the recombination process takes place in a filamentous complex, in which the protein monomers are arranged in a helical manner around a single-stranded dna (ss-dna). in the presence of atp the filament can accommodate a second, double-stranded dna (ds-dna) and the strand exchange reaction can occur. the three-dimensional structure of reca itself and its complex with adp have been determined by x-ray crystallography. the active nucleoprotein filament, however, has only been studied at low resolution. both electron microscopy (em) and small-angle neutron scattering (sans) indicate significant differences between the structures of the active nucleoprotein filament and the compressed, inactive filament of only reca. we have presented a structural model of the reca protein in its active filament with ss-dna, using data obtained by linear dichroism (ld) polarized-light spectroscopy, based on a technique, we call "site-specific linear dichroism", which allows the orientation of a set of amino acids to be determined from ld data by systematic modification of the protein. here, we show that ld data of the nucleoprotein filament with ds-dna is over all similar to the data of the complex with ss-dna, indicating that the orientation as well as internal structure of reca in the active filament is not significantly altered when the bound dna is changed from single-stranded to double-stranded. this result supports the idea that the strand exchange reaction occurs without large conformational change of the reca protein. the choline-binding modules: a powerful biotechnological tool jesús m. sanz instituto de biología molecular y celular, universidad miguel hernández, elche, spain choline-binding modules (chbms) are present in some virulence factors of streptococcus pneumoniae (pneumococcus). the most extensively studied chbm is c-lyta, the carboxy-terminal domain of the pneumococcal cell-wall amidase lyta. the three-dimensional structure of choline-ligated c-lyta is built up from six loop-hairpin structures ("choline binding repeats", chbrs) forming a left-handed ␤-solenoid with four choline binding sites. although the structure of the ligand-free form is not yet known, our folding studies suggest that it is more loosely packed, with a partially unfolded amino-terminal region and a stable carboxy-terminal moiety that is extremely resistant to chemical denaturation (maestro and sanz, ) . the affinity of c-lyta for choline and other structural analogues allows its use as an efficient affinity tag for overexpression, immobilization and single-step purification of proteins of biomedical interest (c-lytag fusion protein purification system). this system presents many advantages when compared to current commercial methods, namely simplicity, compatibility with buffers and robustness. the availability of multiple supports that specifically bind chbms (such as multiwell plates) has recently allowed the development of a new procedure for the immobilization of c-lyta-containing hybrid proteins that may be used in proteomics, diagnostics and peptide display. in this communication, we present our last results about the stability, folding and engineering of c-lyta, together with a compendium of the current biotechnological potential of this protein, and highlight the productive link between basic molecular studies and their application. many of the modern approaches for studying disease compare steady state functions, such as repair, growth, and regulated gene expression within the various biological compartments organised by specialized function, be it mitochondria or blood vessels. the assignment of protein identities, which are linked to key biological mechanisms, which are associated with disease processes and disease progressions are an important area of this work (marko-varga and fehniger, ) . today, the technology available for studying proteome expression and resolving exact protein and peptide identities in complex mixtures of biological samples allows global protein expression within cells, fluids, and tissue to be approached with confidence. this confidence is due in part to reproducible repetitive sampling and analysis technologies including robotics data acquisition and high level mass spectrometry including both laser-desorbtion and electro spray ionisation. the precision in defining differences between normal and diseased steady states is aided by the creation of compiled reference and master data sets and by new methods for multiplexing the analysis of samples in groups. the establishment of key representative reference proteome systems representing the dynamic changes in protein expression during disease will be vital to the interpretation of changes observed in specific samplings of disease states and specific cells obtained from these samples. the creation of reference databases of proteins linked to disease pathways will play an important role in furthering our understanding of the "proteome of disease". examples will be given where protein expression patterns have been generated from compartments within tissue sections. marko-varga, g., fehniger, t.e., . j. proteome res. , - . adaptation of the saccharomyces cerevisiae proteome to nutrient limitations studied by metabolic stable isotope labeling and mass spectrometry albert j.r. heck netherlands proteomics centre and utrecht university, the netherlands. e-mail: heck@npc.genomics.nl. url: www.netherlandsproteomicscentre.nl one of the major aims of proteomics is to provide quantitative data on differential protein expression levels. recently, mass spectrometry-based methods have been introduced that can provide quantitative data on differential protein expression, mostly using stable isotope labeling (goshe and smith, ) . we opted for metabolic labeling as this provides efficient means to quantify differential protein expression, and has the advantage that all proteins are labeled universally (romijn et al., ) . in their natural habitat microorganisms encounter non-optimal growth conditions and often growth is limited by one nutrient. microorganisms need to respond rapidly to changes in the environment in order to survive. in the present study, we investigate the proteome response of chemostat cultivated wildtype saccharomyces cerevisiae to two different nutrient limitations, namely carbon and nitrogen limitation. yeast was metabolically labeled in well-controlled chemostat cultures. n and n labeled proteins were separated using d gel electrophoresis followed by rp-lc-esi-ms on a lc-q. relative quantification was performed by using relex software (maccoss et al., ) . we quantified proteins, using on average peptide peak pairs per protein. this analysis revealed that proteins showed a significant increase/decrease in expression level. the functional annotation of these proteins revealed that the yeast cells change expression levels of enzymes involved in metabolism of the growth-limiting compound. the protein expression ratios were compared with corresponding transcript levels. moreover, we compared the accuracy of quantifica-profiles mainly reflected differences in cellular origins in addition to different functional roles. mass spectrometric analysis identified proteins pertaining to several functional classes, i.e. acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization and in lipid metabolism. several proteins not previously implicated in nerve regeneration were identified, e.g. translationally-controlled tumor protein, annexin a / , vitamin d-binding protein, ␣-crystallin b, ␣-synuclein, dimethylargininases and reticulocalbin. real-time pcr analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. in conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. yeasts plasma membrane macromolecular components involved in stress resistance paola branduardi, paola paganoni, danilo porro dipartimento di biotecnologie e bioscienze, università degli studi di milano-bicocca, piazza della scienza, - milano, italy. e-mail: paola.branduardi@unimib.it (p. branduardi) the plasma membrane is a universal structure of living cells constituting an essential barrier dividing and defining the intracellular from the extracellular environment. it is consequently easy to deduce the crucial role played by said structure for any cell of any living organism, and especially for unicellular organisms, since all the information deriving from the external environment as well as many of the consequent cellular responses have to pass through this barrier. unicellular organisms, thanks to easy manipulation and cultivation techniques, can represent a very useful model for studying the plasma membrane function and response. in addition microorganisms, and among them yeasts, can be considered advantageous cell factories for recombinant productions. in this contest, the implementation of any process of production has to take into account, among others, the response and the tolerance of the host to the external environment. from these considerations derives the interest of our group to analyse the main macromolecular components of yeasts plasma membranes (proteins, lipoproteins and lipids), isolated from cells grown under different stress conditions, with particular attention to acidic environments. here, we present our recent data about separation and identification (by sequencing analyses) of lipoproteins isolated from the conventional yeast saccharomyces cerevisiae as well as from the non-conventional and acid tolerant yeast zygosaccharomyces bailii cell cultures grown in different conditions. in parallel, the protein fraction is under evaluation through a differential d proteomic approach and consequent analyses. effect of fungal polysaccharides on the expression of pancreatic proteins in streptozotocin-induced diabetic rats sang woo kim , hye jin hwang , kwang bon koo , jang won choi , jong won yun * : department of biotechnology; department of bioindustry, daegu university, kyungsan, in an attempt to search novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of pancreatic proteins in streptozotocin-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited excellent hypoglycemic effect, lowering the average plasma glucose level of the diabetic rats to . %. pancreatic proteome were analyzed by -de system, which separated more than individual spots. the -de analysis demonstrated that thirty-four proteins from a total of about matched spots were differentially expressed, of which spots were identified as the proteins whose expression has previously been associated with diabetes. twenty-two overexpressed and twelve underexpressed proteins were significant (p < . ) between the healthy and diabetic rats, and the altered proteins were restored (p < . ) upon eps treatment. it was first found that carbonyl reductase ( . -fold, p < . ) and mawdbp ( . -fold, p < . ) were surprisingly upregulated upon diabetes induction, and then those two protein concentrations were completely restored by eps treatment. moreover, we obtained eight unidentified proteins that have not been reported to be related with diabetes mellitus. these results evidenced the effect of fungal eps on searching potential markers for diagnosis and therapeutic manipulation of diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. the model established in our experiment is expected to mimic human diabetic status, which will help us to interpret the roles of biomarkers in diabetic state. the use of polyol-responsive monoclonal antibodies in immunoaffinity chromatography and as a probe for unfolding of wild-type and altered (t i) amidase from pseudomonas aeruginosa s. martins , j. andrade , a. karmali , a.i. custódio , m.l. since immunoaffinity chromatography is a powerful protein purification technique of interest in proteomics, monoclonal antibodies (mabs) against mutant (t i) amidase from p. aeruginosa were raised by hybridoma technology. in order to identify mabs that bind t i amidase tightly but release under gentle conditions, hybridoma clones secreting polyol-responsive mabs (pr-mabs) were previously screened. nearly % of elisa assay-positive hybridoma produced clones secreting pr-mabs with potential application as ligands for immunoaffinity chromatography. to select the optimal conditions for amidase elution, an elisa-elution assay was carried out, with two of these clones (f g ; e a ). the dissociation of ag-ab complex required % of propylene glycol and either . m (nh ) so or . m nacl. the binding of purified mab of igm class (e a ) to wild-type and mutant amidases was investigate by direct elisa, which revealed that it recognised specifically a common epitope on both amidases. conformational changes on antigen molecule were studied. mab e a showed a higher affinity for heat denatured forms than for native forms as revealed by affinity constants suggesting that the mab recognizes a cryptic epitope. the effect of mab e a on amidase activity was also investigated. the binding of mab to wild-type and mutant amidases exhibited an inhibition and activation of % as a function of time, respectively. this pr-mab is useful as a probe to detect conformational changes in native and denatured amidases as well as a ligand in immunoaffinity chromatography, which is of great interest in protein purification and proteomics. fragility and solubility of non-classical inclusion bodieš s. peternel , a. ristič , , v. gaberc-porekar , v. menart , : national institute of chemistry, ljubljana, si- ; lek pharmaceuticals d.d., ljubljana, si- . e-mail: spela.peternel@ki.si (Š. peternel) human granulocyte colony stimulating factor (g-csf) is a pharmaceutically important cytokine. when overexpressed in escherichia coli, it is usually accumulated in the form of inclusion bodies (ibs) . when produced at • c classical insoluble ibs are formed while at • c non-classical ibs containing a high amount of correctly folded g-csf are formed. as higher fragility and solubility of non-classical ibs were noticed, we decided to check whether bacterial cell disruption method has any influence on their mechanical stability and solubility. enzymatic lysis, sonication and homogenization, methods often used for disruption of bacterial cells during the isolation of ibs were compared. lysozyme treatment of bacterial cells appears to be mild enough disruption method not influencing the integrity of ibs. homogenization of bacterial cells at high pressure ( . - . kpa) shows no impact on classical ibs while some loss of target protein from the non-classical ibs is observed. sonication seems to be most harmful as even at rather low sonication altitudes, noticeable disassembling and solubilization of non-classical ibs occurs while no effect on classical ibs is perceived. our studies show that non-classical ibs are much more fragile and soluble than classical ones. therefore, one should extremely carefully choose the method for cell disruption to avoid undesirable loss of the target protein. the danish tick ixodes ricinus parasitize three different hosts both mammals and birds during the -year life cycle. the aim of this study was to identify the last blood host being the host, which the nymph had parasitized before molting to the adult instar. the reason for the study was to reveal the origin of the host contributing the most to the life cycle of the tick and thereby the maintenance of tick-borne diseases in denmark. the most common tick-borne diseases are lyme borreliosis and tick-borne encephalitis (tbe) causing illness in both animals and humans. we analyzed adult ticks, which were collected from known hosts. the analysis was performed at different heat stable proteins, which could be detected during the off host period by elisa. we found that heat stable proteins could be used as identification markers for host recognition. mushroom polysaccharides alter the expression of diabetesassociated proteins in the liver of streptozotocin-induced diabetic rats hye-jin hwang , sang-woo kim , kwang-bon koo , jang-won choi , jong-won yun * : department of biotechnology, daegu university, kyungsan, kyungbuk - , korea; department of bioindustry, daegu university, kyungsan, kyungbuk - , korea. e-mail: jwyun@daegu.ac.kr (j.-w. yun) in the present study, we investigated the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of liver proteins in streptozotocin (stz)-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in eps-fed rats to . %. in the next step, we analyzed the differential expression patterns of rat liver proteins from each group, to discover potent candidates for diabetesassociated proteins. a total of proteins of the -de gel were expressed differentially between diabetic and healthy rats. among them, proteins were upregulated and proteins were downregulated upon diabetes induction. many of these changes were in accordance with observations in previously published studies. surprisingly, the altered levels of most proteins in diabetic group were fully or partially restored to those of non-diabetic control group by eps treatment. moreover, we obtained unidentified proteins that have not been reported to be related with diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. potential is still limited by the differences observed in the structure of plant and mammalian n-glycans. indeed, theses differences and particularly the presence of ␤ , -xylose and ␣ , -fucose glycoepitopes are responsible for the immunogenicity of plant n-glycans. in order to reduce the structural differences between plant and mammalian n-glycans, current strategies are to knock out plant-specific glycosyltransferases or to humanize plant n-glycans by expression of mammalian glycosyltransferases in plants. in the present study, we have expressed a human ␤ , -galactosyltransferase in alfalfa. in order to further increase the efficiency of the human ␤ , -galactosyltransferase in the plant golgi apparatus, we have exchanged the endogenous targeting signal of this human glycosyltransferase for the ones from plant glycosyltransferases recently characterized in our laboratory. we will illustrate this approach of targeted expression with the results obtained by fusion of the catalytic domain of human ␤ , -galactosyltransferase with the n-terminal sequence of a plant glycosyltransferase that targets the fusion to the very early compartments of the golgi apparatus. the efficiency of natural versus targeted expression of human ␤ , galactosyltransferase in alfalfa will be compared in term of n-glycan humanization. altogether, our results clearly illustrate that we are now on the way to get perfect copy of mammalian glycoproteins in alfalfa plants. construction of recb-recd gene fusion and analysis of fusion enzyme activities oytun portakal , gerald r. smith , pakize dogan : department of biochemistry, hacettepe university medical school, , ankara, turkey; divisions of basic sciences, fred hutchinson cancer research center, seattle, wa - , usa. e-mail: oytun@hacettepe.edu.tr (o. portakal) protein folding is a fundamental process to gain protein function. in an oligomeric protein, the interaction between polypeptides affects the folding process and assembly to the holoenzyme. recbcd is a heterotrimeric and multifunctional enzyme that plays an essential role for major pathway of homologous recombination in e. coli. it is composed of recb, recc and recd gene products. recd is the fast motor unit of the recbcd enzyme. recd also plays a role for high affinity dsdna binding, nuclease activity and chidependent regulation. this study was designed to test the hypothesis that recd polypeptide regulates the essential reca loading activity. the approaching of the study was to fuse recd gene to subsequent recb gene and to observe the changes in enzyme activity and structure. for these purpose two genetic fusion mutations, two-nucleotide deletion and three-codon substitution were created at the overlap sites (ta) of recb and recd genes. fusion mutations were constructed by phage-mediated recombination system, which is called recombineering. this technology requires red function but not host reca protein function. here, we showed the recbd fusion polypeptides in crude extracts. genetic characterization tests were revealed that both fusion enzymes are recombination proficient and have wild-type phenotype. biochemical assays demonstrated that recbdc fusion heterotrimers have dsdna exonuclease, unwinding and chi cutting activities. they were also resistant to dna damaging agents. western blot analysis also detected a wild type length recd polypeptide together with recbd fusion polypeptides and a decreased heterotrimer compared to wild type. our findings suggest that recb-recd genetic fusions may affect recd assembling to the heterotrimer, but not affect it's native folding. sandwich immunoassay-a simple strategy for enhancement of the sensitivity and the specificity in prostate specific antigen detection based on surface plasmon resonance cuong cao, sang jun sim department of chemical engineering sungkyunkwan university, chunchun-dong, jangan-gu suwon, prostate cancer is a deadly disease in men. prostate specific antigen (psa) has been proved to be the most reliable and specific biomarker in preoperative diagnosis, monitoring and followup of patients with prostate cancer. in this study, a biochip based on surface plasmon resonance was fabricated to detect psa at concentrations ranging from to ng/ml. to reduce nonspecific binding, the chemical surface of sensor was constructed by using various ethyleneglycol mixtures of different molar ratios of hs(ch ) (och ch ) cooh and hs(ch ) (och ch ) oh. we also biotinylated the sams surface to enhance the orientation of protein immobilization. by using this surface, spr-based psa detection gave a positive ru value at the fist response in the whole range of psa concentrations. however, this ru value could get better and more reliable by simply applying a secondary interactant, the psa polyclonal antibody, in sandwich immunoassay. the results shown this approach could satisfy our purpose without modify the secondary interactant, which has usually been done by the other report. expression of epitopic domains of human coagulation factor viii in escherichia coli amir amiri yekta , , naser amirizadeh , alireza zomorodipour * , fariba ataei : department of mol genet. national institute for genet eng & biotechnol tehran-iran p.o. box: - , tehran, iran; islamic azad university of jahrom, jahrom, iran; department of hematol, faculty of med, tarbiat modarres university, tehran, iran. e-mails: amir amiriyekta@yahoo.com (a.a. yekta), * zomorodi@nrcgeb.ac.ir (a. zomorodipour) human factor viii (hfviii) plays major role in the intrinsic pathway of blood coagulation and is used to treat individuals with hemophilia a for bleeding episodes. many researches have been focused on the molecular aspects of this protein. in this regard, epitopes of hfviii as well as their corresponding antibodies have many important applications. bacterially produced fviii-epitopes are capable to neutralize the alloantibodies that inhibit hfviii activity. the purpose of present study was to over-express two epitope-containing fragments of fviii in e. coli under t promoter (novagen). two dna fragments from light-and heavy-chains of hfviii ( bp-c c and bp-a a , respectively) were subcloned in the expression vector. the use of his -tagged tail was also considered for detection and purification purposes. in each of the examined clones, a protein of expected size was detectable. in the c c -expressing clone the specificity of the over-expressed protein was confirmed by its reaction with the rabbit serum directed against native hfviii as well as anti-his-tag antibody. in the heavy chain-related-expressing clone, the expression level was low, but it was detectable by immunoblotting experiments. manipulations of the growth as well as induction may be required. the over-expression of the other epitopes reported in the heavy chain may be achievable by the expression of (a) sub-fragment(s) of this region. the over-expressed his-tagged c c -related protein was appeared to be trapped in the cell as non-soluble inclusion bodies. therefore, after homogenizing of the induced recombinant cells, the nonsoluble fraction was dissolved in a solution of denaturant (guanidine hydrochloride) and subjected for the purification, using a ni-nta resin (qiagen) followed by protein measurement. accordingly, an expression level of mg/l (of culture) of the purified c c -related peptide was obtained. the recombinant hfviii c c -derived peptide has provided useful mean for further experimental and medical applications. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with . % w/v epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph . ) or chloride (ph . ) as leading ion and -amino-caproic acid (ph . ) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic danalysis. development of strategies for heterologous expression of glucose dehydrogenase from the halophilic archaeon halobacterium sp. nrc- juan carlos cruz-jiménez , lorenzo saliceti-piazza , rafael montalvo : chemical engineering, university of puerto rico-mayagüez campus, mayagüez , puerto rico; biology, university of puerto rico-mayagüez campus, mayagüez , puerto rico. e-mail: juancruzj@hotmail.com (j.c. cruz-jiménez) halophilic archaea are excellent model organisms and valuable for biotechnology applications; they are easy to culture in the lab, genetically tractable, and exhibit a variety of interesting and useful characteristics. most halophilic archaea require . m nacl to sustain growth and structural integrity. among the . genes in halobacterium, we are studying the gene encoding a glucose dehydrogenase, gene id is , located between the and bases (halobacterium sp. nrc- genome project). this extremozyme is bioengineerable, and its use as a model for studying biocatalysis in aqueous/organic and nonaqueous media has not been explored to date. the utilization of enzymes in organic solvents has several potential advantages over aqueous systems. a major benefit is the increased solubility of many substrates, resulting in higher concentrations of reactant and products, hence reducing and purification costs and simplifying recovery protocols. cells were grown aerobically during seven days at • c in a complex medium, harvested by centrifugation and their genomic dna extracted. for cloning of the gene, primers were designed based on the sequence recently published by the halobacterium sp. nrc- genome project. the forward ( -ccgcatgcgcc cacagtccc- ) and reverse ( -ccggcctctagaacggcctgg- ) primers were designed to incorporate restriction sites for sph i and xba i, respectively (in bold). we are pcr amplifying the genomic dna and developing methods for the heterologous expression using the mesophilic escherichia coli, as well as purifying the enzyme. the purification procedure will be carried out using high resolution methods based on the protein's halophilicity. bioinformatics methods will be used to facilitate conforming of protein function and for comparison with a native enzyme. quantitative measurements by mass spectrometry of hundreds of proteins simultaneously using the new proteinchip systemseries p. iversen, e. fernvik ciphergen biosystems inc., symbion research park, fruebjergvej , dk- copenhagen, denmark. e-mail: piversen@ciphergen.com (p. iversen) most mass spectrometry methods used in proteomics allow for the identification of multiple proteins in a limited number of complex samples, but lack the ability to assess the quantity of the proteins and their modifications. however, mounting evidence shows specific cleavage of well-known proteins as being strong candidates for specific biomarkers, and in order to discover these biomarkers one has to be able to monitor the quantity and mass of hundreds of proteins from hundreds of complex samples reproducibly. the new series instrument in connection with proteinchip arrays ® from ciphergen biosystems enables this. the new series instrument is optimized for sensitivity, reproducibility and quantitation. new ion optics allows the use of higher acceleration voltages thus increasing the sensitivity, but without lowering the resolution. a new method of blanking the detector in connection with a non-linear gain of the detector also increases the sensitivity to the effect that igg can be detected down to . fmol. furthermore, the unique design of the instrument permits the detection of proteins with great variation in both mass and concentration and thus making it ideal for proteomics studies. a unique feature of the series instrument is the possibility to normalize the output by controlling the laser and detector so that results can be read with equal precision on different instruments, which is not often possible in mass spectrometry where individual instruments yield different results. this feature is vital in the validation of research results beyond individual laboratories. the coupling of liquid chromatography with mass spectrometry is now firmly established as a routine method for the identification of proteins that have been subjected to enzymatic digestion. in an on-line lc-ms experiment, the column eluent is coupled to the electrospray source via an emitter and any tryptic peptides present in the mixture are mass analyses as they elute from the hplc column. should there be any co-eluting species in the eluent, these will be separated in the mass analyser by their mass-to-charge ratio. it has become increasingly clear that relative quantification of protein expression changes is important in modern biology and medicine. several current approaches have been developed that utilise stable isotope labelling of samples in combination with separation and subsequent analysis by mass spectrometry. however, we have recently described an lc-ms strategy where quantification is achieved via normalisation of the ms datasets and comparison of the peptide intensities (of the observed tryptic peptides) across samples is performed. in this case, it is desirable to perform replicate injections and hence reduce statistical errors. this approach places a requirement upon good chromatography, especially in terms of retention time reproducibility. in addition exact mass measurement of the eluting ions is required as well as the ability to generate reproducible and reliable peak intensity, or area, calculations for the eluting tryptic peptides. the ability to measure the mass to charge ratios of ions accurately, across injections and across samples, increases confidence that the same ions have been matched from each sample injection. in this presentation our current strategy for the relative quantification of proteins will be discussed using, as examples, complex protein mixtures from salmonella enterica, eschericia coli and human serum. proteomic analysis for the production of rhctla ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen -immunoglobulin (rhctla ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using -d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy , cy and cy dyes and run within a single dige gel. using decyder tm software, spots were detected with two-fold thresholds with % confidence and it was found that proteins underwent significant change during the production of rhctla ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. study of substrate specificity of rnr-exoribonucelases using hybrid proteins ana barbas, mónica amblar, cecília m. arraiano instituto de tecnologia química e biológica, ean, - oeiras, portugal. e-mail: ab@itqb.unl.pt (a. barbas) the ribonucleases are essential enzymes responsible for the regulation of gene expression and have shown to be important for biotechnology purposes. for instance, commercial mutants deficient in ribonucleases have been quite relevant for the over-production of recombinant proteins. escherichia coli rnase ii is a processive - exoribonuclease prototype of the rnr family that has homologues widespread in the majority of the sequenced genomes. by sequence alignment it has been proposed for the rnr type proteins the existence of three different domains: an n-terminal cold shock nucleotide binding domain (csd), a rnb catalytic domain, and a c-terminal s nucleotide binding domain. we have constructed several rnase ii deletion mutants to enable the characterization of each domain. these studies have allowed us to determine that both csd and s are involved in the binding of the enzyme to the rna substrate, being the s domain the most important. in rna-binding proteins it has been shown that the s domain's conformation is highly conserved. however, it is not known whether the substrate specificity is s -dependent. in order to characterize the s domain and verify if it is directly related to substrate specificity, we have constructed rnase ii hybrid proteins in which the s domain was substituted by the s of two other exoribonucleases, rnase r (rnii-rnr) and pnpase (rnii-pnp). preliminary results have demonstrated that both quimeric proteins are capable of binding and degrading various rna substrates. in addition, studies are currently being carried out to verify the possibility that s domain of pnp in the hybrid protein might be involved in multimerization and/or interaction with other proteins. the murine monoclonal antibody igg , anti-digoxin was produced in a rolling bottle fermentor. purification was performed on a protein g column. cd spectra were recorded on a jasco- spectropolarimeter. protein concentrations of - g/ml and path length of cm were used for measurements in a far uv region. all measurements were performed in a cell holder thermostand with an accuracy of ± . at • c. at this temperature the predominance of ␤-strands is indicated. large conformational changes occur at • c. at this temperature the spectra tense to irregular ␤-strands and unordered structures. these evidences confirming temperaturedependent conformational changes of protein and also high thermal stability of mentioned monoclonal antibody. generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling janni kristensen, kim kusk mortensen, hans peter sørensen laboratory of biodesign, department of molecular biology, aarhus university, gustav wieds vej c, dk- aarhus c, denmark. e-mail: hans.peter.sorensen@teknologisk.dk (h.p. sørensen) we recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of escherichia coli. recombinant proteins were covalently coupled to the e. coli ribosome by fusing them to ribosomal protein (rpl ) followed by expression in an rpl deficient strain of e. coli. this allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpl and the target protein. to assess the efficiency of separation of target protein from ribosomes, by site specific proteolysis, we required monoclonal antibodies directed against rpl and gfp. we therefore purified rpl -gfp-his, rpl -his and gfp from e. coli recombinants using affinity, ion-exchange and hydrophobic interaction chromatography. these proteins could be purified with yields of , and g per gram cellular wet weight, respectively. however, rpl -gfp-his could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. the purified rpl -gfp-his fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by elisa using rpl -his and gfp to select for monoclonal antibodies specific for each protein. this resulted in antibodies directed against rpl and antibodies directed against gfp. antibodies were screened for isotypes and their efficiency in western immunoblots. the most efficient antibody against rpl and gfp were purified by protein g sepharose affinity chromatography. the purified antibodies were used to evaluate the separation of ribosomes from gfp, streptavidin, murine interleukin- , a phagedisplay antibody and yeast elongation factor a by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. proteomic analysis for the production of rhctla ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen -immunoglobulin (rhctla ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using -d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy , cy and cy dyes and run within a single dige gel. using decydertm software, spots were detected with two-fold thresholds with % confidence and it was found that proteins underwent significant change during the production of rhctla ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. similarity searches and multiple alignment of s and s protein of sars-cov for modeling d structure and its evolution (origin) mohammad soltany rezaee rad, iman tavassoly, negar mottaghi, banafsheh rezaee. e-mail: mohammad.soltany@gmail.com (m.s.r. rad) aims: the exact origin of the cause of severe acute syndrome (sars) is still an open question. nowadays recombinant origins for this virus have been found. s and s subunit of spike protein of this virus are the most important proteins responsible for severe acute respiratory syndrome. in fact they are glycoproteins of this virus exist on its surface. they are responsible for mediating fusion of viral and cellular membrane. the classification and modeling d structure of this virus can help us to suggest new ideas about its charististics and function, which may lead to new therapeutic and preventing modalities. methods: we used nucleotide sequence of s and s subunit of s (spike) protein for multiple alignments. we have done multiple alignments with different bioinformatics software (clusterx, entrez) for comparing the sequence with the other viruses and, we used weblab view software for modeling and identifying d structure of these proteins. findings: the similarity searches on nucleotide sequence of this protein with the single strands rna (ssrna) shows the virus belong to a known classification named coronaviridae. these d structures show the responsibility of s protein in this syndrome. another findings based on these alignments is an important similarity between these subunits and genome of hiv- showing they have familiar mechanism in pathogenesis. discussion: multiple alignments are powerful tool in classification of new recombinational virus and emerging infection. d structure model of this virus is an important guide to understand the mechanism of this virus. the shape of glycoprotein that modeled with bioinformatics software can help us in understanding mechanism of binding this virus to human cells. this fact can be used in designing drug and vaccine to cure and prevent the sars. blocking these origins and sites leads to inhibiting the virus attachment. also the similarity between this virus and hiv- shows us that both of them have similar proteins that cause pathogenesis of these viruses. the simulated moving bed (smb) technology is a continuous countercurrent chromatographic separation technique that has been applied successfully in the last years to a number of significant problems. an smb consists of a series of fixed bed chromatographic columns connected in a loop, and outperforms column chromatography in terms of productivity and solvent consumption. the use of smb instead of batch processes for bioseparations, i.e. separations involving large and rather complex molecules with multiple d configurations depending on parameters such as ph, temperature, etc., is becoming of greater and greater interest. examples of these are therapeutic proteins, antibodies and plasmid dna among others. for all chromatographic processes in this field, one of the most crucial issues is the cleaning of the chromatographic media with a special solvent system, an operation usually referred to as cleaning in place (cip). in single column chromatography this is easily done off-line, but this is not compatible with standard smb operation. in order to overcome this limitation, the standard smb configuration has been modified by adding a dedicated section plus an additional section for the re-equilibration of the freshly cleaned column with the working solvent before it is re-inserted into the smb loop. in such a way, cip according to gmp criteria can be incorporated into the smb unit and operation, which is then called cip-smb. this new smb configuration is also related to the three fraction separation unit called f-smb that has been recently introduced and applied to the separation of nucleosides. in this work we apply cip-smb using a size exclusion stationary phase to the separation of plasmid dna from the filtered cell lysate solution. plasmid purification has become a key issue in the last years as a result of the advances in gene therapy, whereas traditional laboratory methods are not always suitable for therapeutic purposes. we report about separation performances, which are then discussed in the light of smb design criteria and compared to column chromatography performance. computer guided optimization of adsorptive bioseparation processes bernt nilsson department of chemical engineering, lund university, lund, sweden. e-mail: bernt.nilsson@chemeng.lth.se separation processes like chromatography can be highly nonlinear and the behavior can sometimes be hard to predict. optimization of preparative chromatography is often done experimentally, which is both time consuming and expensive. a model-based approach to optimization is therefore an attractive and challenging way to overcome some drawbacks in the traditional working procedure in biotechnical industry. efficient model-based optimization for industrial needs requires three parts; models, methods and tools. model-based methodology requires a set of chromatography column model structures, which can capture the phenomenon of interest. for instance they have to capture column load variations, elution profile changes, operation condition disturbances, column configurations and stationary phase properties. to derive a reliable model for optimization it has to be calibrated and validated to experimental data, which requires an efficient calibration procedure. different calibration procedures are discussed and compared. after validation the model can be used in the design of a separation step. to do a robust design a set of requirements have to be fulfilled. the column size and operation conditions are used to optimize the performance of the step, which requires a constraint nonlinear optimization method. the choice of objective function for optimization and corresponding constraints are not obvious and the resulting operation conditions are often not robust. therefore there have to be additional methods available for analysis of the performance, like sensitivity and robustness analysis. optimization of the purification of antibodies is discussed and exemplified. the work with mathematical models and numerical methods has to be supported by a set of computer tools of different kinds in order to solve industrial problems effective. there is a need for different kinds of tools; customized tool to solve specific problems by a non skilled user and general toolbox for the expert. an example of a toolbox is presented. tina tarmann, alois jungbauer department of biotechnology, university of natural resources and applied life sciences, vienna, austria plasmids and viruses are the contemporary vehicles for genetherapy and genetic vaccination. extremely promising results have been reported from in-vitro, in-vivo and clinical studies. currently a lot of these compounds are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering. membrane based separations, chromatographic separations and precipitation have been employed for separation of plasmids and viruses. chromatographic separation have been designed with aim of protein separation. thus such processes suffer from either mass transfer limitations or low capacity. monoliths without intraskeleton mesopores and chromatography particles with giga pores are excellently suited for adsorption and separation of plasmids and viruses. low mass transfer resistance and high capacity compared to conventional beaded materials can be observed. adsorption kinetics were derived from infinite and finite bath methods and isotherms were constructed. these data also suggest that a conformational change of the plasmids takes place upon adsorption. discussion of the mass transfer properties and an example of scale up of a chromatographic separation process using these novel materials will be shown and discussed in respect to already existing processes. the recent developments in molecular therapies such as non-viral gene therapy and dna vaccination have fostered the development of efficient plasmid dna (pdna) purification processes. the separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large scale purification of pdna vectors intended for a therapeutic use. although escherichia coli produces mainly the more compact sc pdna isoform, oc, linear and denatured pdna isoforms are usually present and are likely to be less efficient in transferring gene expression. for this reason, regulatory agencies specify that more than % of pdna in a therapeutic product is in the sc isoform. in this work histidine-base recognition is explored as a mean to separate pdna isoforms. the agarose gel used here combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. chromatographic profiles were obtained by injection of native plasmid (sc + oc) samples in the histidine-agarose support showing an efficient and baseline separation of both isoforms. the high resolution obtained with this support indicates that the method is potentially applicable to the separation of pdna at preparative and analytical scale. affinity ligand development with a novel encoded bead screening technology ib johannsen, versamatrix a/s, gamle carlsberg vej , dk- valby, denmark. e-mail: www.versamatrix.com the presentation describes a new invention for fast development of affinity ligands, where up to , ligands can be screened onbead and identified in a few hours. combinatorial synthesis by the split and mix procedure is a powerful technique for generating vast numbers of diverse chemical compounds on polymer beads with relatively little effort. traditionally, the technique is hampered by the laborious spectroscopic and chemical analysis, needed to determine the exact structures of the ligand on selected beads. in this way, - month analysis time could easily be spent just to analyze a tiny fraction of the library. in the versaffin tm technology each bead is encoded, individually tracked, and identified during the synthesis and screening. this decreases the whole ligand development time from months to weeks and increases the amount of information significantly. the bead code further enables evaluation of the ligandprotein binding under varying binding and elution conditions. the instrument for reading the encoded beads and for quantifying the amount of bound protein is presented. the encoded beads we use are based on functional cross-linked polyethylenglycol (peg), which is compatible with water as well as most organic solvents. thus, the combinatorial synthesis can be carried out in organic solvents and the resulting compounds can be evaluated, still bound to the parent beads, under aqueous conditions. a further advantage of using peg based beads for on-bead screening is the fact that peg is biologically inert and therefore does not interfere in a bioassay. in the biopharmaceutical industry, pressure is mounting to shorten development times and thereby time to market, e.g. in the field of monoclonal antibodies, generic processes have been established which allow for more rapid development from gene to production of pre-clinical and proof of concept/phase i material. for non-mab products from various expression systems, productspecific approaches still prevail. however, for most product types similar issues like clearance of process-and product-related impurities, overall purity and yield, or manufacturing issues have to be dealt with in downstream process development. integrated and timely approaches based on process science and developed orthogonal analytical tools are often hampered by tight time frames and limited resources available. on the other hand, thorough understanding and analytical characterization of product characteristics but also of (process-related) impurities are pre-requisites for fully exploiting separation power and for achieving final purities way above % in a robust and cost-effective manner. from primary separation to polishing steps, we have made several attempts to improve the efficiencies of process steps themselves but also of ways to develop them. the strategies applied comprise implementation of innovative processing tools, rational streamlining and optimization of a sequence of unit operations, tech transfer and scale up considerations. also in this context, the applicability of scale down and ultra scale down models for process development and optimization purposes, their potential for speeding up and their limitations will be discussed. chromatographic monoliths are rather new chromatographic stationary phases. they consist of a single piece of a highly porous material. the pores are interconnected forming a network of channels. since the transport mechanism is predominantly based on convection, mass transfer between mobile and stationary phase is significantly enhanced resulting in short separation times. because of that they seem to be an ideal support for separation and purification of extremely large molecules like proteins, dna or even viruses. in this talk, various features of the monoliths like high porosity, fast mass transfer, surface accessibility and dynamic binding capacity will be described. effect of each feature on the separation and purification efficiency will be discussed in terms of molecular size and properties. while chromatographic monoliths are already widely accepted in microchip fluid devices, capillary columns as well as analytical columns, very few reports about preparative monolithic columns can be found. reasons for lack of preparative chromatographic columns will be elucidated and preparation strategy for construction of several liter volume methacrylate based monoliths will be presented. finally, several examples of biomolecule purification like large proteins, plasmid and genomic dna and viruses on cim convective interaction media ® monolithic columns will be given. further, their application as bioreactors and supports for solid state synthesis will be demonstrated. hubbuch institute of biotechnology, forschungszentrum juelich, juelich, germany. e-mail: m.schroeder@fz-juelich. de (m. schroeder) the intraparticle diffusion coefficient is an important parameter for modeling of chromatographic separation processes. a new method based on dynamic measurements of intraparticle concentration profiles of proteins in process chromatographic media with a confocal laser scanning microscope is presented. the diffusion coefficient is determined by fitting experimental data to a spherical diffusion model. excellent agreement of experimental data with simulation results is obtained. the diffusion coefficient is measured for seven proteins in sepharose ff, spanning molecular weights from . to kda. in addition, multicomponent diffusion processes for combination of differently sized proteins are analyzed and the influence of adsorbed proteins on the diffusion coefficient is measured in sp or q sepharose ff. taken together the presented method allows measuring the diffusion coefficient of proteins in process chromatographic media in a packed column. use of automated docking for predicting chromatographic behavior of proteins in hydrophobic interaction chromatography andrea mahn, m. elena. lienqueo contreras university of chile, santiago, chile in the present work, we have extended and automated the methodology proposed by mahn et al., for predicting protein behavior in hydrophobic interaction chromatography (hic). this methodology is based on the good correlation level between the average surface hydrophobicity of the interfacial zone (local hydrophobicity lh) and protein retention time in hic, for only three different ribonucleases. for determining the lh it is necessary to select the most probable protein-ligand conformation. in this work, we have determined the most probable conformation, of more than proteins, using (i) first, the module insight ii affinity by accelrys for providing automated docking (grid method) of ligands (phenyl) to the proteins ( conformations); (ii) then, the different probable docked protein-ligand conformations were automatically scored using the module insight ii ludi by accelrys; (iii) after that, each conformation was clustered and each cluster was scored by using the average score of each cluster (iv) finally, the most probable conformation was selected using a function based on the number of cluster components, and the average score value. then, when the most probable conformation was selected, the local hydrophobicity (lh) was calculated using the graphical representation and analysis of structural properties (grasp) program. the results have shown an acceptable correlation level (r > . ) between lh and the experimental dimensionless retention time (drt). in view of these results, we consider that this methodology could be used to adequately represent the chromatographic behavior in hic for all kinds of proteins (with a heterogeneous and homogeneous surface hydrophobicity distribution) and without a large number of tedious experiments, but only using computational simulation and adequate score criterion. potato tuber proteins are nutritious and show potential as functional ingredient in food systems. however, the present bulk processing technology can only recover byproduct protein for animal feed use. an expanded bed adsorption (eba) process for isolating functional food-grade protein from crude potato starch effluent was previously developed. moderate capture efficiency ( - %) of the total crude protein was most likely caused by diffusion limitations and aggregated protein, inaccessible for adsorption. we employed the same adsorption ligand attached to agarose-tungsten carbide beads to create stable beds of . - . × expansion using flow rates at - cm h − . a pilot scale eba process was run in a commercial processing plant over a three month campaign of starch production from potatoes of mixed variety. fresh crude effluent ( - l/cycle) was applied to a column ( cm × m) containing l of resin. protein capture by eba was reliable in operation, producing a refined protein material, which after dewatering and gentle drying, showed improved functionality over heat-coagulated protein produced at the same plant. overall productivity increased. however, finding a robust operating window of predictable productivity is challenging since the potato fruit water is complex and deteriorates easily. from breakthrough curves, it is observed that the major bulk protein, patatin, displays non-langmuir adsorption behavior. this may indicate a range of interactions for different species of the same protein. chlorogenic acid (ca), the main polyphenolic substance in potato tuber, causes enzymatic browning and undesirable flavor changes, but polyphenols can also react with protein. assessing the effects of interacting cell components therefore applies to the bioprocessing of plant material. at present the acceptance of biochip technology for on site use, e.g. diagnosis or environmental control is hindered by rather expensive and complex instrumental systems. there is a need to provide reliable and cost-effective systems that can be operated with minimal training. the construction of electronic biochip microarrays using semiconductor technology enables the construction of compact systems with high integration at acceptable production costs. the key feature of the fully electrical biochip technology are micro arrays made in advanced si-technology and carrying several array positions with interdigitated nanometer gold electrodes on its surface. the chips are fabricated by standard silicon fabrication methods allow-ing high volume production and to minimise the cost per chip. the advantage of fully electronic microarrays is the intrinsic high spatial resolution and direct signal coupling of the biosensing element and the transducer. the function of fully electronic biochips is also based on the electrochemical transduction and quantification of the formation of affinity complexes on the chip surface. a portable device for field applications and point of care diagnosis have been designed and manufactured. the amperometric device enables the recognition of biomolecular interactions by measuring the redox recycling products of elisa enzyme labels. the highly sensitive signal transduction is achieved with a -channel interdigitated ultramicroelectrode array. one major advantage of fully electronic microarrays is the direct signal coupling of the biosensing element and the resulting robustness and opportunity for miniaturisation. those electrical biochip arrays have been adapted for the detection of all types of affinity complexes, such as for dna, rna, proteins and haptens. self assembling of capture oligonucleotides via thiol-gold coupling have been used to construct the array chip nucleic acid interface. thus e.g. pathogenic micro organisms have been identified and quantified via their genomic dna or ribosomal rna respectively. another application based on immobilized antibodies is shown to sense extreme low concentration of bioagent toxins. for processing the assay formats and the electrical read out of the detection of affinity complexes a modular fully automated measurement system has been developed. it is manufactured in industrial lines and available at market. dynamics and self-assembly of organic molecules on surfaces revealed by high-resolution, fast-scanning stm flemming besenbacher interdisciplinary nanoscience center (inano), university of aarhus, dk- aarhus c, denmark. e-mail: fbe@inano.dk in the interdisciplinary area of nanoscience and nanotechnology, the adsorption and self-assembly of organic molecules on singlecrystal surfaces have attracted much attention lately due to the potential applications in fields ranging from molecular electronics to biocompatible interfaces. the supramolecular structures formed upon deposition of molecular species on solid surfaces depend on the molecular architecture and the distribution of functional groups on one hand, which determines the thermodynamically stable molecular arrangement, and on the other hand, on kinetic factors like thermal diffusion, spontaneous rotations and conformational dynamics. i will show how the unique aspect of our aarhus stm and the time-resolved, high-resolution stm imaging can be used to obtain important new insight into the dynamics, and can provide very important new information on the atomic-scale realm and on the dynamics of molecular nanostructures. the time-resolved stm data are visualized in the form of stm movies (see www.inano.dk/spm) which can subsequently be analyzed in order to extract quantitative information on the activation energy, the prefactors and the adsorbate-promoted diffusion. i will specifically discuss: (i) the self-assembly of guanine quartets on au( ) and the influence of cooperative hydrogen bonds, and (ii) the molecular recognition in binary mixtures of dna bases. g molecules are found to self-assemble into a hydrogen-bonded network of g-quartets, whose structure corresponds perfectly with the quartet structure of telomeric dna determined by x-ray crystallography. the strong preference of g molecules to form quartets can be explained by a cooperative effect that strengthens the hydrogen bonds within the g-quartet network over the hydrogen bonds in isolated dimers. by means of a combination of stm experiments and dft calculations we compare the d molecular networks formed on deposition of the binary mixtures g-c (purine-pyrimidine pair of complementary bases) and a-c (purine-pyrimidine pair of non-complementary bases). we find that the non-complementary bases segregate into islands of pure a and a network of pure c, whereas the complementary bases g and c form a network that cannot be separated by annealing up to the desorption temperature for c. high-resolution stm images allow us to identify the structures for the enhanced thermal stability as structures that contain g-c bonds, possibly with the same structure as the watson-crick pairs in dna molecules. kühnle, r., et al., . nature , . otero, r., et al., . angewandte chemie int. ed. , . otero, r., et al., . nat. mater. , . otero, r., et al., . angewandte chemie int. ed. , . rosei, f., et al., . science , . schunack, m., et al., . biomedical and pharmaceutical companies are using an increasing number of carbohydrate polymers in the formulation of drugs. one such polymer with highly attractive features is chitosan that can be produced from crustacean shells. chitosan is non-toxic, biocompatible and biodegradable. chitosan can be formulated as nanoparticles or membranes and have enhanced several bioprocesses. among the well-documented features are enhanced drug uptake by tight junction relaxation and enhanced in vivo uptake and protection of nucleic acid formulations. chitosan research has increased throughout this decade. research programs are addressing the potential of chitosan applications but preparations with variable molecular size and charge are not easily available. specifically companies working with the development of new drugs and enhancement of drug functionality are in need of formulation technology that provides well characterized biocompatible material. in the chitosan innovation consortium the danish companies, coloplast, novozymes biopolymers, pipeline biotech, and zgene, together with the research center inano (aarhus university) and bioneer a/s have developed a series of chitosan preparations suitable for research of biopharmaceutical applications (www.chitosan.dk). the ability to obtain functional formulations is currently being tested in both in vitro and in vivo experiments. the consortium participants have established a platform from which chitosan processing, characterization and formulation technology can be extracted. by providing specified chitosan preparations the polymer feature can be adjusted to fit specialized biopharmaceutical applications. high throughput bioprocessing govind rao center for advanced sensor technology, umbc, baltimore, md , usa the post genome era holds a great deal of promise. an enormous number of new proteins await study. these will require sophisticated culture techniques, as cells will have to be grown under large numbers of environmental conditions to elucidate expression triggers. unfortunately, unlike molecular biology, bioreactor technology is little changed since its inception. the primary reason has been a lack of sensor technology that can be readily employed to monitor the cellular environment. we will take a look at the current status of the technology and report on promising optical sensor technology that permits low-cost high throughput cell culture and fermentation. noninvasive sensors that monitor ph, po and pco and high sensitivity solutions for glucose and glutamine measurements will be presented. in addition, the mixing characteristics that determine bioreactor performance will be examined at the small scale and their relevance to the large scale will be demonstrated. on-line monitoring and fed-batch operation in shake flask and micro titre plate cultures jochen büchs , frank kensy , markus jeude , tibor anderlei , barbara dittrich , doris klee : biochemical engineering, rwth aachen university, aachen, germany; ac biotec, jülich, germany; textile chemistry and macromolecular chemistry, rwth aachen university, aachen, germany although methods of molecular biology has led to rational design of micro-organisms to suit our requirements, screening of large numbers of strains and media is still one of the most important tasks in biotechnology. batch operation of shaken bioreactors and absence of on-line monitoring is still the general state of the art for that purpose. it is also a very common practice in screening projects that only the final product titre is measured at the end of the culture for the evaluation of the "best performers". in the recent years several approaches were introduced to follow microbial cultures also in shaken bioreactors like shake flasks or micro titre plates (mtp's). it became obvious that the cultures can behave quite unexpected and most relevant and essential information is lost, if only the final product titre at the end of the cultures is utilised for evaluation. as a result, the screening may be directed to an unknown and non-intended direction or may even fail. this is demonstrated with some examples in this contribution. new methods and techniques are introduced to measure the oxygen and carbon dioxide transfer rate and the respiratory quotient in shake flasks and the optical density, nadh fluorescence, ph and do in mtp's. if the desired product can be fused to a fluorescence protein, like gfp or yfp, also the product formation can be monitored on-line in mtp's. it is of utmost importance that the operating conditions of the applied shaking bioreactors are suitable and shaking motion is not stopped during the measurement. otherwise, e.g. power input, mixing and oxygen supply is interrupted and the micro-organisms will ongoingly rearrange their metabolism to cope with these disturbances of their environmental conditions. another problem of screening is the commonly applied batch operation mode. a lot of microbial systems display an overflow metabolism, substrate or osmotic inhibition or are characterised by a catabolite repressed product formation. in all these cases, batch operation is not the preferred operation mode and, therefore, these cultures are run in fed-batch in larger scales. in particular, it is nearly impossible to screen systems, which are catabolite repressed by the carbon source, in batch mode in defined mineral media. after initial growth has led to a nearly complete consumption of the carbon source, the product formation is derepressed. but then no more carbon source is available to continue with production. it is quite questionable whether in batch operation mode suitable strains can be selected for later fed-batch operation or not. this consideration has resulted in the development of a new technique which allows to run the screening in fed-batch operation mode. this technique is applicable in shake flasks as well as in mtp's. dramatic increases in product titre between -and -folds were observed under these conditions compared to conventional batch screenings. mikkel nordkvist, john villadsen center for microbial biotechnology, technical university of denmark, dk- lyngby. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) efficient mixing and mass transfer are highly important in the chemical industry and in the fermentation industry. poor mixing can result in low yield and variable product quality in a number of cultivation processes, and mass transfer can easily become the limiting step in aerobic cultivations, especially at high cell density. we have tested a new tank reactor system, where liquid is withdrawn from the bottom of a tank, rapidly circulated, and injected back into the bulk liquid through the nozzles of rotary jet heads. liquid feed as well as gas is added in the recirculation loop and thereby distributed via the rotary jet heads. solid feed in powder form can also be added in the loop with advantage, and heat is efficiently removed in a plate-type heat exchanger, which is part of the loop. the system has a very simple design with no internal baffles or heat exchange area, and between batches the rotary jet heads are used for cleaning in place. a number of applications ranging from dispersion of liquid and powder to mass transfer will be presented. mass transfer applications include baker's yeast cultivation and oxidation of lactose to lactobionic acid by a carbohydrate oxidase. fast and accurate analytical information that can be used to rapidly evaluate the interactions between biological systems and bioprocess operations is essential for optimization of biological production processes. we have researched and developed a multiplexed microbioreactor system for the parallel operation of multiple microbial fermentations. the microbioreactors have working volumes from to l, and are instrumented for real-time monitoring of dissolved oxygen, ph and optical density. the growth profiles obtained with escherichia coli compare favorably to results obtained from conventional ml batch bioreactors. we also demonstrate the use of our microbioreactors coupled to dna microarray analy-sis, as a tool for accelerated discovery and elucidation of metabolic pathways and gene expression profiles. the multiplexed system represents a significant step towards high-throughput data acquisition and has the potential to replace current instrumented bioreactors, which are bulky, expensive to run, and require many mechanical manipulations. design of a laboratory scale bioreactor to study solid-state tobacco fermentation m. di giacomo, l. nappi, d. silvestro, m. paolino, d. parente r&d biology department, british american tobacco italia, naples , italy italian toscano cigar production is based on the fermentation of dark fire-cured tobacco. the process starts with the rise of leaf moisture to levels of water activities assuring development of the wild phylloplane microflora in the absence of free water. the intense growth of microorganisms modifies leaf characteristics (ph rise from acidic to alkaline condition) contributing to define the toscano typical smoke profile. tobacco fermentation takes place in great bulks of kg which cause considerable amount of heat evolution as a function of the metabolic activities of the microorganisms. this heat accumulates and temperature can rise to as high as • c. a laboratory cylindrical packed-bed bioreactor was designed to work under isothermal conditions. the reactor was ideal to ferment small quantities of tobacco ( g) and was made up of a column aerated from the bottom with humidified air and placed in a thermoregulated room. experiments were conducted with constant temperature and air flow. moreover, bioenrichment experiments were conducted in the presence of different microbial starter cultures. fermentation courses were monitored by measuring microbial counts and chemical/physical modification of the substrate. with this laboratory scale system we obtained different kinds of information on the role and dynamics of the microorganisms involved in the fermentation process and on the influence of different environmental conditions. for the future, the design of an adiabatic device for tobacco fermentation is planned. on-line liquid chromatography as a process analytical technology for monitoring and control of biotech processes rick e. cooley , : process analytics center of excellence, dionex corporation, sunnyvale, ca, usa; eli lilly and company, indianapolis, in, usa (retired) biotech processes, used to produce an active pharmaceutical ingredient (api), generally differ from small molecule api manufacturing processes in that the starting materials tend to be more variable, more complex, and the product of interest in lower concentration due to the fact that they originate from a biological rather than a chemical process. this complexity and low starting concentration has generated a high level of interest in developing technologies that can be utilized to increase yield and reduce variability in the initial bioreactor phase, as well as, downstream isolation and purification operations. the use of process analytics, or on-line analytical measurements, is a technology approach that can contribute to increased process understanding and control. this presentation will provide examples of how various analytical measurement technologies have been utilized to monitor typical bioprocess unit operations leading to increased automation and control. examples of the use on-line liquid chromatography to monitor bioreactors, process scale chromatography columns, and enzymatic reactions will be presented in more detail. application of advanced monitoring strategies for recombinant protein production karl bayer department of biotechnology, university of natural resources and applied life sciences, vienna a- , austria high yield in combination with the required quality are the key objectives of large-scale production of recombinant proteins using different host/vector systems. however, in the past the efficient production of recombinant proteins was frequently limited due to inadequate exploitation of the cell factory and deficiencies in process design. to achieve optimal exploitation of microbial cell factories the key requirement is to enhance the monitoring capabilities to improve the insight into the host metabolism dynamics and to cope with limited understanding of the interaction of recombinant protein synthesis with host cell metabolism. since each protein exerts an individual influence on the host/vector system, the selection of appropriate analytical methods is even more important. in order to overcome these problems high throughput technology platforms, such as dna microarrays for transcriptome and differential -d electrophoresis (dige) for proteome analysis provide extensive data to screen for significant analytes and provide an appropriate basis for in silico modelling and reverse engineering of regulatory networks. taking advantage from such an iterative process experimental design will be improved and further aid to increase the performance of modelling. in addition, transcriptome data are used to screen fast stress responsive promoters to set up gfp based reporter gene fusions for in-situ monitoring of the metabolic load due to recombinant gene expression. moreover chemometric methods are frequently applied to model complex data from easily obtainable on-line data sets to overcome the limited monitoring capabilities due to the high complexity and nonlinearity of biological reactions and reaction networks. this strategy has been successfully applied to model bdm (bacterial dry matter), pcn (plasmid copy number) and the amount of recombinant protein using data sets acquired from off-gas analysis (o consumption, co evolution), alkaline consumption rate, in-situ capacitance and multi-wavelength fluorescence measurements. at-line monitoring of bioprocess-relevant marker genes using an electric dna-chip britta jürgen , daniel pioch , le thi hoi , jörg albers , rainer hintsche , stefan evers , karl-heinz-maurer , michael hecker , thomas schweder : institut für pharmazie, ernst-moritz-arndt-universität, greifswald, germany; ebiochipsystems, itzehoe, germany; vtb-enzymtechnologie, henkel kgaa, düsseldorf, germany; institut für mikrobiologie, ernst-moritz-arndt-universität, greifswald, germany. e-mail: britta.juergen@uni-greifswald.de (b. jürgen) the gram-positive bacteria bacillus licheniformis and bacillus subtilis represent important industrial hosts for the production of enzymes (e.g., proteases and amylases) or antibiotics (e.g., bacitracin). both organisms are attractive for this purpose because of their apathogenity and their classification as gras organisms (generally regarded as save). moreover, their easy cultivation and their high natural capacity to secrete proteins into the growth medium qualify them for the industrial overproduction of homologous or heterologous proteins (simonen and palva, ) . for the control of the physiological state and the productivity of the production cells efficient analysis tools are of great interest. a prerequisite for the evaluation of the physiological state of cells during industrial fermentation processes is the analysis of so-called process-relevant marker genes, the expression of which indicates unfavorable growth and production conditions (schweder and hecker, ) . by means of proteome and transcriptome analyses we have identified critical process-relevant genes of b. licheniformis and b. subtilis cells under different nutrient limitation conditions and during industrialclose bioprocesses. dna-chips with probes for such process-relevant marker genes could be valuable diagnostic tools for the monitoring of the cellular physiology during microbial bioprocesses. in order to provide reliable tools for the monitoring of the cell physiology during microbial bioprocesses, we have developed a fast mrna analytical approach, which allows an at-line monitoring of the transcriptional activity of selected marker genes during bioprocesses. this approach is based on an easy, fast and reliable rna isolation procedure and the measurement of specific mrnas by means of an electric dna-chip barken et al., a robust bioprocess is crucial to ensure the consistent process performance and provide the high quality of product for drug manufacturing in biopharmaceutical industries. existing methodologies for bioprocess design do not involve establishing mechanisms to achieve the desirable robust bioprocesses and have low capacity in handling uncertainty in the product manufacturing. also, the solutions are often obtained step wise and do not account for interactions between the steps. despite its importance, the robustness of a bioprocess has not been properly defined and studies carried out in statistic sense are often retrospective. in addition, the computational cost is expensive due to using a line search algorithm for finding an optimal operating solution. finally, the existing methodologies are difficult to apply to the whole bioprocess in biopharmaceutical industries. this paper attempts to define rigorously a measure for process robustness and presents a new methodology for evaluating the robustness of bioprocess operations and their performance. the methodology is based on the concept of 'windows of operation' which shows the whole range of possible operating regions. the methodology also establishes a lower bound for the largest variation of a design variable to ensure the performance. these bounds are achieved by min-max optimization techniques. a direct search algorithm has been developed and its computational cost is much lower than the line search algorithm. results include visualization of robust operating regions and a set of indices which compare the performance of different operating strategies. the capabilities and efficiency of this methodology are illustrated by applying it to the centrifuge selection for the clarification of high solids density cell broths. the research work will impact considerably upon robust bioprocess operation. when grown on methanol, pichia pastoris is able to synthesize proteins to high titres as well as secreting and glycosylation, thereby making this organism a very interesting host for the production of recombinant drugs at large scale. the methanol residual concentration has been reported to strongly influence the specific productivity, the optimum concentration being around g/l. a suitable monitoring and control technique is therefore necessary to study and improve the productivity of p. pastoris fermentations. the current research aims at showing how a mid-infrared spectrometer (atr-ftir) can be calibrated in-situ in order to monitor and control p. pastoris fermentations. this method is simple and fast, and eliminates the need of both standards preparation and off-line calibration. it is based on the observation that during fed-batch processes, only substrate and biomass concentrations vary significantly. the method therefore consists in adding a known amount of methanol at the beginning of the process, just after inoculation, and subsequently calibrating the instrument. financial support for the swiss national science foundation is gratefully acknowledged. implantable biomaterials are subjected to inflammatory responses mediated by adherent phagocytes such as monocytes and macrophages. these cellular responses and behavior have been shown to be dependent on the type of protein that adsorbs to the surface. surface modification is necessary to control and prevent protein adsorption, and thus modulate the inflammatory responses. hydrophilic surfaces that adsorb minimal amounts of protein are considered useful for minimizing the inflammatory reactions to biomaterials. in this study we have used two routes to modify polyethylene terephthalate (pet) films: ( ) a wet-chemical method for attachment of linear polyethylene glycol chains (mpeg); and ( ) gas-phase plasma polymerisation of diethylene glycol vinyl ether (degve) to generate peg-like surfaces. the surface chemistry was assessed by x-ray photoelctron spectroscopy (xps), fourier transform infrared spectroscopy (ftir) and time-flight secondary ion mass spectrometry (tof-sims). the two pegylated surfaces were compared for their ability to minimise both fibrinogen adsorption and the adhesion and activation of macrophage-like human leukocytes. adsorbed fibrinogen has been shown to be one of the key proteins in stimulating inflammatory responses to biomaterials. adsorption was investigated quantitatively using i-radiolabeled human fibrinogen. in addition, the conformation of the adsorbed protein was tested using an antifibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. the results showed that pegylated surfaces adsorbed up to % less fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mpeg layers than on the peg-like plasma polymer surfaces. adhesion of in-vitro differentiated macrophage-like u cells was reduced on both the peg-like plasma polymer surfaces and the linear mpeg layers compared to the unmodified pet surface, but cells adhering to the peg-like plasma polymer surfaces secreted less tumor necrosis factor-␣ (tnf-␣) than cells adhering to the linear mpeg layers. thus, the linear mpeg surface is relatively efficient at reducing adhesion of macrophage-like cells, but those cells that do attach are in a more activated and proinflammatory state. analysis of ceramides from biological samples m. budvytiene , j. liesiene , b. niemeyer , n. ceramides in human skin play an important role in the regulation of cell growth, differentiation and apoptosis. moreover, they are involved in the numerous signaling pathways. the growing interest in the investigations of ceramides physiological functions requires efficient separation methods of ceramides from biological resources and sensitive analytical methods. in this work some sensitive and selective methods, involving thin layer chromatography (tlc), high performance liquid chromatography (hplc), mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) were employed for the separation and characterization of ceramides from human foreskin. epidermal lipids were extracted from human foreskin for h at room temperature using three solvent mixtures (chloroform/ethanol/water : : . , v/v/v); (chloroform/ethanol : , v/v), and (chloroform/ethanol : , v/v). ceramides retention char-acteristics in tlc and hplc were compared with the retention of commercial standards. the best separation was obtained using normal phase column packed with hilic silica m. the elution was performed using mixture of chloroform and ethanol / (v/v) as an eluent. two commercial standards n-stearoyl-sphingosine cer(ns), r f = . , and n-palmitoyl-sphingosine cer(np), r f = . , were selectively separated with hplc system in above mentioned conditions. the retention time of cer(np) and cer(ns) was . and . min, respectively. the same lipids were detected by hplc in the human foreskin extracts. the structure of the lipids from collected fractions was confirmed by means mass spectrometry and nmr. the physiological functions of the separated ceramides are investigated. production recombinant human thymosin-␣ overexpressed as intein fusion protein in e. coli roman s. esipov, vasily n. stepanenko, anatoly i. miroshnikov, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya / , moscow, russia. e-mail: esipov@ibch.ru (roman s. esipov) medicines based on polypeptides consisting of and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin-␣ production system intein mediated purification with affinity chitin (impac system)-binding tag has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin-␣ . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl and we have found that, in case of intein-thymosin-␣ , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of . - mm zinc chloride in buffers on all stages of thymosin ␣ isolation. the structure of recombinant thymosin-␣ of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. we present a microbioreactor with thermoelectric cooling to inactivate cellular metabolism by cell culture freezing. small-scale cultivation methods have gained increased importance and their development has been supported by advances in bioprocess monitoring methods. yet efficient sampling methods for off-line analysis remain important where in-situ real-time measurements are difficult, for example for intracellular metabolite concentration or enzyme activity measurements, and for all methods which are invasive by nature, such as protein purification. freeze-stop measurements of metabolite levels are ideal, because they are inert, i.e. do not require the addition of a chemical. in large systems, the chilling time is often the limiting factor, and alternative methods for cell metabolism inactivation are required, such as the spraying of the cell suspension in % methanol at a temperature of − • c, or the use of boiling buffered ethanol. due to the small thermal mass of microsystems, shorter chilling times can be expected. sample cooling to • c in a microbioreactor has been presented previously. in this contribution, we investigate sample freezing to completely inactivate the cell metabolism from microbioreactor working volumes of approximately l. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic , alvin w. nienow , ian w. taylor , ryan hicks , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w ). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. when choosing an expression host for production of a specific recombinant protein, one can essentially select from a multiplicity of different systems. while e. coli bacterium is usually the starting point for any cloning and expression effort, there is no universal expression host that would work optimally for all proteins. practical issues to consider include e.g. need for post-translational modifications and protease activity of the host. we have produced recombinant hiv- nef in different host cell systems: e. coli, p. pastoris yeast and stable drosophila s insect cells. using strain/cell line development, production and purification data from practical experiments, we were able to conduct a techno-economical comparison of the different host cell systems. the annual production goal was set at mg of high-purity nef. this was supposed to be produced campaign-wise in - batches using laboratory-scale bioreactors and other equipment. in this study, it was shown that although the production costs of the different systems were in the same range, the production in e. coli was most inexpensive, and the s cell system was the most expensive. regardless of the selected host system, the labour costs incurred the most expenses. when comparing different stages of the work (strain/cell line development, bioreactor production and down-stream processing), the strain development, the most man-hours demanding stage, involved approximately half of the costs of every production system. although e. coli was the most inexpensive host system for producing nef, it has some definite disadvantages: e.g. the production of endotoxins and the disability to perform post-translational modifications. if these disadvantages are of importance, the production must be done using more expensive system. modelling and control of industrial fermentation j.k. rasmussen, s.b. jørgensen capec, department of chemical engineering, technical university of denmark, dk- lyngby, denmark. e-mail: jkr@kt.dtu.dk (j.k. rasmussen) fed-batch processes play a very important role in chemical and biochemical industry. fermentations in biochemical industry are most often carried out as fed-batch processes. present control schemes do not utilize the full potential of the production facilities and may fail to achieve uniform product quality and optimal productivity. the introduction of model based control strategies is considered difficult because suitable models are not readily available. first principle engineering models can be used but the usually limited knowledge of the regulatory network in the micro-organism makes model development very time consuming. another strategy is to use a purely data-driven approach where only limited prior knowledge of the process is required. a framework for generation of such blackbox models is used in this project. this framework is called "grid of linear models" (golm), it uses a large number of linear models which each describes the behaviour of the process within a certain time interval. the combination of these models results in a model which covers the entire time span of the fermentation and approximates the nonlinear time varying behaviour. a procedure for deriving golm models from operational data has been developed and because they consist of a large set of linear models it makes them suitable for model predictive control implementation with iterative learning capabilities from batch to batch. iterative learning model predictive control (ilmpc) based on a golm model is being implemented on a fermentor at novozymes a/s. the results will be evaluated in terms of the controller's capability to ensure uniform product quality and reject both intra and inter batch process disturbances. the model based approach renders optimization of the process recipe possible by using the ilmpc capability. this opportunity provides a great potential for increase of productivity and reduction of cost. j.m. viader-salvadó, j.a. fuentes-garibay, l.j. galán-wong, m. guerrero-olazarán institute of biotechnology, biological science school, autonomous university of nuevo león, san nicolás de los garza, n.l., méxico, the production of recombinant trypsin and trypsinogen has been reported as difficult due to a probably toxicity on host or its instability. in an effort to attain high-level production of shrimp trypsin for aquaculture applications, we have evaluated shrimp trypsinogen production by recombinant pichia pastoris strains in -l bioreactors. a p. pastoris gs mut+ containing in its genome the litopenaeus vannamei trypsinogen cdna fused in frame to saccharomyces cerevisiae ␣-factor secretion signal, previously constructed in our laboratory, was used. four three-step fermentations (glycerol batch, glycerol and methanol fed-batch) were carried out. the glycerol batch step ( l of basal salts medium, g/l glycerol, . ml biotin . %, . ml ptm , l antifoam, ph adjusted to with % nh oh) was carried out until glycerol was completely exhausted ( h). the glycerol fed-batch step was carried out feeding with % glycerol ( ml/l biotin . % and ptm ) at . ml/min by h and min of posterior starvation. the methanol fed-batch step was carried out feeding with % methanol ( ml/l biotin . % and ptm ) by h using a methanol concentration on/off feedback control to maintain constant the methanol concentration in the culture medium to g/l. in all the fermentations the air flow rate and the agitation were set at l/min and - rpm, respectively. with the four fermentation assays, the influence of the ph and temperature in the production phase to the recombinant shrimp trypsinogen production were evaluated. in the four fermentations, at the end of the second step a biomass of g/l wet weight were obtained (od ). the methanol demand in the four fermentations surprisingly was not increasing rather initially it was . ml/min, after h decreased . times for h, increased to . ml/min for h and afterwards it was decreased manually to a constant value of . ml/min for that the dissolved oxygen will not decrease to values less than % (last h). the total protein amount in the culture medium supernatant increased during the production step until values of . g/l (assay at ph ), . times more than the worst assay (ph ) recombinant shrimp trypsinogen production was confirmed by sds-page (about mg/l) and trypsin enzymatic activity was detected using bapna as substrate after trypsinogen activation with shrimp hepatopancreas extract. large conformational change on giant dna induced by ascorbic acid: a nobel scheme on its antioxidative activity yuko yoshikawa, emi sakai, yoshiko oda department of food and nutrition, nagoya bunri college, nagoya - , japan ascorbic acid is often regarded as an antioxidant in vivo, where it protects against dna damage by scavenging reactive oxygen species. in the present, we will show another potent scenario on the protective effect of ascorbic acid through a significant structural change of giant dna. recently, we examined the effect of ascorbic acid on the higher order structure of dna through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant dna molecules, where elongated and compact parts coexist along a molecular chain. the results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. as the extension, here we study the protective effect against double-strand breaks by reactive oxygen at different concentrations of ascorbic acid, in relation to the change of the higher order structure of giant dna. we have performed a real time observation on the doublestrand breaks on individual dna molecules by use of fluorescence microscopy. we have found that the double-strand break is markedly protected when ascorbic acid exists over millimolar concentrations. it is found that such a protective effect of ascorbic acid corresponds well to the above mentioned change on the higher order structure of dna. it has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on dna may be of physiological significance. , y., et al., . febs lett. , - . yoshikawa, y., et al., . plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. there are many process both industrial and food based which employ protein hydrolysis and hydrolytic products have a wide variety of applications from industrial fermentation media to food additives. traditionally, proteins are hydrolysed by chemical means. acid hydrolysates of protein are used to produce food ingredients and flavour compounds. however, hydrolysis by chemical reagents produce potentially hazardous byproducts and these non-selective hydrolysis products cannot easily be defined. the use of enzymes allows for selective hydrolysis of protein and thus produces a potentially safer and more defined material. the present investigation describes the effects of substrate, enzyme and hydrolysate concentration on the hydrolysis of corn gluten. the corn gluten was hydrolysed by a commercial protease preparation neutrase. the protein hydrolysis reactions were carried out in . l of aqueous solutions at the temperature of • c and ph and were monitored by using ph-stat method. the degree of hydrolysis (%) and soluble protein concentration depending on time were investigated by using , , , , and % (w/v) substrate concentrations; and . , . , . , . and . , % (v/v) enzyme concentrations; and , , and % (v/v) this paper describes medium optimization for urease production by aspergillus niger ptcc by one-factor-at-a-time and orthogonal array design methods. the one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. the effects of nutritional components for urease production by a. niger ptcc in the first and second stages were in order of urea > niso > sucrose >kh po > k hpo > cacl > yeast extract > mgso and yeast extract > sucrose > k hpo > kh po > urea > cacl > niso , respectively. the optimal concentrations of nutritional components for improved urease production were determined as g/l sucrose, . g/l urea, . g/l yeast extract, . g/l niso · h o, . g/l mgso · h o, . g/l cacl , . g/l kh po , and . g/l k hpo . these results showed that urea, niso , yeast extract and sucrose had significant effect on urease production by a. niger ptcc . tween and mgso had negligible effect on urease production by this strain. the subsequent confirmation experiments determined the validity of the models. maximum urease activity in optimized media by onefactor-at-a-time and orthogonal array methods were about . and . times greater than with the basal medium, respectively. carbon sources create fingerprint fermentation characteristics pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre lab, department of chemical engineering, ankara university, ankara, turkey; ib lab, department of chemical engineering, metu, ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. Ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e. glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e. serine alkaline protease (sap; ec . . . ),) and ␤-lactamase (ec . . . ), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. . - -fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide Ç alık bre lab, department of chemical engineering, ankara university, ankara, turkey ␣-amylase (e.c. . . . ) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣- , glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b- ) which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in . dm airfiltered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs ). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled . and . dm systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc s ultracentrifuge, ␣-amylase activity was measured by the dns method (bernfeld, ) . amino acid concentrations were determined with a hplc (waters), protein and organic acid concentrations were measured with a hpce (waters, quanta e) (Ç alık et al., ) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method (rainer, ) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources i.e., glucose, fructose, maltose, lactose and soluble starch; n sources i.e., (nh ) hpo , (nh ) so , and nh cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and byproduct concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar Ç alık iblab, department of chemical engineering, metu, ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc ::bal carrying recombinant e. coli on a defined medium with . kg/m glucose were investigated in order to finetune the bioreactor performance, in v = dm batch bioreactors at five different conditions with the parameters at, i.e. q /v r = . vvm and n = , , and min − and; q /v r = . vvm and n = min − . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph . are optimum for maximum bal activity, i.e. u/cm at h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains metabolites and reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e.coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. the detection and diagnosis of pathogenic bacteria causing many diseases to the human body is an area of important research to public welfare. food is the most important energy source to humans, but it can give rise to disease caused by pathogenic bacteria not performing adequate detection tests. oligonucleotide-based microarrays are becoming increasingly useful for the analysis of expression profiles and polymorphisms among interested genes. here, we checked the possibility of development of oligonucleotide-based microarrays for detection and diagnosis of foodborne pathogenic bacteria. the oligonucleotide chip technology was applied to one control strain and seven foodborne pathogenic bacteria strains. it was designed repeated spots of eight hyperspecific and two highly conserved (control) capture probes from s rdna sequences. in order to validate experimental quality and to certificate specificities among specific spots at a glance by d and d views, quantitative visualization tool was developed. using the proposed oligonucleotide chip, we could classify and diagnose species and even subtypes of some pathogens. induction of in vitro neuro-muscular junctions using neuroblastoma and fibroblast cell lines for facilitating receptor-binding studies with botulinum toxin arindam chaudhury, bal ram singh department of chemistry and biochemistry, university of massachusetts dartmouth, old westport road, north dartmouth, ma - , usa. e-mail: g achaudhury@umassd.edu (a. chaudhury) botulinum toxin (bont), the most potent biological toxin known, is responsible for botulism, a fatal paralytic disease of the neuromuscular transmission. it blocks the release of acetylcholine at the neurotransmitter junction of the synapse. the objective of the current study was to induce in vitro neuro-muscular junctions through co-culturing of nerve and precursor-muscle cell lines. presently no known primary cultures or cell lines are available for nerve-muscle co-culture, thus validating the current work. j - t fibroblast cell line was first adapted to grow in media conducive for growth of sh-sy y neuroblastoma cell line. the two cell lines were then splitted and co-cultured and observed for junction formations. light and fluorescent microscopic studies revealed en plaque (twitch-type) and en grappe (bulbous nerve endings) nerve-muscle junctions. growth rate of j - t cells decreased substantially when the media was initially changed. structurally they were more spindle-shaped than the normal reticular shapes of j - t cells, when grown in a media tailor-made for them. the formation of nerve-muscle junctions were confirmed using markers specific for each cell type. future work is focusing on receptor identification for the botulinum toxin in the established in vitro neuro-muscular junctions and also the transcellular translocation of the toxin. fourier-transform infrared (ftir) spectrometers have recently enjoyed widespread popularity in bioprocess monitoring applications due to their non-invasiveness and in-situ sterilizability. their online applicability creates an interesting opportunity for process control and optimization. however, the precision and accuracy of the predicted analyte concentration values directly depend on the quality of the measured signal and the robustness of the calibration model. instability and time drift in the measured spectra are currently one of the main obstacles in ftir monitoring. the intensity of the detected signal is influenced both by random noise and structural drifts and offsets. as a result, it is often necessary to scale the measured spectrum with respect to a constant reference spectrum, a technique similar to the internal standard approach used in analytical assays, such as hplc. applying this technique has lead to a noticeable decrease in the standard error of prediction in the monitoring of an anaerobic s fermentation of the saccharomyces cerevisiae yeast. in order to test the robustness of the calibration model and to increase its resistance to signal instability, random spikes of known amounts of analytes were introduced into the measured medium. this approach can be used to fine-tune the calibration model on-line and is currently one of the aspects investigated in this laboratory. the effect of the stringent response induction on l-valine biosynthesis by corynebacterium glutamicum ilze denina , , longina paegle , liga zala , , maija ruklisha : faculty of biology, university of latvia, kronvalda blvd. , riga lv- , latvia; institute of microbiology and biotechnology, university of latvia, kronvalda blvd. , riga lv- , latvia. e-mail: ilzede@hotmail.com (i. denina) the present study was focused on methods of the stringent response induction and on investigation of its effect on valine overproduction by isoleucine auxotrophs of corynebacterium glutamicum. the intracellular level of guanosine -diphosphate diphosphate (ppgpp) increased and bacterial growth rate (µ) decreased during the short-term experiments when the exponentially growing cells were exposed to isoleucine limited conditions. the induction of the cellular stringent response resulted in a drastic increase in the activity of acetoxydroxy acid synthase (ahas), also by a significant increase in valine production. in contrast, an increase in ahas activity and valine synthesis by c. glutamicum was not achieved when bacterial growth was down-regulated in a ppgpp-independent manner. these results demonstrated that induction of the ppgpp-mediated stringent response might be significant in order to increase valine overproduction by c. glutamicum. infections with human cytomegalovirus (hcmv) continue to be an important health problem in certain patient populations, such as newborns, graft recipients of solid organs, or bone marrow and aids patients. in these groups, hcmv is a major cause of morbidity and mortality. the complex biology of hcmv necessarily begins with an initial interaction between the envelope of the infectious virus and the host cell. glycoprotein b (gb) is the major antigen, on the envelope of hcmv, for the induction of neutralizing antibodies. the region between aa and of hcmv gb (termed antigenic domain , ad- ) has been identified as the immunodominant target for the humoral immune response following natural infection. screening methods for detection of neutralizing antibodies have not been used because they are costly and labor intensive and thus far are not feasible for use on a large scale. for the development of reliable and inexpensive serodiagnostic tests the ad- of hcmv glycoprotein gp , which are known to bind neutralizing antibodies, was expressed in prokaryotic systems. in this work, one prokaryotically expressed fusion protein which codifies ad- with ␤-galactosidase was used. the influence of different process conditions, on the production of the fusion protein containing the ad- as well as sugars addition to the fermentation medium was investigated. in order to analyze the expression of fusion protein, the ␤-galactosidase activity was followed throughout the fermentation. lysis process was also optimized and some final confirmation tests about protein antigenicity were performed. polyenzymic systems for the preparation of drugs based on modified nucleosides d. chuvikovsky, r. esipov, t. muravyova, a. miroshnikov shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya / , moscow , russia. e-mail: esipov@ibch.ru (r. esipov) considerable progress in the preparation of nucleoside analogues was achieved by combination of chemical and biochemical transformations. enzyme-catalyzed chemical transformation is now widely recognized as practical alternative to traditional organic synthesis in pharmaceutical and chemical industries. pentofuranosyltransfer reaction catalyzed by nucleoside phosphorylases was successfully employed for the synthesis of a variety of base-and sugar-modified nucleosides. enzymes involved in the metabolism of ribose phosphate and deoxyribose phosphate, such as ribokinase and phosphopentomutase, were used for the preparation of sugar-modified nucleosides. nucleosides phosphorylases (thymidine phosphorylase (tp), uridine phosphorylase (up) and purine nucleoside phosphorylase (pnp)), ribokinase and phosphopentomutase from e. coli have been cloned and overexpressed in e. coli. fast and efficient methods for the purification of nucleosides phosphorylases have been developed. the amount of purified protein was about mg/l of cell culture, corresponding to , , and units, respectively, of the up, tp and pnp. synthesis of medicinal drugs ribavirin ( -(␤-d-ribofuranosyl)- , , -triazole- -carboxamide), cladribine ( -chloroadenine- -␤-d- -deoxyribofuranoside) and fludarabine ( -fluoroadenine- -␤-darabinofuranoside) with the use of recombinant enzymes were studied. several important factors affecting the modified nucleosides production (ph, temperature, enzyme concentration, donor/acceptor ratio) were investigated and optimized. under optimum conditions ribavirin, cladribine and fludarabine produced in the reaction mixture in yields of , and %, referred to , , -triazole- -carboxamide, -chloroadenosine and -fluoroadenosine, respectively. aggregation and adsorption of fibroin molecules in aqueous solution won hur school of biotechnology and bioengineering, kangwon national university, chunchon - , korea. e-mail: wonhur@kangwon.ac.kr fibroin, the structural protein from bombyx mori, is composed of heavy chain (generally called 'fibroin') and light chain polypeptides of about and kda, respectively. this study investigated the aggregation of fibroin and the adsorption between fibroin and surfaces. the variations of particle size and zeta potential were investigated by electrophoretic light scattering spectrophotometer (els). the adsorption of fibroin on surface was investigated in a continuous flow system by biacore applied surface plasmon resonance(spr) technique. the particle size and zeta potential range of aqueous fibroin were nm, ± mv, respectively. iso-electric point(ph iep )of fibroin was ph . . the amount of fibroin adsorbed on a gold surface was less than . g/ml even in the presence of high concentration of fibroin. the modification of gold surface was accomplished by applying chemicals known to form self-assembled monolayer, those are carrying nh + , coo − , benzene ring and peptide that similar structure with fibroin. the adsorbed amount of fibroin on the self-assembly monolayers (sams) increased in the following order: nh + > benzene ring > coo − > peptide surface. the deposition of fibroin in aqueous solution on non-waven fabric was affected by nacl and high temperature. ph influences metabolite profiling of ␤-lactamse producing b. licheniformis nazarİleri , pınar Ç alık , ali Şengül : ib lab, department of chemical engineering, metu, ankara, turkey; gülhane sch med, dept immunol, ankara, turkey. e-mail: e @metu.edu.tr (n.İleri) ph conditions in the bioreactor affect product and by-product formations by influencing metabolic pathways and changing metabolic fluxes, based on its influence on, i.e. dna transcription, protein synthesis, transport mechanism, atp generation and cellular energetics. whereupon, some fermentation processes favours uncontrolled-ph conditions while others favours controlled-ph conditions. on the bases of the interactions between the cell and the bioreactor through a process, carried out at either uncontrolled-or controlled-ph conditions, intracellular ph can be widely different and variable during the fermentation process. consequently, if one aims towards a quantitative understanding of the cell metabolism, one has to take into account the time variations of the intracellular ph and its effects on the in-vivo kinetics of the metabolic steps involved. moreover, since the presence of dormant or dead cells in the cultivation medium have negative effect on the synthesis of the production of desired product; the physiological state of the culture has great importance. in this context, the effects of ph on the regulation of intracellular ph, transport mechanism, and metabolic activity of b. licheniformis during production of ␤-lactamase (ec . . . ), an industrial enzyme catalyzing the hydrolysis of beta-lactam ring in beta-lactam antibiotics, was investigated. in addition, the physiological state of the organism and its effect on the production were observed. the optimal controlled-ph operation was found to be ph . with u/cm enzyme activity. the intracellular and extracellular na + , k + ion concentrations increased significantly throughout the process with increasing ph. on the other hand, the intracellular nh + ion concentration was relatively constant. isolation and characterization of angiotensin-i converting enzyme inhibitory peptides by use of anti-peptide antibody fida hasan , megumi kitagawa , yoichi kumada , naoya hashimoto , masami shiiba , shigeo katoh , masaaki terashima : graduate school of science and technology, kobe university, nada, kobe - , japan; department of human science, kobe college, okadayama, nishinomiya - , japan inhibitory peptides against angiotensin-i converting enzyme can be promising bio-functional peptides as natural alternatives for the non-peptide ace inhibitory drugs. these peptides are inactive within sequences of parent proteins and can be released during enzymatic digestion or food processing. immunointeraction is very effective for the purification of proteins and peptides with high purity. in this study, ace inhibitory peptides from hydrolysate of bonito meat were isolated by an anti-peptide antibody column and hplc, and kinetics of production of these ace inhibitory peptides was studies. an anti-peptide antibody against an ace inhibitory peptide, which was found by kohama et al. from tuna was obtained by immunization of the antigen peptide pc-iace (kkpthikwgd). water extract of bonito meat was digested at • c in a modified gastric juice, . mg/ml nacl containing g/ml pepsin (ph ). peptides in hydrolysates were purified by use of an affinity column coupled with the antipeptide antibody and separated by hplc equipped with a reverse phase column (cosmosil c -ms-ii, . cm × cm). amino acid sequences and ic values of the potent ace inhibitory peptides were determined. sds-page and western blotting experiments clarified that bonito protein contained peptides having similar sequence to the antigen peptide. a fraction of retention time - min in hplc purification samples showed high inhibitory activity, and several peptides in this fraction were separated. after h digestion, two major inhibitory peptides, herdpthikwgd and pthikwgd, were found to be relatively stable in the gastric juice. kluyveromyces marxianus physiology on several levels of carbon, nitrogen sources and oxygenation during inulinase production silva-santisteban yépez, o. bernardo, francisco maugeri department of food eng./unicamp, - , campinas, sp-brazil. email: maugeri@fea.unicamp.br (f. maugeri) inulinase produced by yeasts is an interesting alternative compared with the one produced by filamentous molds, as culture conditions can be better controlled. during the assays, it was observed that inulinase production levels varied with nutritional conditions in batch culture. kluyveromyces marxianus atcc culture is described by two main phases, the first one being the growth phase, where substrate consumption and basal inulinase production were performed, and the second one being the phase where some metabolites are uptaken and high inulinase production is observed. the metabolic fluxes analyses were used to describe the cell physiology in the first phase, in a variety of conditions of sucrose and ammonium sulfate concentration and aeration condition. the metabolic network included the main metabolic pathways such as glycolisis, pentose phosphate pathway, krebs cycle, oxidative phosphorylation and biomass biosynthesis. the physiology in this phase was correlated with high inulinase production in the second phase. it was also noticed that inulinase production diminished when sucrose was in high concentration, leading, additionally, to ethanol production. in these terms, it was unveiled a kind of crabtree effect performed by this strain. forward extraction of l-aspartic acid from fermentation broths by reverse micelles and backward extraction by gas hydrate methodÖ. aydogan , e. bayraktar ,Ü. mehmetoglu , m. parlaktuna , t. mehmetoglu : department of chemical engineering, faculty of engineering, ankara university, tandogan, ankara , turkey; department of petroleum and natural gas engineering, middle east technical university, ankara , turkey. e-mail: mehmet@eng.ankara.edu.tr (Ü. mehmetoglu) recently gas hydrate method has been applied as a technique for backward extraction of amino acids from reverse micelle systems. in this study, backward extraction of l-aspartic acid was investigated by gas hydrate method. at the first stage, production of l-aspartic acid was carried out using ml of . m ammonium fumarate (ph . ) as substrate at circc in an orbital shaker at rpm for h. e. coli (atcc ) was used as biocatalyst. at the end of reaction excess fumaric acid was extracted in reverse micelle phase. then forward extraction of l-aspartic acid was carried out with injection method in reverse micelle phase. for back extraction, co is used to form gas hydrates crystalline structure. back extraction experiments were carried out between - bar g pressure and at circc. at the end of the back extraction l-aspartic acid was obtained in crystalline form. the results indicate that recovery of l-aspartic acid from reverse micelles by forming gas hydrate can be achieved with a yield of . %. consequently, gas hydrate method can be used as a new technique for backward extraction of amino acids from reverse micelles. aerobic and anaerobic cultivations of aspergillus niger on different nitrogen sources susan meijer, gianni panagiotou, lisbeth olsson and jens nielsen center of microbial biotechnology, biocentrum-dtu, technical university of denmark, dk- lyngby, denmark in this study, we aim at creating a succinic acid producing strain of a. niger. a. niger is known to be a strictly aerobic organism, meaning it is not able to use the reductive part of the tca cycle to produce succinate. during aerobic growth a. niger uses oxygen as electron acceptor in the respiratory chain, thereby reoxidizing the produced nadh to nad + . however, under anaerobic conditions other compounds than oxygen, such as no − are required as electron acceptor (denitrification). this process consists of no − reduction to nh + coupled to substrate-level phosphorylation that supports growth under anaerobic conditions. in the present study, our aim was to investigate the effect of different nitrogen sources on the physiology of a. niger during growth under aerobic and anaerobic conditions. aerobic growth experiments on three different nitrogen sources; ammonium, nitrate and nitrite, showed that ammonium and nitrate could be consumed by the filamentous fungus. nitrite on the other hand could not facilitate growth, indicating the absence of a nitrite uptake system. however, under anaerobic conditions notable growth was only observed on nitrate. these data support the hypothesis of the existence of an alternative electron acceptor that might facilitate anaerobic growth of a. niger. among the therapeutic proteins derived from mammalian cells, recombinant antibodies received a great deal of attention as a prominent product through biotech pipelines toward the marketplace. they now occupy about % of the estimated medicines in clinical development and many more antibodies which lead the value of the market going forward are reported. there are various environmental factors affecting rcho cell cultures such as medium components, temperature, ph, and byproducts (ammonia, lactate, and, etc.) . because most of mammalian cells are very sensitive to their environmental change, appropriate control of environmental parameters is a very important consideration to enhance cell growth and production of target proteins. balanced addition of limiting medium components plays an essential role on improvement of cell density and product concentration. temperature and ph are easily adjustable process parameters, being reported to influence cell growth and recombinant protein production. ammonia and lactate are well-known byproducts which have an inhibitory effect on cell growth when their concentrations exceed a specific level. in this work, effects of various environmental factors including temperature, ph, amino acids, vitamins, hormones, and metabolic byproducts on cell growth and recombinant antibody production were investigated in the cultivation of recombinant chinese hamster ovary cells. the most suitable condition of each environmental condition was proposed for enhancement of the cell growth and the productivity of recombinant antibody. the present study was carried out in order to assess the protective effects of calycosin- -o-␤-d-glucopyranoside isolated from astragali radix (ar) on hyaluronidase (haase) and the recombinant human interleukin- ␤ (il- ␤) induced matrix degradation in human articular cartilage and chondrocytes. we isolated the active component from the n-butanol soluble fraction of ar as haase inhibitor and structurally identified as calycosin- -o-␤-d-glucopyranoside by lc-ms, ir, h nmr, and c nmr analyses. the protective effect of arbu on the matrix gene expression of immortalized chondrocyte cell line c- /i treated with haase was investigated using a reverse transcription polymerase chain reaction (rt-pcr). its effect on haase and il- ␤-induced matrix degradation in human articular cartilage was determined by using a staining method and calculating the amount of degraded glycosaminoglycan (gag) from the cultured media. pretreatment with calycosin- -o-␤-d-glucopyranoside effectively protected against matrix degradation of the human chondrocytes and articular cartilage. therefore, it would appear that calycosin- -o-␤-d-glucopyranoside from ar is a potential natural ant-inflammatory or anti-osteoarthritis agent and can be effectively used to protect against proteoglycan (pg) degradation. catechol-o-methyltransferase (comt) is an enzyme that catalyses a variety of endogenous and exogenous catechol substrates by transferring a methyl group from s-adenosylmethionine (sam) to either the meta-or the para-hydroxyl group of the catechol ring. the enzyme has a physiological role in the metabolism of the catechol estrogens, inactivation of the catecholamine neurotransmitters such as dopamine and epinephrine and detoxification of a variety of xenobiotic catechols. comt activity has been identified in various tissues; however with the developments in molecular biology and gene technology, the production and purification of large amounts of recombinant comt is a good option for biochemical, pharmacological and structural studies. in this work, cultures of recombinant e. coli harbouring a model plasmid were grown in a ml shake-flask containing ml of complex medium. the influence of medium composition and induction time on comt production, recovery and clarification by sonication, ammonium sulphate precipitation and purification by hydrophobic interaction chromatography onto a butyl-sepharose column will be presented and discussed. bioactive bacterial exopolysaccharides corinne sinquin , karim senni , jacqueline ratiskol , farida guéniche , jean guézennec , gaston godeau , sylvia colliec-jouault : ifremer, nantes cedex , france; ea université rené descartes, montrouge, france. e-mail: corinne.sinquin@ifremer.fr (c. sinquin) interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce in an aerobic carbohydrate-based medium, unusual extracellular polymers (guezennec, ; colliec-jouault et al., ) . these exopolysaccharides (eps) present original structural features that can be modified to design innovative bioactive compounds and improve their specificity. with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of one eps produced by vibrio diabolicus have been undertaken (raguenes et al., ) . the structure of the native eps has been described (rougeaux et al., ) the potential of the eps derivatives as therapeutical agents will be presented. physiological responses of e. coli to glucose and oxygen shifts in fed-batch fermentations jaakko soini , christina saarimaa , arne matzen , peter neubauer : bioprocess engineering laboratory, department of process and environmental engineering, university of oulu, oulu fi- , finland; sanofi-aventis, germany. e-mail: jaakko.soini@oulu.fi (j. soini) in high-cell density fermentations e. coli cells are often subjects of transient changes in microenvironment around them. these changes can be, for example, medium component gradients or differences in oxygen availability. we have studied the physiological response of e. coli w cells to simultaneous oxygen limitation and overfeeding of glucose. the aim is to obtain more information of physiological changes for better understanding of the bottlenecks in such processes. the response of the cells for glucose and oxygen shifts was studied by analyzing key metabolites and proteins and mrna transcript levels. the transcript levels were measured using a sandwich hybridization technique (rautio et al., ) proteomic analysis was carried out by d-electrophoresis and the metabolite analysis by hplc. the main focus of this study is to monitor the expression patterns of marker genes involved in mixed acid fermentation, glycolytic pathway and tricarbonic acid cycle. rautio et al., . sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates. microb. cell fact. influence of ner on genetic instability of the (ctg/cag) tracts in bacterial chromosome sylwia szwarocka , paweł parniewski : department of microbiology and immunology, university of Łódź, - Łódź, banacha / , poland; centre for medical biology, polish academy of sciences, - Łódź, lodowa , poland many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy and friedreich's ataxia, are associated with expansions of triplet repeat sequences (trs) (cgg/ccg, ctg/cag and gaa/ttc) in or near specific genes. mechanisms that mediate the expansions and deletions of trs include dna replication, repair and recombination. many investigations suggest that the structural properties of the trs play a consequential role in their genetic instabilities. nucleotide excision repair (ner) is the major cellular system in both prokaryotes and eukaryotes and recognises damages due to distortion of the dna helix. involvement of ner in the hairpin loop repair that can form within ctg tracts has been reported. the participation of this repair systems in the trs instability was investigated in e. coli only on multicopy plasmids. the results showed that deficiency of some ner functions dramatically affects the stability of long (ctg/cag) inserts. in this work we present a chromosomal model to study the instability of the trs in e. coli. we introduced the (ctg/cag)n tracts into the chromosome of e. coli and used strains with some deficiency of the ner and investigated genetic stability of these tracts after multiple recultivations. in general, our results show that the (ctg/cag)n repeats are much more stable in the chromosome than in plasmids. these data may suggest that instability of trs in plasmids is associated with interaction between repetitive tracts on different plasmid molecules inside the cell. however, mutations of ner genes may increase (uvra and uvrb mutants) or decrease (uvrc and uvrd mutants) stability of the trs in the e. coli chromosome. this study was partially funded by the kbn grant p a . performance analyses of a multi-stage integrated fermentation process for lactic acid production hsun-tung lin, feng-sheng wang department of chemical engineering, national chung cheng university, chia-yi - , taiwan. e-mail: chmfsw@ccu.edu.tw (f.s. wang) in this work, we considered a multi-stage integrated continuous fermentation process for producing lactic acid. each stage consists of a mixing tank, a fermenter, a cell recycle unit and an extractor. the generalized kinetic model is first applied to formulate the integrated process. we have compared the overall productivity and conversion of the integrated process with those of two simplified processes. from the design equations, we obtain that three processes have the identical overall conversion. however, the proposed process has the greatest overall productivity. the specific kinetic model for lactic production (youssef et al., ) was applied to the integrated process in order to find the maximum overall productivity. two optimization problems are respectively considered to determine the optimal stages, operating conditions and design variables. the first problem supposes that the integrated process has the equal working volume ratio for each fermenter. such a process requires four stages to yield the maximum overall productivity and the nearly complete overall conversion. however, if the working volume ratio for each stage is considered as the decision variables in the second optimization problem, three stages is enough to achieve the identical overall productivity. youssef, c.b., guillou, v., olmos-dichara, a., . contr. eng. pract. , - . modelling of the binding of ligands to macromolecules jørgen m. mollerup department of chemical engineering, building , dtu, lyngby, denmark a variety of factors that govern the properties of proteins are utilized in the development of chromatographic processes for the recovery of biological products including the binding and release of protons, the non-covalent association with non-polar groups (often hydrophobic interactions), the association of small ions (ion exchange) and the highly specific antigen-antibody interaction (affinity interactions). the fulcrum point in the understanding and modelling a chromatographic separation is the adsorption isotherm that determines the peak shape at preparative load. to enable an efficient chromatographic process development strategy it is necessary to conduct theoretical and experimental investigations of the adsorptive behaviour of proteins. thermodynamically consistent models for ion exchange chromatography and hydrophobic interaction chromatography have been developed and can be utilised in the simulation of a chromatographic separation. besides, measurements on hic media can be utilised to determine the cohn salting-out coefficient. the lig-and binding process can frequently be coupled to associated structure changes in the protein, the ligand or both. this gives rise to nonlinear adsorptive behaviour known as cooperativity which cannot be modelled using conventional models which displays convex behaviour. examples of cooperative behaviour are the reversible binding of oxygen and carbon monoxide to haemoglobins and the binding of nad + to yeast glyceraldehydes -phosphate dehydrogenase. in the paper we discuss the modelling of reversible binding of mobile as well as immobilised ligands to macromolecules and compare modelling to experiment. comparative analysis of the temperature policy for processes with a deactivating native enzyme i. grubecki, m. wojcik department of chemical and biochemical engineering, university of technology and agriculture, - bydgoszcz, ul. seminaryjna , poland a comparative analysis of the temperature policy for an enzymatic reaction with michaelis-menten kinetics in a batch reactor has been carried out. both isothermal and optimal temperature policies for processes with deactivating native enzyme have been considered. in the model, the thermal deactivation was described by a first-order reaction, and the arrhenius-type dependence between rate parameters and temperature was assumed. as an indicator for a direct comparison between the isothermal and optimal temperature policies the quotient of conversions under identical initial and final condition was used. a method was presented to calculate this indicator, which is based on the analytical and numerical solutions. this method can be of great importance for the industrial practice. application of changeable temperature policy could result in significant increase in conversion when ratio of activation energy for deactivation and activation energy for reaction is high. studies on the impact of mixing during brewing using near and mid-infrared spectroscopy georgina mcleod , alvin w. nienow , graham poulter , reg wilson , henri tapp , christopher j. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w ). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. template refolding utilizing biospecific interactions shigeo katoh , yoichi kumada , nanae maeshima , daisuke nohara : graduate school of science and technologykobe university, kobe - , japan; department of biomolecular sciencegifu university, gifu - , japan. e-mail: katoh@kobe-u.ac.jp (s. katoh) recombinant proteins over-expressed in e. coli are often accumulated as insoluble particles called inclusion bodies. proteins in inclusion bodies must be solubilized by a denaturing agent, such as urea and guanidine hydrochloride, and refolded to recover their native structures having biological activities. in bioprocesses it is important to obtain high refolding efficiencies and high throughputs at high protein concentrations. in refolding operation, a denatured protein solution is usually added batch-wise into a large volume of a refolding buffer in order to start refolding by reducing the concentration of a denaturant and to prevent aggregate formation of renaturing molecules. thus, a large volume of a stirred tank is required, and the concentration of protein after renaturation becomes low. biointeractions between a pair of biomolecules, such as enzyme-inhibitor, antigen-antibody and hormone-receptor, are highly specific and have been used for detection and separation of biomolecules. these interactions may be used as templates for refolding of target molecules, which can be captured with the templates and are prevented from aggregate formation and, in the case of proteases, from autoproteolysis. the specific interaction might promote refolding of the target molecules. these might improve the refolding efficiency. the biointeractions between antigen-antibody and enzyme-inhibitor were used for efficient refolding in packed columns, in which template ligands (antibody, inhibitor) were coupled on gel support. denatured solutions of target molecules (carbonic anhydrase and s. griseus trypsin) were mixed with refolding buffer and supplied to the affinity column coupled with the template ligands for refolding. with refolding in the column, higher refolding efficiencies were obtained than those by the batch dilution method with relatively low concentrations of denaturants. by increasing the adsorption capacity of the column, throughput of refolding can be increased without decrease in the refolding efficiency. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic , alvin w. nienow , ian w. taylor , ryan hicks , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. carbon sources create fingerprint fermentation characteristics pınar Ç alık , güzide Ç alık , tunçer h.Özdamar bre lab, department of chemical engineering, ankara university, ankara, turkey; ib lab, department of chemical engineering, metu, ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. Ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e., glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e., serine alkaline protease (sap; ec . . . ),) and ␤-lactamase (ec . . . ), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. . - -fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. kinetic resolution of racemic benzoin with different lyophilized microorganisms Ç . babaarslan ,Ü. mehmetoglu , a.s. demir : ankara university, faculty of engineering, department of chemical engineering, , ankara, turkey; middle east technical university, department of chemistry, ankara, turkey. e-mail: barslan@eng.ankara.edu.tr (Ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the -hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at • c and rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs - as ee s = % and ee p = % (conversion = %) using thf as solvent and vinyl acetate as acyl donor. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box , campinas, cep - , são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during , and days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the mediun was composed by, cwb:cr were mixed with distilled water and transferred into ml capacity erlenmeyers flasks and autoclaved at • c for min. the medium was then inoculated with spores ( . × ) and the flaks were incubated at • c. tannase was assayed according to the methodology of mondal et al. ( ) . acording to the statist analyses, the optimum conditions to produce tannase was the range of temperature ( - • c); tannic acid ( . - %); residues % (coffe: wheat bran) ( : ) and days fermentation time. the enzyme production increased . times more enzyme production than that was obtained before this optimization. how to cope with fda's pat-initiative with respect to fermentation process monitoring and control marco jenzsch , andreas luebbert , rimvydas simutis : institute of bioengineering, martin-luther-university halle-wittenberg, halle (saale), germany; process control department, kaunas university of technology, kaunas, lithuania with its pat initiative, fda forces drug manufacturers to increase their activities in innovative manufacturing techniques, and, more than previously, to focus on quality assurance. the agency particularly places emphasis on making use of modern process supervision and control techniques such as up-to-date process analytics, multivariate data acquisition and analysis tools in order to improve process monitoring and control. in this contribution we show by means of practical examples how this guidance can be applied to cultivations of genetically modified microorganisms. a comparison of different multivariate state estimation techniques will be presented and compared with more knowledge-based techniques such as the extended kalman filter. the comparison was made for the model system gfp expressed from e. coli bacteria (bl /de /gfp) for which more than full data sets are available. all these techniques have already been used during real protein formation at productionscale fermenters, with the same success. hence, the requirements expressed in the pat initiative can immediately be put into practice. feedback control of the recombinant protein production processes based on such estimations is show for several cultivation systems. simple parameter adaptive controllers are compared with model supported controllers, for instance, generic model controllers and model predictive controllers. the results clearly show that we have at hand a rather extended arsenal of feedback control procedures that can be used successfully to tightly control the processes even along set-point profiles of physiological variables such as the specific growth rate (µ). again, fda's suggestion with respect to "control in the engineering sense" can be applied immediately to reduce batch-to-batch variances and thus to increase process quality. extending life by alternative respiration? alexander kern, franz hartner, anton glieder institute of biotechnology, graz university of technology, a- graz, austria. e-mail: a.kern@tugraz.at (a. kern) alternative oxidase transfers electrons directly from the ubiquinol pool in mitochondria to oxygen, allowing cell respiration in presence of complexs iii and iv inhibitors like antimycin a or cyanide. electron transfer by alternative oxidase is not coupled with proton transfer across the mitochondrial membrane, thereby uncoupling the supply of small metabolic intermediates by the central metabolic pathway from energy production in the cell. alternative oxidase is present in mitochondria of plants, many fungi and a few, mostly crabtree-negative yeasts, but not in p. angusta (hansenula polymorpha) and s. cerevisiae. alternative oxidase has multiple functions in different organisms. it is involved in stress answers, in programmed cell death, maintenance of the cellular redox balance, and also citric acid accumulation in a. niger. we isolated the alternative oxidase gene from the methylotrophic yeast p. pastoris in order to study its effects on the cellular energy content, respiratory activity, its protective role against oxidative stress. our results indicate the importance of an exact regulation of the alternative oxidase due to its impact on many cellular functions. new types of energy efficient fermenters with better mass transport, mixing and cooling properties than the current crop of rushton turbine derived tank bioreactors are likely to be required in the future. such fermenters will be needed in order to meet the increasing pressure on costs for low price commodity type products such as single cell protein or food and technical grade enzymes, and to meet the demands of the new wave of white biotech, in which bio-produced chemicals must be made at prices competitive with those of the traditional chemical industry. with this in mind, a prototype pilot scale ( l) u-loop fermenter has recently been commissioned at biocentrum-dtu. in this fermenter, liquid circulation is driven by a propeller pump through a vertical u-shaped pipe, which is connected at the top with a de-gassing tank. we present here a study of liquid mixing and dispersion in the prototype u-loop fermenter. sub-sequently we show that the results can be described with the tanks in series model. mixing was characterised using pulses of nacl tracer, which were detected with a conductivity probe in various parts of the fermenter. bodenstein numbers (bo) were determined for flow rates corresponding to a linear fluid velocity of . m/s in the 'legs' of the reactor and showed that the majority of the mixing occurred in the top degassing part (bo = ) rather than in the u-loop section (bo = ). it was also observed that the time for mixing to % homogeneity after tracer pulse addition was a function of the number of cycles through the reactor ( - . ) within the range of flow velocities (u) studied (u = . m/s to u = . m/s). the mixing time to % homogeneity was between . s (at u = . m/s) and s (at u = . m/s). today many biotechnological processes are operated at suboptimal conditions and according to best practice. however, the current industrial development is towards analyzing more parameters and in particular there is a large interest in analysis of biological/biochemical variables. the quality of products and also the possibility to optimize production in submerged cultivations would be greatly enhanced if more on-line/real-time information were at hand. the present investigation was undertaken with the aim of evaluating the potential in using multi-wavelength fluorescence for monitoring and control of filamentous fungi fed-batch cultivations. a recombinant a. oryzae expressing a heterologous lipase was applied as model system. spectra of multi-wavelength fluorescence were collected every five minutes with the bioview ® system (delta, denmark) and both explorative and predictive models, correlating the fluorescence data with the important biological parameters cell mass and lipase activity, were built. the models will be presented, furthermore, advantages and disadvantages of multiwavelength fluorescence for monitoring of cultivation processes will be discussed. moving from r&d to pharmaceutical development is a costly process. it is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. therapeutic recombinant proteins designed for human administration should be as close to the authentic product as possible. here, the use of a scalable process and an economically sound affinity tag can be a relevant choice. the tagzyme tm system has been designed to allow for the precise removal of amino terminal affinity tags. the system is based on the use of recombinant aminopeptidases including dipeptidyl peptidase i (dapase tm ). dapase tm is currently produced under cgmp providing a suitable strategy for its use in pharmaceutical production. the tagzyme tm system is superior to other methods since: ( ) it is based on exopeptidases, precluding, e.g., unspecific protein cleavage reported when using so-called site-specific endoproteases. ( ) it has been tested for production of more than recombinant proteins. ( ) it is easily scalable from lab scale to kg of processed protein. ( ) it allows the use of his-tags for commercial production without patent infringment, due to our ipr position. ( ) the commercial use of tagzyme tm does not require any licensing, only purchase of the enzyme(s). ( ) the use of aminopeptidases for pharmaceutical production has been extensively documented for approved drugs. ( ) a number of therapeutics is currently being developed using tagzyme tm . ( ) unizyme can assist in the optimization of the dsp to enable further cost reduction in the process. these aspects will be discussed and illustrated in the presented poster. website sphingolipids are biologically active molecules involved in the regulation of a large quantity of biological responses. they function in cell proliferation, survival and death (apoptosis) as messengers. dysregulation of apoptosis has significance in numerous pathological conditions including cancer. several anticancer agents act by increasing tumor cell ceramide (a kind of sphingolipid) content. so, a novel approach to cancer therapy would be the pharmacological manipulation of sphingolipid metabolism. in this study, sphingolipid metabolism in baker's yeast s. cerevisiae is used as a model system as many of its sphingolipid related genes and proteins have been characterized. gepasi-biochemical kinetics simulator was used for metabolic control analysis (mca) of the above-specified system. the concentration control coefficients (ccc), flux control coefficients (fcc) and elasticity coefficients were calculated, and their significance in identification of anticancer drug targets is determined. elementary flux modes were also identified and metabolic pathway analysis (mpa) was performed. quantitatively, control effective flux (cef) values were used for potential drug target identification. the results from mca and mpa indicate almost the same potential drug targets: serine palmitoyl transferase, ceramide synthase and ceramidase. drugs against these targets are in preclinical and clinical development. for the identification of new potential drug targets, the cccs, fccs, cefs and elasticity coefficients were examined with an objective function of maximizing the cell ceramide concentrations. it was found that manipulation of inositol- -phosphate synthase and phosphoinositide kinase activities have considerable effects on ceramide concentrations. if a drug targeting the two enzymes at the same time is designed, it might give a better outcome in terms of cancer therapy. in recent years, there is a growing interest in utilization of airlift reactors (alrs) to biotechnological processes. nevertheless, their industrial application still remains limited because of a lack of reliable studies on transfer phenomena and mixing enabling a suggestion of suitable scale-up procedure. the way to more widely utilization of alrs to biological processes lies in experimental research (on a model medium as well as on a real fermentation medium) followed by mathematical modelling and scaling-up of the processes. this paper deals with a modelling of a glucose-gluconic acid fermentation by a. niger in an internal loop airlift reactor. knowledge of the stoichiometric relationship in the key reaction provides a good opportunity for estimation of substrate and product concentration. the model is based on material balance equations and has been adjusted to experimental data obtained from three internal loop airlift reactors ( . , and l) . in the model, the alr is divided into ideal stirred tanks in series. in each zone (tank) of the alr the material balance is calculated in two phases (the gas and the liquid phase). this work was supported by the slovak scientific grand agency, grant number vega / / alkaline phosphatase (ap, e.c. . . . ) is a thermolabile enzyme which is indigenous to all dairy products. it has an inactivation temperature slightly above the value that is required to destroy the most resistant pathogenic microorganism likely to be found in milk. due to that feature, this enzyme is used as an indicator of proper pasteurization. the effect of temperature treatment on the activity of ap was investigated in raw cow's and goat's milk. the stability of alkaline phosphatase in raw milk was compared with the stability of this enzyme in a . m potassium phosphate buffer with ph . . the ph value of the buffer was approximately the same as that of raw milk. the inactivation curves were measured in the temperature range from to • c. ap in cow's milk was completely inactivated at • c during s but approximately % of activity remained at • c after min of treatment. the time required for a complete inactivation of the enzyme in the raw cow's milk was reduced from to min as the temperature increased by • c. heat treatment of goat's milk caused the decrease of activity of the enzyme in the same temperature range as in the case of cow's milk. the increase of temperature from to • c reduced the inactivation time from min to s. the study of thermal stability of the alkaline phosphatase in the buffer solution showed that the time required for inactivation of enzyme was significantly shorter than in milk. milk thus had a protective effect on the activity of alkaline phosphatase. the experi-mental curves were fitted simultaneously using kinetic models where the initial heating period was considered. this work was supported by a grant of th framework program of eu, project foodpro, no. sme- - - . during the process of separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale, the chromatographic unit operations have an important role. three different protein-binding modes are employed: ion-exchange, hydrophobic and affinity binding. two adsorbent properties are of uppermost importance: a high selectivity and adsorption capacity. in the case of ion-exchange/hydrophobic chromatography, the binding of charged proteins can be affected by ph and ionic strength. in this work, the adsorption capacity of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros a, prosep-va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was measured as a function of ph. as a model mab and contaminant proteins, human immunoglobulin (igg), human serum albumin (hsa) and horse skeletal muscle myoglobin (myo) were used. the resin properties were investigated within the range of ph - . the experiments were conducted in a batch-mode, individually for each model protein. the results showed that ion-exchange and hydrophobic resins provided the best selectivity for igg at ph . the selectivity of affinity adsorbents was essentially unaffected by ph, however, the highest capacity for igg was at ph . another investigated aspect was the dynamics of protein binding. the solution of individual protein in contact with tested adsorbent was circulated through an uv spectrophotometer, what enabled the measurement of time-dependent decrease of protein concentration. the results indicated that affinity adsorbents with a rigid matrix needed approximately four times shorter time to reach the adsorption equilibrium with igg in comparison with a gel. the gels, however, provided higher adsorption capacity. at ion-exchange resins, the time necessary to adsorb % of total amount of igg was about . - h. the affinity adsorbents were highly selective and therefore they adsorb very small amount of tested contaminant proteins (hsa, myo). the adsorption capacity was saturated by % in less than min in all cases of dynamic adsorption measurements. this work was supported by a grant of th framework program of eu, project aims, no. nmp -ct- - . microtechnology has for several years been applied within chemical reaction engineering. the advantages of microtechnology are that it makes it possible to develop light weight and compact systems, and the systems enable large surface-to-volume ratio, which results in low mass-transfer distances. in addition, parameters like pressure, temperature, residence time, and flow rate are more easily controlled. the use of microtechnology is also beginning to find its ways into the field of biotechnology. what we are aiming at is the development of a microreactor that can be applied as a production tool in industry as an alternative to conventional enzymatic reactors. our strategy is to use a small plate of a suitable material with microchannels fabricated into its surface, and the approach is to covalently couple enzymes into the microchannels. substrate can then be pumped through the channels and the enzymatic conversion will take place within the channels. as model enzyme in the development of the microreactor we are applying celb, a thermostable ␤-glycosidase from pyrococcus furiosus. kinetic resolution of racemic benzoin with different lyophilized microorganisms Ç . babaarslan ,Ü. mehmetoglu , a.s. demir : ankara university, faculty of engineering, department of chemical engineering, , ankara, turkey; middle east technical university, department of chemistry, , ankara, turkey. email: barslan@eng.ankara.edu.tr (Ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the -hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at • c and rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs - as ee s = % and ee p = % (conversion = %) using thf as solvent and vinyl acetate as acyl donor. chromatography is one of the most important unit operations at separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale. since these proteins belong to the group of immunoglobulins, their molecular weight (about , g/mol) or hydrodynamic radius ( . nm), respectively, is relatively large. the adsorbents used in ion-exchange/affinity chromatography of these biomolecules should thus provide a high pore accessibility coupled with a high value of specific surface area in order to ensure a sufficient ligand density and a high binding capacity. in this study, the pore accessibility of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros a, prosep -va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was investigated via size exclusion of standard-sized molecules (glucose, sucrose and dextrans with molar weight range - × g/mol) at nonbinding conditions. the experiments were conducted in a batch and column-mode. the batch experiments provided absolute partition coefficients, which were calculated from a mass balance and represent the fraction of pore water accessible to a solute. it was found that several adsorbents contained a small fraction of very small pores (less than nm), whereas some adsorbents contained a significant fraction of pores larger than nm. the column measurements provided relative partition coefficients, which were calculated from the retention volumes of solutes and represented the relative accessibility of pores, scaled between the accessibility of the smallest and largest solute used. when the absolute partition coefficients were recalculated into the relative form, it was found that the coefficients obtained by both methods correlated very well. the relative partition coefficients of solutes with the hydrodynamic radius of nm (corresponding to mabs) was about . - . at ion-exchange and hydrophobic adsorbents and . - . at affinity adsorbents. the relation between hydrodynamic radius of the solutes and their partition coefficients was successfully described with the giddings random plane model. this work was supported by a grant of th framework program of eu, project aims, no. nmp -ct- - . the transfer of laboratory results to a larger scale is often a critical step in process development to industrial application. the objective of this study was to scale-up the bioreactor for the fructosyltransferase production based on the results obtained in a dm stirred bioreactor. the investigations made in this bioreactor provided a clear picture of the effect of medium composition on the obtained fructosyltransferase (ftase) activity but the influence of mixing intensity was unequivocal. the increase of agitation rate had a positive effect up to a certain level where both fructosyltransferase and biomass production increased. the final optimal yield factor of ftase per dry cell mass obtained in the laboratory bioreactor was , u g − . we studied the effect of oxygen transfer on the process of ftase production at a larger scale, in and dm mechanically stirred bioreactors and in air-lift bioreactors, and dm whilst the medium composition was kept constant. the yield factors of ftase were comparable in both mechanically stirred bioreactors and they were about u g − . this decrease compared to that in the laboratory bioreactor could be explained by a slower cell growth. this fact was also confirmed by that glucose was not depleted till the end of fermentation and free fructose concentration was also lower. the yield factor of ftase was u g − in the dm air-lift reactor and u g − in the dm air-lift bioreactor. the lower yield of ftase in dm bioreactor was caused by a better biomass growth. this work was supported by slovak scientific grand agency, grant numbers vega / / and / / and by science and technology assistance agency, grant number apvt- - . the poster gives an overview of the objectives and achieved results of an interdisciplinary project on direct product isolation from crude feedstocks using magnetic micro adsorbents in combination with suitable magnet technology. the project was funded by the deutsche bundesstiftung umwelt and was running between august and november . in the course of the project several milestones could be met, which can be looked at as critical key points on a route towards an industrial realization of the process. among these milestones are: (i) the production of magnetic micro adsorbents with high capacity and selectivity in batches up to - g; (ii) the proof that the micro adsorbents can be reused many times; (iii) generation of a variety of recombinant tagged, active enzymes; and (iv) the design, assembly and operation of a fully automated pilot plant capable of generating approx. g/h (≈ % purity) protein. the process was also simulated by help of the software tool superpro designer and simple mass balance and sorption equilibrium approaches were used to derive rules for estimating optimum process parameters and productivities. finally an environmental performance evaluation was conducted externally by the german dechema. in this study the effect of fed-batch cysteine addition to a culture of a high-gsh-accumulating yeast strain on the metabolism of glutathione was investigated. it is known that cysteine is the rate limiting amino acid in the biosynthesis of gsh. the influence of the consumption rate of cysteine on glutathione metabolism and growth of s. cerevisiae mt- was determined. the results show that for rates of consumption below a critical value the microorganism growth is similar to a culture without feed of cysteine, but glutathione production is increased two-fold. on the other hand, if cysteine consumption rate is above the critical value the changes of cell metabolism implies ethanol accumulation in the extracellular media which diminishes biomass synthesis. the maximum specific glutathione production in this case is maintained at two-fold; however, gamma-glutamylcysteine accumulation is increased. cysteine present in culture media directs cell metabolism to a greater synthesis of ammonia and amino acids. hydrophobic interaction chromatography (hic) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. in contrast to reversed phase chromatography this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. when proteins are injected on hic columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. the higher the salt concentration the lower is the amount eluted protein. the rest can be desorbed with a buffer of low salt concentration or water. it has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. this has been also corroborated by physicochemical measurements. the retention data of five different model proteins and different stationary phases were evaluated. partial unfolding of proteins upon adsorption on surfaces of hic-media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. furthermore we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain. stationary phases for bioseparation of glycoproteins j. aniulyte , j. liesiene , b. niemeyer : department of chemical technology, kaunas university of technology (ktu), radvilenu pl. , kaunas, lithuania; institute of thermodynamic, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg , hamburg, germany d- nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated.three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of atoms) and carbonyldiimidazole activation.cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to . mg per ml support and a high recovery (up to %). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affinity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately × − m, and . × − m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. cell disruption and chromatography are key unit operations in the downstream processing of an intracellular product. the cost involved in the extraction and purification of intracellular products can be reduced by selective release of proteins and reduction in the number of steps involved in the purification. the extent of disruption can be varied to provide a selective release, limiting the release of the contaminant proteins. the particle size distribution of the cell debris in the resulting suspension depends on the extent of disruption. expanded bed adsorption chromatography allows for the direct capture of the proteins from an unclarified suspension. this technique allows for the integration of solid-liquid separation, concentration and preliminary purification in one unit operation. a perfectly stable expanded bed can be obtained by choosing the appropriate flow conditions and a suitable adsorbent. the difference in the density between the adsorbent and the cell debris in the suspension, permits the cell debris to flow through the column without blocking, whilst the protein molecules in the suspension are adsorbed onto the adsorbent. after sample application, the bed is washed with buffer and the proteins eluted from the column in the packed bed mode. the presence of the cell debris in the feedstock influences the expansion of the bed and the adsorption of protein molecules. the physical properties of the suspension obtained after cell disruption depends on the extent of disruption. the particle size distribution of the cell debris, the viscosity and the release of soluble proteins and other intracellular components are influenced by the extent of disruption. the influence of the extent of disruption of e. coli on the expansion of the bed and the adsorption of ß-galactosidase is presented in the current study. e.coli cells were disrupted at different operating pressure using a high pressure homogenizer. the resulting crude homogenate is subjected to expanded bed adsorption chromatography using streamline deae as adsorbent. the disrupted suspension was characterised in terms of viscosity, density, particle size distribution of the cell debris and the extent of protein and ß-galactosidase released. the interaction between cell debris and adsorbent was quantified as the cell transmission index (ratio of the amount of cells present in the sample before and after passing through the bed). the expansion of the bed at a constant settled bed height and flow rate was measured. the influence of the cell debris on the extent of adsorption of ß-galactosidase has been quantified in terms of dynamic binding capacity (dbc) at % of the inlet concentration. the dbc of ß-galactosidase that was released by disruption at psi ( %, w/v, w/w, pass) was found to be u/ml of adsorbent while dbc of samples disrupted at psi ( % w/v, w/w, pass) was u/ml of adsorbent. the extent of disruption of e. coli over a wide range and its effect on the expansion and adsorption will be presented. study of dna binding during expanded bed adsorption and factors affecting adsorbent aggregation ayyoob arpanaei , niels mathiasen , timothy hobley , owen rt thomas , : center for microbial biotechnology, building , biocentrum-dtu, technical university of denmark, , lyngby, denmark; department of chemical engineering, university of birmingham, edgbaston, b tt, uk. e-mail: aa@biocentrum.dtu.dk (a. arpanaei) the adsorption of sonicated calf thymus dna (as a model dna molecule) to biosepra q hyper z adsorbents was evaluated in batch and expanded bed modes. stability of the expanded bed during feedstock loading was also studied. two batches of prototype q hyper z (batch and ) were examined, which had ionic capacities measured to be and mmol cl − /ml support respectively. in all adsorption experiments a mm tris-hcl ph buffer was used. maximum static binding capacities of adsorbent batches and were determined to be . and . mg dna/ml particle, respectively. dynamic binding capacity at % breakthrough (dbc % ) was measured in a -cm diameter eba column containing . ± . cm settled bed with a feed of g/ml dna. dbc % of the adsorbents were . and . mg dna/ml support for batches and , respectively in buffer containing no salt. however, the maximum dbc % for batch ( . mg dna/ml support) and ( . mg dna/ml support) were obtained in buffers containing . and . m nacl, respectively. further increases in salt concentration led to a decrease in dbc % for both adsorbent batches. the bed compression during loading that was observed in experiments at high conductivities (achieved by adding salt) was less than that seen with low conductivity ( ms/cm) solutions. aggregation of adsorbent particles and channeling of flow were not observed in the presence of salt concentrations more than . m. the effect of different concentrations of dna during loading in the presence of . m nacl was studied. it was found that increasing dna concentrations in the feed from to g/ml, to g/ml resulted in a decrease of dbc % by , and %, respectively. the bed compressed slower during loading of feedstock with low dna concentrations compared to that for higher concentrations. the expanded bed showed a partly reversible compression behavior during feedstock loading. this is attributed to the electrostatic interaction between dna adsorbed on the particles surface and rearrangements of dna strands as the number of free ligands on the adsorbent surfaces decrease during loading. large-scale production of plasmid dna for gene therapy and dna vaccination applications has become necessary as a result of the increasing number of approved protocols using non-viral vectors for gene delivery. a major challenge of large-scale plasmid production is to establish a robust cgmp manufacturing capable of producing hundreds of milligrams or grams of a pharmaceutical grade product. alcohol and salt precipitation are operations largely used in the early steps of plasmid downstream processes. however, there are few systematic studies on the influence of these precipitation agents in the final plasmid recovery and purity. in this work, alcohol and salt precipitation steps used in a plasmid purification process developed by our group have been optimized aiming at large-scale production. the optimization of alcohol precipitation indicated that almost % of the pdna precipitated when . vol. of isopropanol were used. the studies also indicated that the precipitation profile was strongly influenced by pdna initial concentration. finally, the final plasmid recovery and purity after a sequential alcohol and salt precipitation were strongly dependent on the concentrations of these precipitation agents. thus, a commitment between high recovery and purity level should be made during the development of the downstream processes. comparison of novel and conventional processes for protein refolding and initial purification h. ferré , , u. jørgensen , l. scale down of downstream processing unit operations is convenient for assessing process alternatives, particularly if feedstock is scarce. in this study it was imperative to use the smallest possible scale for comparison of a new system for continuous protein refolding and direct expanded bed adsorption (eba) capture with a traditional process composed of discrete operations of batch renaturation, centrifugation, microfiltration and packed bed chromatography (pbc). minimisation of the scale was restricted by the eba step: the smallest practical scale being a cm diameter column with - cm of settled bed, expanded two fold. in order to permit a fair comparison a similar column diameter and adsorbent volume was used in the packed bed process. in both alternatives, chelating media charged with cu + was used and a feedstock of denatured hat-tagged human beta- microglobulin (hat-h␤ m). following batch refolding and clarification, the performance of the packed column was severely hampered due to fouling of the top adapter. reducing the protein loaded to the packed bed to % of dbc working lead to a recovery of . % at a purity of % and . -fold concentration. the eba-based process performed unimpeded and productivity was calculated to be % higher than for that employing a packed bed. however, due to the severe scale restrictions placed on the eba process, which limited optimisation, significant productivity improvements of eba over packed bed are expected at larger scale. high gradient magnetic filtration has the potential for rapid processing of large volumes of crude bioprocess liquors when magnetic adsorbents are employed. the binding of a protein to a superparamagnetic solid support provides a unique selective 'handle'. typically the focus is placed on using the magnetic handle for direct capture of a protein from a fermentation broth. however, magnetic adsorbents may provide solutions to a range of downstream processing problems and in this presentation we illustrate this with a number of case studies. using whey as a model system, we show that the extent of the tryptic hydrolysis (ca. . mg/ml added enzyme) of proteins could be controlled by adding benzamidine-linked magnetic adsorbents after a given period ( - min), followed by removal of the loaded adsorbents using a magnetic filter. hydrolysis was stopped effectively and approx. % of the added trypsin could be recovered. a coupled process was devised for the refolding and purification of inclusion body proteins. solubilised (in m urea) inclusion bodies of recombinant histidine affinity tagged human beta microglobulin (hat-h-beta m), were refolded by dilution in a pipe reactor ( s), then captured directly on cu(ii) charged magnetic immobilised metal affinity adsorbents in a second pipe reactor ( s residence time). loaded adsorbents were retained in a magnetic filter, then washed and the protein eluted. a generic framework for the prediction of scale-up when using compressible chromatographic packings r. tran , j. joseph , a. sinclair , y. zhou , n. packed bed chromatography is the pre-eminent technique in the downstream purification of many biological products. the aspect ratio of a packed bed has a significant effect on the column pressure drop by virtue of wall support which is reduced at low aspect ratios. this can result in unexpectedly high pressures during manufacturing caused by the compression of the matrix via drag forces due to fluid flow through the bed. the need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation so that maximum throughput can be achieved. several studies have generated correlations which allow for the prediction of column pressure drops but have either been mathematically complex, which makes their practical use unfeasible, or they have used highly specific empirical constants and hence require a large amount of experiments to be performed before they can be used. in this study, we have established relationships to link the critical velocity of operation, to bed height (l), column diameter (d c ), feed viscosity (µ) and also to the matrix rigidity through the level of agarose cross-linking (a%). the correlation is straight forward to use and involves very few system-specific constants thus significantly reducing the need for any preceding laboratory-scale experimentation. this paper describes the series of experiments that were performed to establish the correlation, using a range of cross-linked agarose matrices ( - %), at various aspect ratios, fluid flow rates and varying viscosities ( . - . mpas). a mathematical model was developed where parameter estimation for multi-variables was achieved by least squares optimisation. the model can be used to predict the extent of compression in industrial chromatography applications and will be useful in the development of chromatographic operations and for column sizing. institute of process engineering, swiss federal institute of technology, zurich. e-mail: makart@ipe.mavt.ethz.ch (s. makart) simulated moving bed (smb) technology receives increasing attention in biotechnology and in the biopharmaceutical industry as it enables an increase in productivity per unit mass of stationary phase, reduced solvent consumption, and fast and reliable scale-up. combining continuous chromatographic separation unit and reactor should enable the production of biopharmaceuticals and fine chemicals with high purity and yield at the same time. due to the increasing demand of enantiopure intermediates in the pharmaceutical industry, biocatalytic processes gain more and more importance because of their excellent enantioselectivity. yet the application of biocatalytic carbon-carbon bond formation on process scale is often hampered by an unfavourable equilibrium position and difficult downstream processing due to substrate/product mixtures. coupling a continuous separation unit to such a process would improve the feasibility by driving the reaction to completion and thus increasing the overall yield. we will discuss the design of such an integrated biocatalytic/smb process, taking the formation of l-allo-threonine from glycine and acetaldehyde, catalysed by the glycine-dependent aldolase glya from e. coli, as a model reaction. the enzyme exhibits absolute stereoselctivity at the c-alpha atom, whereas selectivity is less strict at c-beta. in situ product removal, by the integration of an smb unit, would aid to maintain a high diastereomeric excess as it shortens the residence time of the products in the reactor, in addition to shifting the reaction to the product side. the in-line coupling of the chromatographic unit to the enzyme reactor requires the use of the same solvents for reaction and separation, so the choice is limited to aqueous solutions close to physiological ph, limiting in turn the possible stationary phase materials. in a screening of different cation exchangers, amberlite cg- ii gave promising results: threonine is more retained than glycine, acetaldehyde is poorly and the cofactor plp is not retained. adsorption isotherms were determined by the retention time method and a smb under process conditions was simulated. by improving the packing of the column, i.e. achieving a more even particle size distribution, we tried to further increase the efficiency of the separation step. the application of enzymes for the synthesis of optically active substances is nowadays of growing importance in the pharmaceutical industry. this requires a proper cultivation of the microorganism as well as a posterior isolation process yielding a constant catalyst quality at high purity. goal of this project is the development of an integrated process for the production and isolation of a lipase from trichosporon beigilie and its posterior application for the enantioselective synthesis of pharmaceutical products. the cultivation of the microorganism is optimised in a laboratory and pilot-scale fermenter in a fed-batch mode. parameters like media composition, temperature, ph and aeration rate are set up. taking advantage of the localisation of the enzyme (covalently attached to the cell membrane) the first step of product isolation consists of a continuous cell disruption. optimal results are achieved with the continuous bead mill disruption process ( % enzyme release with a specific activity of . u/mg of protein). the non disrupted cells are recycled as inoculums for a new cultivation, increasing the yield of the overall process (by -times in the pilot-scale fermenter). in order to isolate the product two different process sequences are considered. the first one consists of an extraction (peg and phosphate buffer) coupled to an ion-exchange chromatography (q-sepharose ff). the second one applies a precipitation step with ammonia sulphate followed by a hydrophobic interaction chromatography (sepharose-hic) provid-ing a lipase yield of % ( -times higher than the one provided by combining extraction-chromatography). an ultrafiltration process is used in order to concentrate the lipase and its final properties (molecular weight, isoelectric point, activity, stability and kinetic data) are studied using p-nitrophenylacetate as model substrate. the relevance of the obtained product for its application in the pharmaceutical industry is proven by transforming (r,s)-naproxen-methylester into (s)-naproxen acid with an enantiomeric excess of > % (after h). biotensides (sugar fatty acid esters, sfaes) find nowadays a wide range of applications in pharmaceutical, personal care and food industry because of their biocompatibility, biodegradability and special surfactant properties. goal of this project is the development and optimisation of an integrated process for the enzymatic synthesis of sfaes from renewable sources to be used in cosmetic formulations. the following figure shows the scheme of the overall process. commercial and also new screened lipases are applied in the reaction between sugar and fatty acid. the mixture grade of the initial reaction system is increased by ultrasounds taking into account the influence on the catalyst characteristics and also the necessity of an organic solvent as adjuvant. the reaction takes place in an enzymatic membrane reactor (emr) equipped with an ultrafiltration membrane which retains the catalyst. the separation of the by-product (water) from the rest of the components can be achieved by means of a pervaporation unit which coupling to the emr allows the semi-batch process. in order to separate the esters from the fatty acid a stepwise elution chromatography method is developed using silica as adsorbent and ethyl acetate and methanol as eluents. with this system % of the dimer is isolated with purity (hplc) of %. the application of a dialysis membrane technique allows the separation of % of the fatty acid by building ester micelles changing the polarity of the organic solvent used as eluent. solubility and crystallisation properties of recombinant bacillus halmapalus ␣-amylase cornelius faber centre for microbial biotechnology, biocentrum dtu, building , lyngby, denmark a comprehensive knowledge of solubility properties is a prerequisite for the efficient design and operation of bulk enzyme recovery processes, however, complete phase diagrams are only available for very few proteins, in particular lysozyme of high purity. here, we present the results of detailed solubility studies in aqueous solutions of an industrially relevant ␣-amylase of technical grade. experiments were conducted in small scale batch mode (working volume of ml). the influence of selected cations and anions from the hofmeister series on the stability of the ␣-amylase was examined. the hofmeister series for anions was followed in the correct order at all salt concentrations studied, i.e. from to m, whereas the series was reversed for monovalent cations at concentrations up to . m, with the exception of lithium. to further investigate why the position for lithium was different to the hofmeister series established for lysozyme, the zeta potential of protein solutions at low concentrations of selected salts was determined. the results of these measurements indicate a pronounced effect of lithium on the zeta potential, as compared to other salts. in particular, the ph of zero zeta potential (i.e. the pi) was shifted approximately . ph units towards alkaline conditions in the presence of lithium, whereas the pi stayed almost constant for sodium and potassium. since the solubility exhibits a minimum at ph-values at or near the protein's pi, shifts in ph caused by salt addition are important to identify and quantify to avoid uncontrolled phase separation. the measurement of the zeta potential of proteins in solution holds significant promise as an attractive tool for understanding and controlling processes that are operated close to the solubility limit and which are often plagued by uncontrolled precipitation or crystallisation and thus rely on carefully chosen operating conditions. polyphenolic interactions with potato proteins during industrial expanded bed adsorption processing sissel løkra , knut olav straetkvern , bjørg egelandsdal , gerd vegarud : department of natural science & technology, hedmark university college, n- hamar, norway; norwegian university of life sciences, as, norway. e-mail: sissel.lokra@lnb.hihm.no (s. løkra) in plant extracts it has been shown that polyphenols have a tendency to react with proteins, either by covalent or non-covalent interactions. these reactions can induce changes in the surface properties of the proteins, and, e.g. cause proteins to be insoluble and precipitate at ph-values below their isoelectric point. potato proteins have a high nutritional quality and show interesting functional properties in food systems. moreover, chlorogenic acid (ca) and caffeic acid constitute about % of the total polyphenol content of potato tuber. we have experienced expanded bed adsorption (eba) chromatography to be a method well suited for recovering industrial proteins from potato starch effluent. the process separates proteins from polyphenolic pigments, fiber and minerals. during the adsorption step, patatin, the major potato tuber protein shows complex binding kinetics demonstrated by breakthrough curves. in addition to diffusion limitations in the eba resin, changes in protein structure and surface properties probably are likely to affect this adsorption behavior. reactions between ca and patatin might result in a range of interactions for different species of the same protein. this project therefore aims to assess the interactions between ca, patatin and other major tuber protein fractions and how these changes affect the protein capture in eba. changes in size and charge are screened in -d electrophoresis and analyzed further. samples of different protein fractions are taken from breakthrough curves and dynamic binding capacity experiments in model systems with real feedstock. sandwich hybridisation assay for analysis of brewery contaminants s. huhtamella , m. leinonen , t. nieminen , a. breitenstein , p. neubauer : bioprocess engineering laboratory, university of oulu, finland; scanbec gmbh, halle, germany. email: peter.neubauer@oulu.fi (p. neubauer) here we describe the development of a sensitive, cultivationindependent analytical method for the analysis of brewery contaminants which can be performed within three hours in crude sample extracts. the method is based on s rrna detection by a paramagnetic bead based sandwich hybridization assay (sha) with two oligonucleotide probes designed to either detect the species or a group of contaminants. the signals were read out either by a fluorimeter (rautio et al., ; leskelä et al., ) or potentiometrically with an electric biochip instrument (ebiochip systems) . this assay is advantageous over rt-pcr becasue it only detects viable cells and the method can be directly applied to crude cell extracts without prior purification. we describe the principle of designing and evaluating a series of groupspecific lactobacillus probes and the optimisation towards effective cell lysis and high assay sensitivity. the applicability of the sha was evaluated with real brewery samples and the results were compared to routine tests. in all steps of the evaluation the reliability and usability of the method was prioritised. the optimised method combined with a h pre-enrichment period gave reliable results, had a detection limits of about - cells per assay and was easily applicable in a brewery environment. biodesulfurization is one of the possibilities studied by the researchers to attain the maximum sulfur levels imposed for a near future by governments (european directive, ) . rhodococcus erythropolis igts is a natural and strictly aerobic microorganism able to remove the sulfur atom from dibenzothiophene (dbt) in a selective way ( s route (oldfield et al., )), obtaining hydroxibifenyl (hbp) and sulfate. growth is carried out using the experimental procedure performed in previous works dealing with the inoculum built up, media composition and operational conditions (del olmo et al., a (del olmo et al., , b . this work is focused to determine the oxygen uptake rate during the production of the biocatalyst. four experiments were carried out at a biostat b fermentor (braun biotech.) using as only variable the constant stirrer speed used: , , and rpm. oxygen uptake rate have been determined by means of two methods: dynamic technique at different times during growth for few seconds to ovoid influences and from oxygen profile when the term dealing with oxygen transfer rate is known (predicted by the model proposed in a previous work (garcía-ochoa and gómez, )). our values obtained from the techniques used present the same tendency in all the runs carried out: our values from dynamic technique is always lower than our values obtained from the oxygen profile. these values are modeled and the difference observed is explained due to the cellular economy principle: during the seconds employed in the dynamic technique determinations microorganism do not produce s route enzymes. it was studied different methods to recover a p. salmonis antigenic protein from recombinant e. coli cells. this protein has shown be highly effective in vivo vaccine. it has the ability to stimulate salmon immune system protecting them against of aggressive disease salmonid rickettsial syndrome resolving by this way a great problem of salmonid aquaculture. biomass obtained from iptg induced e. coli bl (de ) codon plus culture was used for soluble and insoluble antigenic protein recovery. it was evaluated recuperation by glass bead mill, freezing and thawing, osmotic shock and lisozyme/edta treatments, all of them applied in single or combined way. biomass was measured by dry weight of cells, soluble protein concentration was quantified according to bradford method, and antigenic protein was identified by sds-page and western-blot analysis. cells treated with lisozyme/osmotic shock and then glass bead mill the soluble protein was a . % of the dry weight cell mass whereas using lisozyme/edta and glass bead mill as a single treatment only a . and . % were obtained respectively. the freezing and thawing disruption treatment released less than % of soluble protein, as well as the osmotic shock procedure too. the sds-page and west-ernblot analysis revealed that the antigenic protein must be purified from the insoluble cell fraction when physical or mechanical disruption methods were employed and from the soluble cell fraction when chemical or enzymatic treatments were used. we propose investigate in further studies the inclusion bodies formation to design an efficient purification procedure for the target protein. the iso-peroxidase pox from garlic bulb allium sativum that represented the major peroxidase activity was purified to homogeneity. the enzyme is monomeric and has a molecular mass of kda, and a pi around . the optimum temperature ranged between and • c, while optimum ph was around . pox appeared remarkably thermostable since it retained % of its activity at • c for at least h. in addition, the enzyme was stable at a ph range from , to . kinetic constants were calculated, the apparent k m values were and m for gaïacol and h o , respectively. the high thermostability of pox may represent clear advantages in a number of processes including immobilizing peroxidase and use it as a biosensor to detect oxidant component as h o and other peroxides. immobilization of pox was achieved by binding covalently the enzyme to a sepharose matrix (bead and membrane va epoxy). the immobilized peroxidase showed great stability at heat and storage than the soluble enzyme. the native enzyme retained % of its activity at • c for mn while the immobilized pox retained full activity for mn at the same temperature. in other side, the free enzyme retained full activity for at least one month and a half during storage at • c, and lost m of its activity after months. the immobilized form of pox retained complete activity for months at the same temperature. the immobilized enzyme was used to detect h o in some food components such as milk and fruit juices. in a second study, same experiments were performed in order to detect the smaller quantity of added h o to the farming milk. purpose: a new research field has been created to begin to address protein function at level of regulation of enzyme activity. this new area has been given the name chemical proteomics, or activity-based proteomics (abps), and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site. the selectivity of the chemically reactive group allows specific proteins or protein subset to be tagged, purified and analyzed. methods: this molecule (abps) has three subsets: tag, linker, and warhead. warhead is a nucleophile and attach to active site. linker is a polypeptide that makes a simple connection between warhead and tag. tag is fluorcent or radioactive material that facilitates the detection of drugs. findings: several diseases such as cancer, rheumatoid arthritis and osteoporosis are associated with elevated levels of protease activity. serine hydrolyses abps have been used to profile enzyme activity in a diverse range of cancer cell lines. in studies comparing metastatic and non-metastatic human breast cancer models, it was shown that the former exhibited a higher activity of a ␥-glutathione-s-transferase, an enzyme that has not previously been associated with breast cancer. discussion: additionally, abps can be used to develop robust screens for small molecule inhibitors of a specific enzyme target within a large family of related enzymes. this method of inhibitor screening allows compounds to be assayed for both potency and selectivity against a set of related in complex biological samples. this technique is able to identify novel enzymatic proteins and drugs and has the potential to accelerate the discovery of new drug target. a cyclodextrin glycosyltransferase (cgtase) from a new isolated strain from bacillus clausii e , was purified through q-sepharose, gel filtration chromatography and deae-sephadex a- . the mw of the pure enzyme was kda with sds-page. the enzyme displayed optimum ph value and ph stability at ph . and in range of . - . , respectively. the optimum temperature and thermostability were at • c and up to • c by h, respectively. the k m and v max were . mg/ml and . mol/min mg and . mg/ml . mol/min mg using maltodextrin and soluble starch, respectively. the isoeletric point was . and the n-terminal region of the pure enzyme was sequencing by maldi-tof-ms. the ratio of ␣-, ␤and ␥-cd was . : . : . and : : with maltodextrin and soluble starch at . %. application of magnetic separation technology for recovery of immobilised lipases nadja schultz , anke neumann , george metreveli , matthias franzreb , christoph syldatk chair of technical biology, university of karlsruhe, engler bunte ring , d- karlsruhe; forschungszentrum karlsruhe, institute of technical chemistry, water-and geotechnology; inst. für wasserchemie, engler bunte ring , ka. e-mail: nadja.schultz@ciw.uni-karlsruhe.de (n. schultz). url: www.fzk.de/itc-wgt (m. franzreb) first results on the development of the magnetic separation technology for the recovery of immobilised lipase from a -phase-system, which should be suitable for a large scale use in future, known as high gradient magnetic separation (hgms) are presented. the application of immobilised lipases makes the reuse of the enzyme in a process possible and is therefore interesting for industrial applications. in this study immobilised lipase is used in a -phase-system. here the new approach to recycle and reuse the lipase, immobilised on magnetic particles, from a -phase-system with the help of the new high gradient magnetic separator (hgms) is examined in ml scale. as model enzyme for the immobilisation on magnetic microparticles (polyvinyl alcohol (pva), - m) the commercially available (novonordisc) lipase a (cala) from candida antarctica was used. necessary for screening of immobilisation methods and characterisation of the immobilised lipase (candida antarctica) was the development of a robust, simple and rapid chromophoric activity assay. therefore the pnpp-lipase assay was optimised for direct application on immobilised lipases (in preparation schultz et al., ) . further more a ph-stat-assay for measuring the activity of free and immobilised cala in a -phase-system of tributyrate and buffer was optimized for this system. another important basis for the realisation of the recovery of immobilised lipases was to optimise the immobilization technique of the lipase. furthermore approaches for the explication of generally empirical based immobilization techniques on insoluble support were made. hereby we successfully applied the zeta potential measurement on the immobilization behaviour of the lipase cala. for to determine the operating temperature for the biomagnetic separation procedure we studied stability analysis of free and immobilised lipase cala at different temperatures ( , , , − • c) and at different ph values (ph , and ). the optimal temperature and ph value for the free and immobilised lipase was determined. presently and constructively on the so far developed methods and techniques we intensively work on the demonstration and feasibility of the recovery of immobilized lipase from a -phase-system. challenging approaches and first results on the recovery of immobilized lipase from a -phase-system in ml scale were shown already. nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated. three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of atoms) and carbonyldiimidazole activation. cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to . mg per ml support and a high recovery (up to %). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affin-ity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately × − , and . × − m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. quantitative methods in high throughput screening of aqueous two phase systems matthias bensch, björn selbach, jürgen hubbuch institute of biotechnologie , forschungszentrum jülich, germany purification of biopharmaceuticals is one of the most expensive and at the same time least understood steps in bioprocesses. during the process development for protein production, short time to market and the demand for cheap processes dominate today's process development. one way of reducing process costs is to implement integrative processes. aqueous two phase systems (atps) combine the advantages of removing cell debris and simultaneously purifying and concentrating the target protein, however, to the cost of highly complex systems which are difficult to predict and optimize. using high throughput screening techniques in the development of atps processes thus seems to be an ideal candidate for achieving both a reduced development time and an economical process without the need for preliminarily well characterized systems. in this study, we use the robotic system tecan freedom evo tm as an automation platform for the evaluation of aqueous two phase systems. central to this workstation are the integrated hardware as liquid handler, gripper, reader and centrifuge. we have created high throughput methods for rapid parameter estimations. as a first step, pipetting and mixing had to be calibrated for the use of highly viscous polymer solutions which are common in atps. the focus of the current work lies on the integration of the automatic preparation and analysis of two phase systems in microtiter plate scale. the robotic platform can now automatically create aqueous two phase systems and measure characteristic values such as binodal curves, protein concentrations and protein distributions between the two phases. the major bottleneck of hts processes, namely the rapid analysis of impure systems, is tackled by using automated elisa tools. depending on the intended use, the high number of measured partition coefficients and yields can be used for modelling or rapid process design. today, most optimisations of chromatography separations are based on experimental work and rule of thumb. the pat initiative has opened up for a model-based approach for downstream processing of pharmaceutical substances. this work uses a nonlinear chromatography model to optimize an ion exchange separation step. the general rate model with langmuir mpm kinetics described the behaviour of the components in the column. the optimal operating points using both productivity and yield as objective functions were found. the optimizations were run with both igg and bsa as target proteins respectively to compare their optimal operating points. the requirement on the optimal operating point was a purity of at least %. this requirement was added to the optimization problem as a nonlinear inequality constraint. flow rate, loading volume, start salt concentration in elution, elution gradient and cut points were used as decision variables in the optimization. the more retained component, bsa, was much easier to separate from igg with a gradient elution than igg from bsa while still retaining a high productivity and yield. the higher load volume at the optimal operating point, with bsa as target protein, causes a displacement of igg and thereby improving the separation. a high productivity at the yield optimum was still possible with bsa as target protein. both a lower productivity and yield was obtained with igg as target protein. optimisation and robustness analysis of a hydrophobic interaction chromatography step niklas jakobsson, marcus degerman, bernt nilsson department of chemical engineering, lund university, p.o. box , se- lund, sweden process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. the present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. factorial experimental design is common practice in industry today for robustness analysis. the method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. in addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. the methodology consists of threes consecutive steps. firstly, screening experiments are performed using a factorial design. secondly a kinetic-dispersive model is calibrated using gradient elution and column load experiments. finally the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. the process studied in this work is the separation of polyclonal igg from bsa using hydrophobic interaction chromatography. department of biochemistry and microbiology, ict prague, technicka , prague cz- , czech republic the display of novel metal binding sites on the surface of the biosorbent represents potent tool to increase its binding capacity and improve selectivity. in this study, the . kda transcriptional regulator merr of mercury-inducible mer operon of tn exhibiting high affinity and selectivity towards hg + , was displayed on the surface of s. cerevisiae. to achieve this, merr was genetically fused with gene encoding c-terminal domain of ␣-agglutinin which resulted in covalent attachment of the of the fusion protein on the cell wall glucan via glycosylphosphatidylinositol anchor. to evaluate the performance of such modified whole-cell biosorbent with specific regard to hg + , we constructed a new biosensor e. coli strain, which utilizes kanamycine resistance gene as a reporter under the control of mer promoter. it allowed determination of hg + in a range of - nm by simply monitoring the growth in the hg + /kanamycine-containing media. the effect of genetic engineering of s. cerevisiae surface by merr became significantly pronounced in biosorption experiments with solutions containing m hg + when modified cells accumulated . -fold more hg + than the control strain expressing mere anchoring domain. sensitivity analysis of amino acids in simulated moving bed chromatography ju weon lee, chong ho lee, yoon mo koo center for advanced bioseparation technology, inha university, inchon - , korea. e-mail: ymkoo@inha.ac.kr (y.m. koo) the difficulty of simulated moving bed (smb) design is that the optimization of the operation conditions relies on the determination of accurate adsorption isotherms. most smb chromatograph is carried out under nonlinear conditions, and the nonlinear behavior should be considered properly in the equilibrium isotherms. the other difficulty is the smb operation which has the characteristics of continuous process, all flow rates and switching time of valves should be maintained during the operation of smb. if the disturbances of operating conditions and isotherm parameters are occurred, it affects the zone flow rates and the migration velocity of the solutes, and these effects change the internal profiles of the solutes. therefore, it is the reason of decreasing the purity and the yield of products the objective of this work is to consider the sensitivity of isotherm parameters and operating parameters in smb chromatography process. two amino acids, phenylalanine and tryptophan, separation by smb process is selected as control system. application of ph and po probes during bacillus caldolyticus fermentation: an additional approach in improving a feeding strategy johannes bader , boris neumann , karima schwab , milan popovic , rakesh bajpai : studiengang biotechnologie, fachbereich v, tfh-berlin, seestr., berlin, germany proteome factory ag, dorotheenstr. , berlin, germany; department of chemical engineering, university of missouri-columbia, w ebe, columbia, mo, usa. e-mail: popovic@tfh-berlin.de (m. popovic) bacillus caldolyticus,a thermophilic microorganism, is a good producer of thermostable liquefying ␣-amylase. during optimisation of initial and feeding media for fed-batch fermentation a two component feeding containing starch and casitone was found advantageous. to approach the optimal feeding rate the method published by akesson et al., was extended to two component feeding. the key idea, discussed in this presentation, was using the po and ph probing signals to determine if the feeding of one or the other component should be increased or decreased. each of the probes offers information of different areas of feeding condition. to prevent excessive feeding of starch the ph probe is preferable. in case of excessive casitone feeding the po probe responds in very authentic way enabling together with the ph signal reliable and reproducible evaluation of feeding strategy. however a congruent response of po and ph probes means the approaching of the optimum feeding rate for both components. akesson, m., hagander, p., axelsson, j.p., . probing control of fed-batch cultivations: analysis and tuning. contr. eng. pract. , - . antibody immobilization by using the plasma polymerized acrylic acid r. jafari, m.tatoulian, f. arefi-khonsari laboratoire de génie des procédés plasmas et traitement de surface, enscp, upmc, rue pierre et marie curie, paris, france the objective of this work is therefore to produce a surface containing a high density of cooh functions on the polymer beads (ps) for the covalent immobilization of antibodies. we have investigated the plasma polymerization of acrylic acid in a fluidized bed reactor the polystyrene (ps) beads. for such application, there is a strong need to obtain stable plasma polymerized acrylic acid (ppaa) coating, resistant to washing with water. different physico-chemical analyses have been used (water contact angle measurements (wca), xps and sem analysis) to characterize the ppaa coating deposited on ps beads under different experimental conditions. the xps results showed that the pretreatment of surface of the beads before deposition of acrylic acid plays an important role on the stability of ppaa layer. the instability of the coating is partially due the fact that under certain conditions the coatings are soluble in water and secondly due to the bad adhesion of the polymer beads which are hydrophobic to the growing ppaa coatings. xps as well as tof-sims gives evidence of the immobilization of the antibody. xps results as well as static sims allows to detect nitrogen on the surface of the treated beads which proves the presence of the immobilized antibodies. under optimum condition the ppaa coatings provides the possibility to show a nitrogen uptake which varies between . and % of the apparent stoicheiometry of the surface. holst department of medical physiology, the panum institute, university of copenhagen, dk- copenhagen, denmark glp- (glucagon-like peptide- ), a peptide of amino acids secreted by endocrine cells in the gut in response to meal ingestion, was discovered during a systematic search for gut factors capable of enhancing insulin secretion. it turned out to be the most efficacious insulin releaser known, and unlike other factors, was shown to retain its insulinotropic activity also in patients with type diabetes. subsequent research has documented that the peptide not only releases insulin from the beta cells, but also enhance all steps of insulin biosynthesis, up-regulates beta cell gene transcription, and has trophic effects on the beta cells. the latter includes both proliferation of existing cells, neogenesis from ductal precursor cells, and inhibition of apoptosis. the peptide also inhibits glucagon secretion, reduces gastric emptying and reduces appetite and food intake. because of these actions, glp- administered to patients with type diabetes dramatically lowers blood glucose as well as glycated hemoglobin levels, and reduces body weight. however, natural glp- is extremely rapidly metabolized in the body, and the problem has been how to convert the unstable peptide into a clinically useful agent. the two main problems are its susceptibility to enzymatic degradation by ubiquitous dipeptidylpeptide peptidase iv (dpp-iv) and its rapid renal elimination. a related peptide, isolated from the saliva of a lizard, exendin- , was found to be a full agonist of the glp- receptor, to be resistant to dpp-iv and to be cleared more slowly by the kidneys. this peptide was highly effective in clinical studies and has now ( / ) been approved for diabetes treatment by the fda. other approaches include acylation of glp- whereby it attaches to albumin in the body and acquires resistance to dpp-iv as well as a slow renal elimination. also this analogue (liraglutide) has favourable clinical effects. fusion proteins of glp- and larger, slowly eliminated proteins in the body are currently being evaluated. small molecule, orally available inhibitors of dpp-iv have been demonstrated to protect endogenous glp- from degradation and to be efficacious in both experimental and clinical diabetes, and numerous inhibitors are currently in clinical development. the incretin hormones are released from gut endocrine cells upon meal ingestion. they enhance glucose-induced insulin secretion and nay be responsible for up to % of postprandial insulin secretion. the incretin hormones are glucagon-like peptide- (glp- ) and glucosedependent insulinotropic polypeptide (gip). in patients with type diabetes ( dm) the incretin effect is severely reduced or absent. in dm patients the secretion of gip is normal, but its effect on insulin secretion is almost completely lost. glp- secretion, on the other hand, may be impaired, but its insulinotropic actions are preserved and it may restore insulin secretion to near normal levels. substitution therapy with glp- might therefore be possible. glp- is a product of the glucagon gene and its actions include: ( ) potentiation of glucose-induced insulin secretion; ( ) stimulation of the expression of ␤-cell genes essential for insulin secretion, including the insulin gene; ( ) stimulation of ␤-cell proliferation and neogenesis (by enhancing endocrine differentiation of duct cells) and inhibition of ␤-cell apoptosis; ( ) inhibition of glucagon secretion; ( ) inhibition of gastrointestinal secretion and motility, notably gastric emptying; and ( ) inhibition of appetite and food intake. these actions make glp- particularly attractive as a therapeutic agent for dm. thus, continuous subcutaneous administration of glp- for weeks resulted in a mmol/l reduction in mean plasma glucose and a reduction in hgba c of . %; a weight loss of kg; improved insulin sensitivity; improved ␤-cell function; and the treatment was associated with no significant side effects. unfortunately, glp- is rapidly destroyed in the body by the ubiquitous enzyme, dipeptidylpeptidase iv (dpp-iv). clinical strategies therefore include: ( ) the development of metabolically stable analogues of glp- viz. activators of the glp- receptor; and ( ) inhibition of dpp-iv. orally active dpp-iv inhibitors have proven successful in experimental diabetes and several companies are now trying to develop clinically suitable inhibitors. so far the clinical experience is limited, but recent clinical studies have provided proof of concept. metabolically stable analogues/activators include the structurally related lizard peptide, exendin- or analogues thereof, as well as glp- derived molecules that bind to albumin and thereby assume the pharmacokinetics of albumin. these molecules are effective in animal experimental models of type diabetes, and have been employed in clinical studies of up to weeks' duration. on the basis of these studies it can be concluded that a therapy of type diabetes mellitus based on stimulation of glp- receptors is likely to be effective and to become a clinical reality within the not too distant future( - ). recombinant activated coagulation factor vii (rfviia) was developed to treat bleedings in hemophilia patients, who have developed inhibitors against fviii or fix, and has been demonstrated to have an efficacy rate of - % in major surgery as well as in serious bleedings in such patients. to use rfviia as a hemostatic agent in severe hemophilia is a new concept of treatment, not being a substitution therapy, but using a pharmacological dose of exogeneous rfviia to compensate for the lack of fviii or fix. the administration of extra rfviia has been found not only to bind to tissue factor (tf), but also to the negatively charged phospholipids surface of thrombin activated platelets. hemostasis occurs on surfaces being initiated on the tf-expressing cells as a result of exposure of tf, not normally exposed to the circulating blood, following an injury to the vessel wall. tf is a true receptor protein with an intramembraneous part and an intracellular tail. its ligand is fvii/fviia. as soon as tf is being exposed to the blood, it forms complexes with fviia already present in the circulation. these complexes activates fx and provide the initial limited amount of thrombin molecules activating the co-factors, fviii and fv, as well as fxi and platelets. following the thrombin activation of platelets, negatively charged phospholipids are being exposed on the outer surface of the platelets. on this surface most coagulation proteins bind tightly, facilitating the conversion of fx into fxa and the full thrombin burst, necessary for the formation of a tight fibrin hemostatic plug resistant against premature lysis. in hemophilia patients the initiation of hemostasis is essentially normal, but, since they lack fviii or fix, they do not form the fviiia-fixa complex necessary for full thrombin generation on the activated platelet surface. as a consequence the fibrin plug formed in hemophilia is loose, fagile and easily dissolved resulting in continuous bleeding. pharmacological doses of rfviia have been demonstrated to mediate direct binding of rfviia to the negatively charged thrombin activated platelet surface, thereby generating thrombin formation in the absence of fviii/fix. through this mechanism hemostasis is generated in hemophilia patients independent of fviii/fix. furthermore, by generating more thrombin at an increased rate the formation of stable, tight fibrin hemostatic plugs are facilitated. such fibrin plugs are more resistant against premature lysis and help not only to initiate but also to maintain hemostasis. based on its capacity of enhancing thrombin generation locally on the activated platelet surface, rfviia has been used to ensure hemostasis also in other situations than hemophilia, such as platelet defects including thrombocytopenia. recently, rfviia was shown to be hemostatically effective in patients with profuse bleedings as a result of vast trauma and tissue damage. in these patients with a complex hemostasis pattern including a host of changes leading to an impaired hemostatic function, extra rfviia seems to help generate a burst of thrombin resulting in the formation of a stable hemostatic plug more resistant against the ongoing lysis. in patients with intracerebral haemorrhage, one single dose of rfviia recently was found to limit the expansion of the haemorrhage and thereby leading to improved functional outcome. institute for medical microbiology and immunology, panum . . , blegdamsvej , dk- copenhagen n, denmark. email: s.buus@immi.ku.dk complete genomes from several species including many pathogenic microorganisms are rapidly becoming available along with the corresponding "proteomes". even at the peptide level, the diversity of proteome is enormous and easily represents a unique imprint of the originating organism. it is perhaps not surprising that the immune system considers peptides as key targets. recent immunological advances have shown that mhc molecules act as peptide selectors for immune recognition. we have proposed to generate accurate predictions of peptide binding to mhc and used these to identify immunogenic epitopes directly from genomic data. we have developed an iterative data-driven immunobioinformatics approach where data is used to generate predictors, and predictors are used to select new and complementary data for the next iteration. we have demonstrated the superior performance of this approach compared to a random data selection approach. we have also developed an efficient approach to select the most informative mhc molecules to investigate. the resulting, immunobioinformatics resource represents an immediate and powerful application and interpretation of genomic data, and will enable a rational approach to immunotherapy in the future. allergen specific immunotherapy is a causal treatment for igemediated allergic diseases such as hay-fever, and it has relied traditionally on preparations derived from aqueous extracts of various natural allergenic source materials. the cloning and production of an increasing number of allergens through the use of dna technology has not only facilitated the characterisation and analysis of the allergenic proteins, but also provided the opportunity to use these recombinant proteins instead of natural allergen extracts for the diagnosis and therapy of allergic disease. detailed physicochemical, biochemical and immunological characterisation are essential for the comparison of natural and recombinant proteins, and also provide a basis for developing derivatives. chemically modified allergens with attenuated ige-reactivity are currently used for immunotherapy in order to enable high doses to be achieved with a minimized risk of inducing allergic side reactions. dna technology provides the opportunity to develop and produce hypoallergenic allergen variants using strategies including gene mutation. the design of such variants must ensure that t cell reactivity and immunogenic activity are retained in order to preserve therapeutic potential. the recombinant allergens and their derivatives have several advantages over natural allergen extracts. they are relatively easy to produce in consistent pharmaceutical quality; the problems of natural extract standardisation can be avoided completely; the relative concentrations of the individual allergens can be controlled to obtain optimal dosages; nonallergenic proteins are excluded; the possible risks of contamination are avoided. the first clinical trials with grass pollen allergens and birch pollen hypoallergenic variants have yielded very encouraging results. the use of recombinant polyclonal antibodies (pabs) may improve the treatment of disease caused by complex targets such as infectious agents, when compared to monoclonal antibody therapy. symphogen has developed a method for reproducible production of target-specific fully human pab compositions, so-called symphobodies. the antibody genes are first isolated from donors with an immune response against the target and antibodies are screened for specificity. subsequently, the pabs are expressed in mammalian cells using the sympress technology, which is based on site-specific integration. this procedure ensures that each of the expression constructs encoding the antibody genes are stably integrated at the same site in each of the host cells, thereby eliminating genomic position effects and differential growth and production rates. further, the sympress technology comprises the generation of a polyclonal working cell bank (pwcb) which is used as inoculation material for the manufacturing. these cells display sufficient genetic stability to enable a controlled gmp production of recombinant polyclonal antibodies. symphogen's first product, sym , is a recombinant human polyclonal rhesus d-specific symphobody preparation consisting of different anti-rhesus d antibodies. this product is intended to be used for treatment of idiopathic thrombocytopenia purpura and prevention of hemolytic disease in newborns. recombinant anti-rhesus d symphobodies were produced and shown to be biologically active against rhesus d. the expression technology provided a compositional reproducibility between batches which is sufficient for manufacturing of such a polyclonal product for clinical use. scaledup production for clinical trials is currently ongoing. stem cells play an important role in renewing tissues such as skin and cornea. they are responsible for the continuous generation of the differentiated epithelium. we have characterized stem cells of the skin and cornea in situ and their fate in vitro in human skin reconstructed by tissue engineering using keratin (k) . in the outer root sheath of the hair follicle, stem cells (label-retaining cells) present in the basal layer of the bulge area express k and present a loosely arranged keratin filament network and low levels of k protein in their cytoplasm. in addition, another stem cell population (also labelretaining) is present in the first suprabasal layers. these cells exhibit a very dense keratin network and express k . three-dimensional tissue constructs (dermis and epidermis) obtained by the self-assembly approach of tissue engineering allow the preservation of k positive stem cells in the basal layer of the epithelium. in the eye, the stem cells are located in the limbal part but not in central cornea and they express k . the epithelium of reconstructed cornea is thinner compared to reconstructed skin and more transparent. the characterization of stem cells in reconstructed tissue is essential to evaluate the long-term survival of these tissues in vitro but also after grafting. these human reconstructed tissues are developed for fundamental (physiological, toxicological studies) and clinical applications such as transplantation for the permanent replacement of damaged organs. lg is holder of the canadian research chair (cihr) on stem cells and tissue engineering. alessandra gliozzi physical department, university of genoa, genoa, italy hollow nanometer-sized capsules can be prepared by means of different techniques. first "nanocapsules" were liposomes, however they are too unstable for many medical or pharmaceutical applications. in contrast, recently developed polyelectrolyte capsules prepared by means of the layer-by-layer technique are much more stable and seem to be a very promising way for coating living cells or tissues in order to prevent or reduce their immune rejection after implantation. several observations on single living cells encapsulated by the alternative adsorption of oppositely charged polyelectrolytes will be presented. the most relevant result is that cell preserve their metabolic activity, are still capable of dividing and performing specific functions. moreover, a technique to immobilize in defined arrays coated cells expressing green fluorescent protein by using a microcontact printing of polyelectrolytes will be presented. finally, tests performed to study the induction of fibrosis and vascularization by nanocapsules implanted in rat kidney and liver will be presented. over the past decade we have developed methods to generate spontaneously and synchronously beating tissue equivalents from neonatal rat heart cells in the culture dish. these tissue equivalents display the key morphological and functional features of intact myocardium and have been termed engineered heart tissue (eht). to generate ehts, heart cells are mixed with freshly neutralized, liquid collagen i, matrigel and growth supplements and grown in a circular casting mold around a central cylinder, which subjects the cells to a continuous mechanical load. this process is enforced by cyclic mechanical stretch. we use eht mainly for two purposes, as a test bed for the effects of pharmacological or genetic manipulations and for cardiac repair. as a cell culture model, ehts compare with standard d monolayer cultures of neonatal rat cardiac myocytes and freshly isolated adult cardiac myocytes. advantages of ehts are their functional similarities with intact heart muscles, the ability to easily measure force of contraction under mechanical load, the pos-sibility to transfect cardiac myocytes inside ehts with adenovirus at high efficiency and the reproducibility in large series. a disadvantage is that contractile function as measured at the end of the culture period also integrates influences on tissue development, cell-cellconnections, extracellular matrix production and on non-myocytes. at present we are working on downscaling the eht method to a well format for screening purposes. to use ehts for cardiac repair we created multi-looped ehts from five circular ehts large enough to cover the infarct scar days after coronary artery ligation in rats. ehts survived and formed a layer of muscle tissue on top of the infarct scar. ehts restored undelayed anterograde impulse propagation over the scar, prevented further ventricular dilatation, normalized enddiastolic pressure and relaxation, and partly restored contraction of the scar. thus, the study provides evidence that implanting ehts onto infarcted hearts can improve cardiac contractile function after myocardial infarction. the goal of tissue engineering is the development of skin, bones and even organs to restore, maintain and improve tissue function within the body. the current paper focuses on the investigation of invitro growth of osteoblast cells in different types of scaffolds. three of the scaffolds were made of pcl (polycaprolactone) % glycerol and % hca(hydroxylapetite). two of the scaffolds were made by compression molding, and one was made by fused deposition modeling utilizing the stratasys. the fourth cerabio was a commercially available product totally ceramic. the pores in compression molding were obtained by putting in % volume of sugar either and m which was later removed by leaching. the fused deposition scaffold was made by placing the filaments in a predetermined arrangement. the stratasys system was a computer designed model. the scaffolds were seeded with hfob . human fetal osteoblast cell line with vigorous shaking overnight and incubating at • c and % co . observation of the seeded scaffolds was made after days and days of incubation. the seeded cells were stained with bcip/nbp at • c overnight. the cell proliferation in the and m scaffolds appeared approximately the same with a possible advantage of m. the cerabio sample demonstrated the greatest proliferation among the four scaffolds studied and the stratasys sample exhibited a different type of cell adhesion with the cells were clustered in the interstices of the structure. denise freimark, ruth freitag, valérie jérôme chair of process biotechnology, university of bayreuth, d- bayreuth, germany. e-mail: denise.freimark@uni-bayreuth. de (d. freimark) tissue engineering is emerging as an alternative to bone grafts for the regeneration of defects that do not heal spontaneously. ultimately, the development of an optimum carrier and the identification of ideal inductive factors and cells may enable tissue engineering to provide an improvement over bone grafts in the future. bone formation and repair require a complex cascade involving growth factors, cytokines and angiogenesis. at present the complexity of the molecular mechanisms that control gene expression in bone forming cells in embryo as well as in adult is not fully understood. several factors like bone morphogenetic proteins (bmps), transforming growth factor beta (tgf-␤), vascular endothelial growth factor (vegf) and insuline-like growth factor (igf) have been identified and their ability to stimulate bone formation in vitro and in vivo has been investigated. while much is known about these factors per se, less is known about genetic regulation of artificially stimulated osteogenesis. interestingly, some in vitro investigations showed that only optimal growth factors concentrations lead to effective bone formation whereas higher concentrations had deleterious effects which suggest some variation in the activated regulation pathways. therefore, one of our goals is to analyze kinetics, dose-dependence and synergistic effects of growth factors and cytokines on regulation pathways of bone formation. moreover, the optimal vascularization of the scaffold is a major hurdle in the development of engineered bone. it is well known that: (i) vascular invasion precedes bone growth and (ii) osteogenesis takes place in the vicinity of newly formed vessels. thus, inadequate bone vascularization is associated with decreased bone formation. further analysis of the intercommunication between endothelial cells and osteoprogenitors in co-culture systems could provide key information that could be thereafter used to solve this problem. we propose to add some new knowledge to this complicated puzzle. a first step in our investigation is the production of some of the growth factors mentioned above in recombinant form. these factors are expressed in a novel vector (ptriex tm ; novagen) which allows recombinant protein production in prokaryotic or in eukaryotic systems with a single plasmid. afterwards, we analyze potential synergistic effects of these factors as well as kinetic and dose-depend parameters on the genetic regulation of downstream pathways in osteoblasts culture. in parallel, we develop an in vitro system allowing us to investigate the intercommunication between endothelial cells and osteoprogenitors. there has been significant interest in the therapeutic and scientific potential of human embryonic stem (es) cells since they were first isolated in . if human es cells could be differentiated into suitable cell types, stem cells might be used in cell replacement therapies for degenerative diseases such as type i diabetes and parkinson's disease, or to repopulate the heart following myocardial damage. however, there is a significant shortage of high quality human es cell lines and few research groups have experience in the propagation and manipulation of these cells. we are addressing this important issue using the combined expertise of the stem cell biology laboratory and the assisted conception unit at king's college, london. with local ethical approval and under licence from the uk human fertilisation and embryology authority, we have been establishing high quality human es cell lines from a novel source of human embryos. to date, we have derived three human es cell lines and are now focused on the generation of therapeutically important cell populations, including cells that may have clinical application in degenerative and traumatic injury to the brain and spinal cord, heart, retina and other target organs. wallenberg neuroscience center, department of physiological sciences, lund university, bmc a , s- lund, sweden cell replacement therapy for parkinson's disease is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. in rodent and primate models of parkinson's disease it has been shown that transplanted dopamine neuroblasts can re-establish a functional innervation and restore dopaminergic neurotransmission in the area of the striatum reached by the outgrowing axons; that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites; and that they can reverse or ameliorate some of the parkinson-like motor impairments induced by damage to the nigrostriatal system. clinical trials in patients with advanced parkinson's disease have shown that dopamine neuroblasts obtained from fetal human mesencephalic tissue can survive and function also in the brains of pd patients, restore striatal dopamine release, and ameliorate impairments in motor behavior. the principal limitation of this approach is the problems associated with the use of tissue derived from aborted human fetuses, and the large numbers of donors needed to obtain good therapeutic effects. until now, transplantation of dopamine neurons has focused primarily on differentiated neuroblasts and young postmitotic neurons, at the stage of neuronal development that is optimal for survival and growth of the grafted cells. however, progenitors taken at earlier stages of development might prove more effective. efforts are now made to expand multipotent neural stem-or progenitor cells in vitro, and control their phenotypic differentiation into a dopaminergic neuronal fate. initial results suggest that in vitro expanded cells can survive and function after transplantation to the striatum in the rat pd model, but the overall yield of surviving dopamine neurons has been very low. with further development, expanded progenitors or dopamine neuron precursors, possibly in combination with cell engineering techniques, may offer new sources of cells for replacement therapy in pd. stem cell therapy has been very much in vogue for several years now. like gene therapy before it, it has raised unrealistic hopes of cures being available imminently. unlike gene therapy, it can cite proof of principle in the well established practice of bone marrow transplantation which is actually a good example of stem cell therapy. however, most of the uses that are now touted as targets for stem cell therapy, envisage the conversion of the stem cells into lineage restricted progenitor cells or more commonly, the final differentiated cell type. such conversions are extremely difficult to initiate and control. the procedures involve the manipulation, differentiation, and expansion of cell cultures in the laboratory, with unknown long term effects on the genetics and physiology of the cells. these issues are compounded when one considers as source material, human embryonic stem cells, where not only the final cell product requires significant scrutiny, but also there are safety issues surrounding the persistence of undifferentiated cells. on top of all these challenges are commercial (for stem cell companies), clinical, and regulatory pressures which will impact heavily on the pace of progress. nonetheless, despite all these hurdles, various academic groups and companies are making significant progress and examples of such developments in diabetes and cardiovascular repair will be given stem cells have the unique ability to perpetuate themselves while continually replenishing tissues throughout the life of an organism. the era of cellular and tissue regeneration for the treatment of disease and the effects of aging has indeed begun. it has been known that mechanical factors play an important role in the regulation of cell physiology. it is therefore reasonable to believe that mechanical factors also play a significant role in the metabolic activity and differentiation of mscs. in this study, we investigated the viscoelasticity of individual bone marrow-derived adult human mesenchymal stem cells (hmscs), and the role of specific cytoskeletal component -f-actin microfilaments on the mechanical properties of individual hmscs. the mechanical properties of hmscs were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. for the hmscs under control conditions the instantaneous young"s modulus e was found to be ± (pa), the equilibrium young"s modulus e ∞ ± (pa), and the apparent viscosity ± (pas). after exposed to m of chemical agent-cytochalasin d that disrupt the f-actin microfilaments, the young's moduli of hmscs decreased by up to % and the apparent viscosity increased by %. these findings suggest that microfilaments are crucial in providing the viscoelastic properties of the hmscs, and changes in the structure and properties of them may influence the mechanical properties of hmscs significantly. pharmacologic transcription control of desired transgenes is essential for gene-function analysis, drug discovery, biopharmaceutical manufacturing, design of complex artificial regulatory networks, precise and timely reprogramming of key cell characteristics for gene therapy and engineering of preferred cell phenotypes for tissue engineering. capitalizing on our recent advances in the design of small molecule-responsive transcription control modalities we have used conditional molecular interventions for (i) improvement of specific productivity in biopharmaceutical manufacturing, (ii) transdifferentiation of therapeutically relevant cell phenotypes and (iii) design of artificial microtissues. we will also report on a completely new dimension of transgene control as well as engineering of hysteretic and epigenetic gene networks in mammalian cells. chronic diseases are a growing burden for the individual and society alike. one key factor driving this increase is an ageing population. currently, there are million persons aged years or over and this number is predicted to triple by the middle of the st century. effective prevention of irreversible damage to major organ systems requires early diagnosis and treatment yielding significant quality of life to the individual and sparing valuable health care resources. even when safe and effective medicines are available, a remaining problem to successful therapy are issues of patient compliance. historically, vaccines have been one of the major advances towards the longevity we enjoy today, with compliance rates close to %. hence, vaccines for early treatment of chronic diseases are ideally positioned for long-term therapy, and will take away the burden of self-medication associated with orally active drugs. here we will discuss a new generation of therapeutic vaccines based on virus-like particles (vlps). by displaying target molecules in a highly repetitive manner on vlps, it is possible to break b cell unresponsiveness in experimental animals as well as in humans. using such vaccines in animals, chronic diseases such as hypertension, alzheimer's disease, obesity and rheumatoid arthritis could be treated. furthermore, vaccination against nicotine resulted in high nictotine-specific antibody titers in humans, greatly facilitating smoking cessation in immunized individuals. interdependence of the impact of methanol and oxygen supply on protein production with recombinant pichia pastoris n.k. khatri, f. hoffmann martin-luther-university halle-wittenberg, institute for biotechnology, halle d- , germany. e-mail: f.hoffmann@biochemtech.uni-halle.de (f. hoofmann) the methylotrophic yeast pichia pastoris is a potent expression system for secretion of recombinant proteins. methanol as inductor of the foreign gene expression is also a substrate with high oxygen demand, which can lead to sudden oxygen depletion upon induction. thus, supply rates of methanol and oxygen are major process parameter during protein production with recombinant pichia pastoris. limiting dosage of methanol allowed maintenance of oxygen sufficient conditions during production of a single chain antibody fragment, but the product was degraded from the c-terminal end. in contrast, full-length product accumulated with controlled methanol concentrations despite oxygen limitation. the volumetric methanol uptake rate are limited by the oxygen transfer capacity of the reactor. higher methanol concentrations decreased the biomass yield and thereby increased the specific methanol uptake rate. this enabled prolonged production and yielded fivefold higher product concentrations. at the same time, the accumulation of small molecular weight contaminants was reduced. dosage of pure oxygen accelerated methanol uptake, grow and production. switching to dostat mode upon oxygen depletion led to an arrest of product accumulation, in contrast to persistent methanol feeding. the productivity was tenfold higher than without oxygen. combined with high methanol concentrations, however, fast methanol uptake led to toxication of the cells and early stop of production. flow cytometry revealed that perturbation of oxygen metabolism was followed by partial lysis of the culture. recombinant production of therapeutic proteins poses severe challenges due to their complexity (cystines, subunits, size). formation of the correct disulphide bridges is a prerequisite for activity but difficult to achieve in a prokaryotic host. there proteins mainly fold post-translationally as opposed to eukaryotic co-translational folding. thus the recombinant products are often not soluble and/or active. that is why different production parameters (e.g. host, induction conditions, temperature, compartment, proteinaceous fusion partners, co-expression of chaperones, foldases) are applied in order to gain functional recombinant proteins. the impact of all these strategies cannot be predicted and every problem of the production (expression, solubility, activity) might need to be solved for every target protein separately. nevertheless, much effort has been put into this for almost decades, because they are important targets for the pharmaceutical industry. human growth factors influencing cellular proliferation and/or differentiation are one example. this case study gives an overview of strategies tested within the development of two processes leading to an optimised yield of active protein. murine wnt- (wnt family) possesses conserved cysteines (most likely all involved in disulphide bridge formation and one in posttranslational modification) and could only be successfully produced in e. coli (fahnert, ) recently despite various attempts for many years. the other target protein is human collagen prolyl- -hydroxylase being a heterotetramer consisting of two ␣-subunits and ␤-subunits each. the ␣-subunit strongly aggregates if produced separately whereas the ␤-subunit is pdi and is suggested to have a chaperone function. therefore a sequential induction strategy was proposed for this protein . the moss physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. due to the elaborated post-translational capabilities of moss cells, the glycosylation patterns have been manipulated to obtain similar proteins to those found in animal cells. the protein expression using moss in suspension offers important advantages in comparison to other systems e.g. cho cells. the recombinant proteins can be targeted into the mineral medium, simplifying the down stream processing. moreover, there are neither known moss viruses nor plant viruses that are pathogenic for humans. the moss are cultivated axenically in a filamentous stage, the so called protonema. a pilot l tubular photoreactor is used to characterize the response of p. patens to variations on the culture conditions. the phototrophic culture in bioreactors is systematically investigated, where light quantity and quality, stress, concentration of phytohormones, and moss morphology influence the differentiation, growth, and protein expression. a tight control of the moss morphology in suspension, quantified by image analysis, has shown to be advantageous in order to delay the cell differentiation and maintain the carbon dioxide uptake in long bioreactor runs. the introduced perfused culture system by means of cross flow filtration allowed for a continuous product separation and concentration, and feed back of the productive cells. the characterization of this highly controlled culture system is presented and the potential of p. patens as an alternative tool for molecular farming is discussed. (rnai) is an evolutionarily conserved, endogenous mechanism for sequence-specific gene silencing that uses small double-stranded rnas (called short interfering rnas or sirnas) to direct cleavage or prevent translation of homologous mrnas. harnessing rnai for therapy presents an opportunity for potentially treating a wide variety of diseases. the main obstacle is delivering sirnas into the cytosol of target cells in vivo. although we were able to protect mice from autoimmune hepatitis by hydrodynamic tail vein injection of sirnas targeting fas, this delivery method is unlikely to be adaptable for human use. alternate strategies to deliver sirnas in vivo as small molecule drugs using currently available, clinically acceptable injection methods that have shown promise in mouse models will be discussed. these include local delivery to mucosal surfaces and delivery into specific cells via cell surface receptors using an antibody fragment fused to protamine. these sirna complexes silence gene expression in vivo only in cells bearing the targeted receptor. experiments showing efficient, effective and cell-specific delivery in a mouse tumor model will be discussed. in addition, encouraging preliminary data using rnai for a microbicide to prevent sexually transmitted infection will be presented. rna interference (rnai) holds significant progress as a therapeutic approach to siolence disease-causing genes, particularly those that encode "non-druggable" targets. the key hurdle for rnai therapeutics is in vivo delivery. a critical requirement for achieving systemic rnai in vivo is the introduction of "drug-like" properties, such as stability, cellular delivery and tissue biodistribution, into synthetic sirnas. our progress in achieving in vivo silencing of endogenous genes with chemically modified sirnas will be discussed. hiv- replication in human t cells can be inhibited by stable expression of a short hairpin rna targeting the viral nef gene (shrna-nef). however, hiv- escape variants emerge after prolonged culturing, and all but one escape mutant acquire a mutation in the shrna-nef target sequence. we observed single and multiple nucleotide substitutions, but also partial or complete deletion of the target sequence. these results demonstrate the sequencespecificity of this antiviral approach. we observed an inverse correlation between the level of resistance and the stability of the shrna/target-rna duplex for most of the escape mutants. however, two escape variants did not follow this pattern, including an escape mutant with a single point mutation at position − upstream of the target sequence. these mutants provide a much higher level of resistance than expected based on duplex stability, which is obviously not affected in the − mutant. we demonstrate that these mutants adopt an alternative rna secondary structure that occludes the target sequence. this results in reduced shrna-nef binding and provides a novel mechanism for rnai-resistance. to avoid viral escape, one should ideally target hiv- with multiple effective shrnas against conserved genome sequences. we performed a large scale screening to identify such targets, and we have identified at least nine genome segments that can be targeted effectively with shrnas. these potent antivirals are currently being assembled in a lentiviral vector for gene therapy applications in hiv-infected individuals. furthermore, we will describe approaches to forecast viral escape routes and to effectively block such evolutionary paths with additional rnai measures. in this study we analyzed the effect of antibodies against electronegative ldl on the development of atherosclerotic lesions in low-density receptor-deficient (ldlr −/− ) mice. two groups of (ldlr −/− ) mice (eight females) fed . % cholesterol-enriched chow were used. the first group received a monoclonal antibody against electronegative ldl ( g) and the second one received pbs (controls). additionally, other two groups (eight males) of (ldlr −/− ) mice were treated with a polyclonal antibody against electronegative ldl ( g) or pbs (controls). antibodies were administered by intravenous route one week before starting the hypercholesterolemic diet and then every week over an experimental time of days. afterwards, quantification of atherosclerotic plaque area of heart and aortic arch was done by analysis of the slices stained with oil red/hematoxolin/light green with the image propus software. the passive immunization with either monoclonal or polyclonal antibodies against electronegative ldl significantly reduced the atherosclerotic plaque areas in atherosclerosis-prone ldlr −/− mice. in conclusion, antibodies against electronegative ldl administered by intravenous route may play a protective role in atherosclerosis. supported by fundação de amparoà pesquisa do estado de são paulo (fapesp, scholarships to d.m.g., l.s. and a.b. and grants to m.h.k. and d.s.p.a.). the enzyme asparaginase from the procaryote escherichia coli or erwinia crysanthemi is used for the treatment of lymphoblastic leukaemia. the drug causes immunological reactions in despite of the treatment efficiency. asparaginase may also be obtained from saccharomyces cerevisiae and this enzyme could provide an alternative to its bacterial counterparts. in this study, the periplasmic nitrogen regulated asparaginase ii from s. cerevisiae, that is coded by the asp gene, was cloned and expressed in the methylotrophic yeast pichia pastoris under the control of the aox gene promoter. the recombinant p. pastoris strain was cultured in shake flasks and in a l instrumented bioreactor. in both cases it was observed specific enzyme yields seven fold higher in comparison to that using a nitrogen derepressed strain of s. cerevisiae, reaching u/g dry cell mass. high cell density cultures carried out in the l bioreactor, in which it was attained g dry cell mass/l, resulted in a dramatic improvement in asparaginase fermentation. as such, it was measured enzyme yields of , u/l and productivities of u/l h. department of biochemistry and food chemistry, biotechnology, university of turku, tykistokatu , biocity th floor, turku, finland. e-mails: lorenzo.galluzzi@utu.fi; deadoc@libero.it; deadoc@aliceposta.it (l. galluzzi) the bacterial luciferase operon from the bacterium photorhabdus luminescens has been used, since its first description, for exceptionally different applications. these ranged from the environmental monitoring to the cell tagging, from the analysis of cellular metabolism to the high-throughput screening of novel compounds. the wild-type luxcdabe operon has been engineered in countless ways (for instance by changing the order of the constituent genes, by optimizing the codon usage and by coupling it to many promoters) and has been expressed in prokaryotic and eukaryotic organisms in order to meet precise research and commercial needs. upon the operon expression light is emitted as the side product of a chemical reaction catalyzed by the luciferase enzyme, an ␣␤ heterodimer encoded in luxa and luxb genes. the reaction involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (fmnh ) with the liberation of excess free energy in the form of a blue-green light at nm. the luxcde genes code for the polypeptides (transferase, synthetase, and reductase) forming the fatty acid reductase complex that catalyzes the conversion of fatty acids into the long-chain aldehyde required for the luminescent reaction. noteworthy is that for the production of the substrates for the luciferase both atp and nadph are required, while neither is involved in the actual light emitting reaction (wilson and hastings, ) . recently, the coupling of the luciferase operon to regulated promoters lead to the construction of genetically modified bacteria able to sense the presence of chemicals and to respond, in a dosespecific manner, with light emission. this approach has been applied to the detection of antibiotics in samples from the food industry as well as to the detection of heavy metal ions in environmental samples (kurittu et al., ; bechor et al., ) . in addition, it opened the possibility of screening wide libraries of new compounds looking for molecules with pre-determined features, able to induce the bioluminescent response by de-repressing the lux operon transcription when incubated with the appropriate bacterial sensor. the high throughput and low costs are the main advantages of this system, which shows as well a certain degree of specificity (galluzzi and karp, ; galluzzi et al., ) . nevertheless, since the in vivo bioluminescence relies upon a complex network of biochemical reactions, under certain circumstances the light emission is not a direct consequence of the lux operon transcriptional induction but it more likely originates at a posttranslational stage. as a matter of fact, one can suppose that a change in the light emission will be observed whenever the concentration of one or more substrates for the lux␣␤ reaction occurs. consequently, all the molecules sharing the ability to impair the delicate chemical equilibrium regarding the compounds involved in bioluminescence will be sensed by the bacteria as inducing compounds. this, in turn, will result in a loss of specificity of the assay. we investigated this aspect of the whole-cell assays based upon the bacterial luciferase operon for drug discovery by means of a reporter plasmid in which the luxabcde genes, rearranged and optimized for the after min (black downward arrow) of incubation at • c under vigorous shaking the following concentrations of trimethoprim were added to the growing cells: g/ml (triangles), g/ml (circles) and g/ml (rumbles). water was administered to control cultures (squares). light emission was quantified every min by means of the wallac victor multilabel counter (perkin-elmer, turku, finland) and normalized to the absorbance, measured at nm with the same device. multi- white-walled transparent-bottomed plates were used for the assays (nalge nunc, usa). between the measurements the plates were kept at + • c under vigorous shaking. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). expression in gram + organisms, are under the control of the qacr regulatory region from staphylococcus aureus . non-pathogenic s. aureus rn cells bearing the pqaclux plasmid (cultivated in lb broth supplemented with , % d-glucose and g/ml chloramphenicol) emit light upon the specific transcriptional induction with quaternary ammonium compounds, widely used as surface disinfectants and in many over-the-counter drugs . however, the incubation of the same cells with an inhibitor of the dihydrofolate reductase enzyme, trimethoprim (sigma-aldrich chemie, steinheim, germany), enhanced in a dosedependant manner the light emission observed upon the induction with the optimal concentration ( g/ml) of benzalkonium chloride (bc). the extent of this increase in luminescence varied from - % to more than %, according to the trimethoprim concentration and to the measurement time. interestingly, when the same plasmid is carried by escherichia coli xl cells, the lux operon is expressed constitutively (since the qacr regulatory region is not functional in xl cells) and at much higher levels than in induced rn cells. also in this experimental system the incubation with trimethoprim results in a dramatic increase of the luminescent signal from the cultures. the explanation for these observations can be found in the molecular mode of action of trimethoprim. the inhibition of dihydrofolate reductase, indeed, directly leads to the accumulation of its substrates, among which is nadph, deeply entangled in the biochemical network of reactions centred on the light emission from the lux operon. nadph provides the reducing power to restore the reduced flavin mononucleotide pool and it is as well involved in fig. . xl /pqaclux growing cells were incubated with the following concentration of trimethoprim: g/ml (triangles), g/ml (circles) and g/ml (rumbles). water was administered to control cultures (squares). light emission and absorbance measurements were performed as previously described for rn cells. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). the diverse level of growth observed for rn and xl cells can be accounted by the different antimicrobial activity exerted by trimethoprim towards gram− and gram+ cells and by the lower activity of the promoter which in pqaclux plasmid drives the transcription of the selection marker (chloramphenicol acetyl transferase). the production of the long-chain aldehyde, both substrates of the lux␣␤ heterodimer (wilson and hastings, ) . in conclusion, here we demonstrate that the use of light emission from the bacterial luciferase as a transcriptional reporter has to be very carefully controlled, since some molecules (here trimethoprim) could mimic to some extent a specific promoter activation by impairing the delicate intracellular biochemical equilibrium. on the reverse side of the coin, fig. . simplified scheme depicting the bacterial folate metabolic pathway. only part of the reactions and compounds are reported. trimethoprim inhibits the nadph-dependant reduction of dihydrofolic acid into tetrahydrofolic acid catalyzed by dihydrofolate reductase. this results in the accumulation of both substrates, which become available for other reactions, and in the depletion of tetrahydrofolate, the major c carrier in the synthesis of purines, thymidine, glycine, methionine, and pantothenate in bacteria. for antimicrobial purposes trimethoprim is often associated with sulfonamides, with which it displays a synergistic activity (since they act on the same pathway but at an earlier reaction) (scholar and pratt, ) . however, it has to be noted that the lux reporter system could be used in many different in vivo experimental setups, where the change in the intracellular concentration of nadph, atp or other metabolites would be sensibly detected. we have carried out the construction of five pichia pastoris strains harboring in their respective genome three different growth hormones cdna's ( and kda human growth hormones and bovine growth hormone), a shrimp (litopenaeus vannamei) trypsinogen cdna and a bacterial (bacillus subtilis) phytase gene. in all the cases, the same kind of expression vector and the same transformation technique were used. each dna was fused in frame to saccharomyces cerevisiae alpha-factor secretion signal to lead the secretion of the foreign protein into the culture medium. the induction of each recombinant strain, all with his + and mut + phenotype, was carried out in shake flaks using methanol buffered minimal medium (bmm) and growth rates on methanol determined. production level of secreted proteins was evaluated by protein analysis by sds-page. furthermore, the expression with three of the constructed strains was performed in a -l bioreactor and the level of secreted protein determined in each case. both human growth hormones (hghs) were secreted into the culture medium with a high degree of purity obtaining up to % of hghs of total proteins in crude fermentation medium and - mg/l of hghs. neither bovine growth hormone, shrimp trypsinogen or bacterial phytase were detected in methanol induced cultures of the respective strains, in spite of the same culture conditions were used for all p. pastoris strains. the growth rates on methanol were different between some strains. in fermentor cultures the hghs production were increased and shrimp trypsinogen was produced and secreted into the culture medium after improving the culture conditions. bovine growth hormone was detected only when the culture conditions were modified. the protein production level for each strain was affected by the proprieties of each recombinant protein produced. competitive advantages of the diagnostic method for invasive amoebiasis using preserved antigenic extracts without using enzymatic inhibitors m.s. flores , * , e. tamez we have patented a method to diagnose invasive amoebiasis using a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors (ic:mc). the available tests for serologic diagnosis of invasive amoebiasis like elisa and indirect haemaglutination (iha) do not have consistent results in endemic zones. here we show the advantages of the assay and the validation of this diagnostic test for invasive amoebiasis. we demonstrated the reduction of proteolytic activity of ic:mc compared with the proteolytic activity of crude extract and crude extract with enzymatic inhibitors using assays. we displayed the ic:mc sds-page pattern and the western blot (wb) pattern useful for diagnosis. to search the clinical utility of this test we examined the wb obtained with sera from patients with different liver diseases; patients had invasive amoebiasis and patients had other liver diseases. the results were compared with those of iha test. also we have tested the accuracy of wb using sera from people with multiple intestinal parasites, like giardia lamblia, hymenolepis nana, blastocystis hominis, entamoeba coli, etc. the sensibility of the wb using the preserved amoebic antigens was %, specificity was %, positive predictive value was %, negative predictive value was % and accuracy was %. the wb did not exhibit cross reactions with sera from persons with intestinal parasites. our test was better than the (iha) test commonly used in endemic zones. these results show the improvement of using the preserved amoebic antigens in diagnostic tests. also they prove the diagnostic accuracy of our new wb test. antibacterial and antifungal activity of heracleum sphondylium subsp. artvinense yasemin kaçar , sema tan , aysun ergene , perihan güler , semra mirici , ergin hamzaoglu , ahmet duran , sinem yildirim : mersin university, faculty of science and literature, department of biology, mersin, turkey; kırıkkale university, faculty of science and literature, department of biology, yahsihan-kırıkkale, turkey; akdeniz university, faculty of education, department of biology education, antalya, turkey; selcuk university, faculty of education, department of biology education, konya, turkey turkey is covered yearly with a huge number of plant species. about species are condenced on the region that between asia and europe. many plant species have been used in folkloric medicine to treat various ailments. heracleum l (apiaceae) is include over than species on the world. this variety is represent with species in turkey that seven species are endemic. heracleum sphondylium subsp. artvinense is endemic species for turkey. ethanolic and aquous extract of heracleum sphondylium subsp. artvinense were investigated for their antimicrobial activities against six bacterial species (e. feacalis, e. coli, s. aureus, p. aeruginosa, l. monocytogenes, shigella) and two yeast (c. albicans, c. krusei). both ethanolic and aqueous extract of heracleum sphondylium subsp. artvinense showed antimicrobial activity against the gram negative bacterium (shigella) and gave the best activity against c.albicans. to develop artificial vectors allowing nucleic acid to transfect into mammalian cells are crucial for extending gene therapy. synthetic vectors based on lipid molecules are particularly attractive because of their potential safety. however, the low encapsulation efficiency of nucleic acid is one of the problem to be solved. recent advances in dna-lipid complex have improved this drawback, and now some lipid molecules are used as transfection agents. in particular, cationic lipids interact with negatively-charged cell surfaces and nucleic acids. the former interaction results in delivery of the nucleic acids directly across the cell membrane. on the other hand, the latter interaction improves the efficiency of nucleic acids entrapment. in this study, we developed a preparation method of "nanovesicle" containing nucleic acids by using reverse micellar solubilization. since dna interacts spontaneously with cationic amphiphiles, complete extraction of the dna molecules into an organic phase using dimethyl distearyl ammonium bromide ( c ab), an oil-soluble cationic surfactant, is achieved. the re-encapsulation of the reverse micellar droplets solubilizing dna by water-soluble surfactants facilitates the formation of nanovesicles. a high salt concentration at the re-encapsulation step promotes the production of nanovesicles containing dna molecules. eventually, more than % of dna was encapsulated in this nanovesicles under the condition of m nacl and % (w/v) cetyltridecyl ammonium bromide (ctab). in addition, the nanovesicles prepared at • c was smaller than that prepared at room temperature. the resultant c ab/ctab nanovesicle was ca. nm. asymmetric and chemically-modifiable vesicle surface are effective to design gene delivery system. moreover, such a small dna (or rna) carrier has a potential for novel gene therapy application from skin. the key players in clinically important inflammatory diseases, endothelial cells and leukocytes, communicate through membranebound cell adhesion molecules (cam's). obvious strategies for therapeutic intervention include specific means to affect the expression of cam's on the cells involved. rna-interference (rnai) is a well known means to achieve specific gene-inhibition. for this study, two cam's were chosen, namely vascular cell adhesion molecule- (vcam- ) as a target for inhibition, and intercellular adhesion molecule- (icam- ) as a non-target reference. we designed short interfering rna (sirna) oligos for vcam- in order to selectively inhibit the expression of this cam in cultured human vascular endothelial cells (huvec). real-time rt-pcr showed an % down-regulation of vcam- mrna while the expression of icam- remained unaffected. neither cam was affected by non-specific sirna. in order to further substantiate the potential use of sirna for therapeutic purposes, we have set out to investigate two things: ( ) does vcam- -specific sirna affect the amount of vcam- on the surface of huvec? ( ) is adhesion between leukocytes and endothelial cells affected by vcam- -specific sirna? the manufacturing of plasmid dna (pdna) is crucial to obtain a consistent product for gene therapy applications. although flowsheets for pdna production are established on the basis of experience, simulation tools provide a valuable help for evaluating alternatives. a process designed to produce kg pdna/year is analysed with the superpro designer tool. the target pdna is amplified in escherichia coli. after harvest, alkaline lysis is used to disrupt cells and release pdna and impurities. precipitations with isopropanol and ammonium sulphate are performed to concentrate/pre-purify pdna prior to hydrophobic interaction chromatography. experimental data is used as input for simulation. inventory analysis identified water, yeast extract, tryptone, isopropanol and ammonium sulphate as the major raw materials. major raw material costs ( %) are related to fermentation components. economic analysis indicates a unit production cost of $ /g pdna. for a selling price of $ /g, the payback time was . years and the roi was . %. an environmental analysis highlighted the replacement of the isopropanol precipitation for a microfiltration step as a benefit which would: (i) reduce the cost of raw materials ( . %), (ii) reduce the environmental impact associated with isopropanol ( %) and (iii) reduce costs associated with the treatment/disposal of liquid waste ( . %). preparation of chitosan microspheres for controlled release of somatotropin s. simsek , j. introduction: proteins and peptides have received extensive interest for their therapeutic applications in clinical applications. in order to achieve high administration efficacy of proteins, polymeric particulate carriers have been developed as an effective way to control the drug release profile and to protect the protein molecules from degradation. somatotropin also known growth hormone is a protein hormone of about amino acids. growth hormone is of considerable interest as a drug used in both humans and animals. chitosan a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin. properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in biomedical and pharmaceutical formulations. the aim of this study was to prepare chitosan microspheres containing somatotropin and to investigate these microsphere formulations in-vitro release properties. methods: somatotropin-chitosan microspheres were prepared as follows: chitosan was dissolved in acidic solution ( %) containing polysorbate . sodium sulphate solution ( % w/v) containing somatotropin was added into the chitosan solution and mixed rpm for a hour. resulting suspension was centrifuged , rpm min at • c. the formed microspheres were freeze-dried and sieved. in-vitro release studies were performed in ph . phosphate buffer and time interval samples were removed and analysed by bradford protein assay method. results: microspheres were obtained by using chitosan. protein encapsulation efficiency was between and %. average particle size of microspheres was between and m. during to in-vitro release studies burst effect was observed with chitosan microspheres. for decreasing the burst effect gluteraldehit, betacyclodextrin and poly ethylene oxide were added to formulations. conclusion: according to our results modified chitosan microspheres are promising vehicles for controlled release somatotropine delivery. cancer immunotherapy using hyperthermia with magnetic nanoparticles and dendritic cells k. tanaka, a. ito, t. kobayashi, t. kawamura, s. shimada, k. matsumoto, t. saida, h. honda department of biotechnology, school of engineering, nagoya university, nagoya, aichi - , japan. e-mail: h d@mbox.nagoyau.ac.jp (k. tanaka) our hyperthermia system utilizes magnetic nanoparticle covered with lipid layer including cationic lipid (magnetite cationic liposomes, mcls) as a heating mediator and necrotic cell death is induced by means of locally generating heat. in this process, heat shock proteins (hsps) are strongly induced and released. dendritic cells (dcs) are potent antigen-presenting cells (apcs) that play a pivotal role in regulating immune responses in cancer, which is in carrying antigens to apcs and in the maturation of dcs by acting as a danger signal. in the present study, we investigated the therapeutic effects of dc therapy combined with mcl-induced hyperthermia on b melanoma. in an in vitro study, when immature dcs were pulsed with b cells heated at • c for min, mhc class i/ii, costimulatory molecules cd /cd , and chemokine receptor ccr in the dcs were up-regulated, thus resulting in dc maturation. c bl/ mice bearing a b melanoma nodule were subjected to combination therapy using hyperthermia and dc immunotherapy. mice were divided into four groups: group i (control), group ii (hyperthermia), group iii (dc therapy), group iv (hyperthermia + dc therapy). complete regression of tumors was observed in % of mice in group iv, while no tumor regression was seen among mice in the other groups. increased ctl and nk cell activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of b melanoma cells. this study has important implications for the application of mcl-induced hyperthermia plus dc therapy in patients with advanced malignancies as a novel cancer therapy. fermentation of a marine bacterium for the production of cytotoxic compounds vicky webb , els maas , eiichi akaho , hiroto kambara , debbie hulston , anna kilimnik : marine biotechnology, national institute for water and atmospheric research ltd, kilbirnie, wellington new zealand; faculty of pharmaceutical sciences, kobe gakuin university, kobe - , japan marine bacteria are a potential source of novel compounds for the pharmaceutical industry. new zealand marine bacteria were isolated from a variety of sources and initially bacterial supernatants were screened using a cell based mtt cytotoxic assay. two bacteria were chosen for further fermentations in different media to optimise cytotoxic compound production. the media used were selected for their diverse ingredients. they were either carbon rich, nitrogen rich, starch rich or a basic seawater medium. the fermentations were extracted using ethyl acetate and methanol prior to assaying. the results showed that cytotoxic compound production was enhanced ten fold in the starch rich medium compared to the other media. the levels of cytoxicity appeared to be depended on the cell line used, with the epithelial lung carcinoma cell line a being more sensitive to the cytotoxic compounds than the human fibroblast cell line mrc- . isolation and identification of marine bacteria from deep-sea sediments els maas , cara brosnahan , vicky webb , helen neil , phil sutton : marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington new zealand; oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand polystyrene micro plate activated by utilizing a common co- gamma source with a crotonic acid. the well surface have been studied in term of optical quality, protein (h-igg) binding capacity and stability. a significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.the majority routine laboratory tests for the measurement of rheumatoid factor (rf) are semi quantitative and in order to achieve accurate, sensitive and specific rf assay, we are developed enzyme linked immuno sorbent assay (elisa) for human igm-rf kit. stable and reproducible binding of antigen (human-igg) to well of micro titer plate is a prerequisite for rf-elisa kit. the principle of the assay was that the antibodies in serum of patients with rheumatoid arthritis (ra), commonly rheumatoid factor (rf) are directed to the fc part of human igg. in most cases rf belong to the igm class, the well of micro titer plate are coated with antigen (h-igm), and antibodies (h-igg) binding to immobilized antigen is detected by adding enzyme conjugate (anti human-igm-hrp-enzyme) to the wells, substrate was used for color reaction.the performance characteristics of the assay was, the coefficient of variation (c.v) of intra and inter assay was . % and . % respectively, the linearity is ranging from to %, and the recovery for three different sera is ranging from to %. the data presented in this paper indicated that the activation of polystyrene micro titer plate by the gamma rays could be use for preparation of igm-rf kit and may be others immunoassay techniques. overcoming the nuclease barrier to gene expression during trafficking of plasmid dna vectors a.r. azzoni , a. tavares , g.a. monteiro , d.m.f. prazeres : centro de engenharia biológica e química (cebq), instituto superior técnico, - lisboa, portugal; instituto gulbenkian de ciência, rua da quinta grande , - oeiras, portugal. e-mail: azzoni@ist.utl.pt (a.r. azzoni) inefficient nuclear delivery of plasmid dna (pdna) vectors is thought to be a bottleneck to gene transfer in gene therapy and dna vaccination utilizing non-viral delivery systems. one of the main barriers found by pdna vectors during trafficking to the nucleus is degradation by a population of endo/exo-nucleases. this barrier may be partially circumvented by shielding the pdna from the nucleaserich cell environment with adjuvants or by using nuclease inhibitors. a different approach that has been studied at the cebq is the generation of pdna vectors that are more resistant to nuclease action a priori. in this work, the nuclease barriers to gene expression are being studied aiming at the generation of pdna with improved resistance to nucleases and thus higher transfection efficiency. by engineering the plasmid labile sequences, new plasmid vectors with an improved resistance to physical-chemical and biological degradation are being generated. this was indicated by an extended half-life of the supercoiled isoforms during storage and when the plasmids were exposed to nucleases present at eukaryotic cell lysates and mice plasma. the intracellular trafficking of the new plasmid vectors through the cytosol of mammalian cells was then assessed by fluorescence in situ hybridisation (fish) and the expression of the reporter protein (egfp) was detected by fluorescence microscopy. identification and evaluation of antibacterial phytochemicals of fishbone fern (nephrolepis cordifolia) rikhia chakraborty , , promod kumar verma : department of cancer biology, lerner research institute, euclid avenue cleveland, oh , usa, department of biotechnology, guru nanak dev university, amritsar, punjab , india. e-mail: riar @rediffmail.com (r. chakraborty) in this study, different aqueous and organic extracts from the fern, nephrolepis cordifolia, were used for screening tests for antibacterial effects. protein and lipid extracts were first tested for antibacterial activity. subsequently, crude extracts of leaves, roots, and stems were prepared in methanol, ethanol, chloroform, hexane, petroleum ether, diethyl ether, and water using optimized standard protocols. each fraction was tested for anti-microbial effect through agar well-diffusion assay, and paper disc method. the antibacterial spectrum against which the fern is active was thus determined. dosagedetermination for optimum activity was also determined for each of the extracts. bacillus and staphylococcus were used as the indicator test-organisms. the results were very encouraging; being effective even at the th day, thus showing that the antibacterial properties were not due to any changes in external factors and physiological effects. ethanol, methanol and chloroform extracts from the subaerial portions had strong anti-microbial properties. agar-well diffusion assay and paper-disc diffusion assay done for different solvent fractions were giving comparable results. . mg was adequate for maximum effect against b. circulans, s. aureus, s. epididermis, and streptococcus sp. . mg was adequate as effective dosage for kleb-siella pneumoniae. mg was required for mycobacterium bovis, e. coli. pseudomonas was not showing any susceptibility. given the broad spectrum of activity, especially towards the gram-negative bacteria, nephrolepis cordifolia is definitely a promising plant having pharmacological importance. for a full interpretation of the present results further investigations are necessary to elucidate the different physical and chemical parameters of the active principles and also to determine the mechanism of action. the present work highlights n. cordifolia as a plant having a broad spectrum of antimicrobial activity, a phenomenon very rarely observed in the plant kingdom. bacteria (escherichia, salmonella, proteus, staphylococci and bacillus) were isolated from hospital soil using selective enrichment and growth on selective and differential medium, viz. macconkey's agar, clyed medium and baird parkers medium. confirmation and species identification was carried out by biochemical and serological tests. from these isolates, three different pathogens were used to study multiple antibiotic resistances. e. coli bj showed resistance to ampicillin, streptomycin and cefurixime. s. typhi and s. aureus showed resistance to ampicillin and cefuroxime. assay using octadisc using e. coli and s. typhi showed a broader resistance pattern to antibiotics including amoxicillin, clavulanic acid, cephalexin, chloramphenicol, ciprofloxacin, and cotrimoxazole. the r plasmid profile was studied to understand the mechanism of drug resistance. to combat the problem of drug resistance, a strategy of combined antibiotic response of cultures were studied. such a synergistic combination would possibly have the effect of overcoming multiple antibiotic resistances. for e.g. kanamycin resistance strains were inhibited in presence of kanamycin and cefotaxime. further, the bactericidal activities of antimicrobials in honey and garlic were also tested. the mic of honey was observed to be %, while that of garlic was between . and %. honey and garlic were also found to inhibit the growth of organisms in the presence of antibiotics. kanamycinresistant e. coli was unable to grow in presence of kanamycin and . % garlic. these traditional agents, long used in ayurvedic system of medicine in india, could be further explored for potent antimicrobial properties. hepatitis b virus (hbv) infection is s global health problem. assays for hbv antigens and antibodies are widely available and standardized. extremely sensitive qualitative pcr kits are also available for detection of hbv in serum. hbv pcr kit may be useful in assessment of occult hepatitis b in hbcab positive alone subjects and carriers. a positive pcr results show presence of virus articles in serum without considering serologic results. but there are differences in efficiency of hbv dna amplification kits. in this study we compare two commercially available hbv pcr kits for evaluation viremia of hbsag positive carriers and hbcab alone positive subjects. material and methods: of the randomly selected subjects serologically examined for hbv, and were positive for hbsag and hbcab alone respectively. both later groups were tested for hbv dna by two commercial kits, hbv pcr detection kit (cinnagen, iran) and hbv pcr test (pazhohesh azma, iran). dna extraction kits recommended by each manufacturer were used for hbv dna extraction. amplicons in both kits were a highly overlapped fragment in conserved sequence of viral genome. results: of the hbsag positive carriers, hbv dna was detected in . and . % using kit and kit respectively. only . % were positive in both kits. in hbcab positive subjects (n = ), . % were positive by kit and all samples were negative when tested by kit . there was a significant difference between two kits. sensitivity of kit and kit were . and . %, respectively. overall, kit increased the detection rate of hbv dna by . %. discussion: our study show there is a significant variation between these two commercial kits especially in hbcab positive subjects that the copy of virus is very low. from these results, it can be concluded that the unstandardized kits have not compatible results, and pcr test interpretation should be done with great care. . the antibacterial activity of the extracts was evaluated based on the inhibition zone using plate diffusion method. most of the extracts were active against both gram positive and gram-negative bacteria, but pseudomonas aerugenosa, bacillus subtilis and sarcina marcescens, were more susceptible to almost all the extracts. finally, further research will be done to elucidate the nature of the active compound and investigate for peptides by using protein gel immobilization bioassay. short interfering rna delivery and gene silencing using polymeric nanocarrier systems k.a. howard , x. liu , d. oupicky , f. besenbacher , j. kjems : inano, university of aarhus, denmark, department of pharmaceutical sciences, wayne state university, detroit, usa the effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. nanocarriers for delivery of bioactive agents are being developed at inano to maximise drug payload at target sites. the inclusion of "biological triggers" into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction. chitosan and peptide-based polymers were used to formulate nanocarriers in the size range - nm containing small interfering rnas (sirnas) for gene silencing applications. page analysis showed the structural integrity of the sirna was maintained during particle formation. in systems composed of bioresponsive polymers, nanocarrier disassembly and sirna release under cellular conditions were shown, using atomic force microscopy. the time course for sirna uptake into nih cells was visualised using confocal microscopy. in addition, sirna localisation within cells could be modulated by the composition of the polymer used. the ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (egfp). furthermore, the various delivery systems were tested in a mouse model stably expressing the egfp protein using both nasal and intravenous delivery routes. the use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. however, the presence of xenoreactive antibodies in humans directed against swine gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. the graft of genetically modified organ of a swine depleted of enzyme ␣ , -galactosyltransferase that is responsible for gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. to prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. for this purpose genetic constructs containing inactivated ␣ , -galactosyltransferase gene, human cd , cd and cd genes controlling complement activation and human genes encoding ␣ , -fucosyltransferase and ␣-galactosidase enzymes modifying cell surface proteins were prepared. these genetic constructs were transfected into the pig foetal fibroblast using lipofection method. after selection, molecular and cytogenetic characteristic of cells with transgene integrated into the host genome were performed. introduction: protease a( a-pro) of coxsackievirus b (cvb ) plays major role in viral replication. in case of infection, viral proteins are being synthesized from viral mrna using host biosynthesis machinery. a-pro of virus, after being synthesized, exhibit two critical functions, cleavage of viral proteins and breaking eif g (eukaryotic initiation factor g-formerly called p ) which leads to host cell translational system shot-off. the enzyme plays essential role in viral replication and cellular damage. to understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure apro enzyme. in this study an expression system with efficient and high yields was obtained. methods: cdna of apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pet b(+) and since a-pro is a toxic product, naturally before induction its expression will act on the host and damage the cells. for this different hosts were checked and finally, blr(de ) plyss which carries an extra-plasmid for lysozyme expression that minimizes unwanted target protein production (leakage) was selected. on the other hand for biological activity assay, polyclonal antibodies against antigenic sites of p was prepared by synthesizing small peptides, corresponding to antigenic site of p coupling to klh and injecting subcutanously to rabbit. then, the enzyme and its substrate (hela cells lysate that contain p ) were incubated together for different times intervals. results: the recombinant product was confirmed by sds polyacrylamide gel electrophoresis and immunoblot analysis. also p cleavage by apro was assessed by sds-page and western blot analysis. cleavage of p by r a-pro was prominent after h. so recombinat a-pro with good activity was prepared. application of bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system on production of glycoprotein in larvae of silkworm ayano kageshima, tatsuya kato, misun kwon, enoch y. park department of applied biological chemistry, shizuoka university, ohya , suruga-ku, shizuoka - , japan bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system was applied on production of glycoprotein in larvae of silkworm. the bacmid system of autographa californica nuclear polyhedrosis virus (acnpv) has already been established and widely used. since the acnpv does not have a potential to infect silkworm we developed the first practical bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system directly applicable for the protein expression of silkworm. by using this system, the green fluorescence protein and glycoprotein, human ␤ , -n-acetylglucosaminyltransferase were successfully expressed in silkworm larvae not only by infection of its recombinant virus but also by direct injection of its bacmid dna. three different kinds of signal sequences were tested for the secretion of glycoprotein into hemolymph of silkworm. signal peptides of prophenoloxidase-activating enzyme and bombyxin: insulin-like brain secretory peptide showed the highest secretion ratio, % of total expressed ␤ , -n-acetylglucosaminyltransferase was secreted into hemolymph of silkworm. using bacmid system mu/ml of ␤ , -n-acetylglucosaminyltransferase was expressed in hemolymph of silkworm, which was -three times higher than that of insect cell. silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. this method provides the rapid protein production in silkworm, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. we have established an efficient system for foreign gene expression in lily (lilium longiflorum) pollen in a transient mode. pollen was transformed using agrobacterium via vacuum filtration for min. the pollen germinated for h was analyzed to confirm its foreign gene expression in molecular analysis. mouse fed the transgenic pollen culture for weeks showed immune reaction specific for pollen-derived recombinant protein. and the igg level was highly elevated by one time boosting injection afterwards. the lily pollen system may be suggested as a novel type of biofactory for producing edible vaccine with rapidity. anti-apoptosis engineering with the kc gene obtained from silkworm shin sik choi, won jong rhee, tai hyun park school of chemical and biological eng., seoul national university, seoul - , korea the chinese hamster ovary (cho) cell line producing recombinant human erythropoietin (epo) was manipulated to express the kc gene, which was originally obtained from a silkworm. the expression of kc inhibited serum deprivation-induced apoptosis and increased the cell density and epo expression level per unit cell by five-and two-folds, respectively. an increase in these two factors resulted in a -fold increase in the volumetric productivity of epo. compared with the controls, the oligosaccharide structures of the epo synthesized by the cells expressing kc showed greater homogeneity. the terminal sialylation of the glycans of epo were promoted by the expression of kc . the positive effects of kc expression on the cell viability and productivity were attributable to the stable maintenance of the mitochondrial activity. these results demonstrate that the cho cell line genetically engineered with the kc gene has a great potential for use in the production of therapeutic proteins. evaluation of fucoidan-chitosan hydrogels on superficial dermal burn healing in rabbit: an in vivo study a.d. sezer , f. hatipoglu , z. ogurtan , a.l. baş , j. introduction: healing of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the pharmaceutical biotechnology. fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. although a great number of studies on the different pharmacological properties of fucoidan are present, there is very limited information on the fucoidan-based system used in dermal burns. the aim of this study was to prepare chitosan hydrogel containing fucoidan and to investigate this hydrogel formulation for treatment of dermal burns on rabbits. methods: fucoidan-chitosan hydrogels were prepared as follows: the polymers were dissolved in acidic solution and sonicated for removing the air-bubbles then the gels were stored at + • c for in vivo studies. seven adult male new zealand white rabbits (mean weight, . ± . kg) were used for the evaluation of the gels on superficial dermal burns. the back of the rabbits were depilated and sedated. the wounds were made by circular stamp aluminium caps ( . cm ) at • c. four wounds were formed for each rabbit; (a) was treated with fucoidan-chitosan gel, (b) was treated with fucoidan solution, (c) was treated with chitosan gel (without fucoidan) and (d) as a negative control group. biopsy samples were taken at , and st days at the beginning of the study and each wound site was macroscopically observed and evaluated histopathologically. results: oedema was not observed in all groups after days treatment except controls. after days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan gel and fucoidan solution. the best regenerate dermal papillary formation and the fastest closure of wounds were observed in group a after days treatment. the wound epithel elongation and thickness values were measured; a ( and m), b ( and, m), c ( and, m), d ( and, m) at the end of the study. conclusion: re-epithelization and contraction of the wound area which was treated with fucoidan-chitosan hydrogel were faster than the other groups. the fucoidan-chitosan hydrogel formulations can be suitable for the treatment of dermal burns. aim: to analyze the neutralizing -related activity of antibodies against e region of hcv, specific polyclonal antibody was raised by immunized rabbits with synthetic peptide that had been derived from e region of hcv with the amino acid sequence e antibody [ghrmawdmm). materials and methods: hyper-immune hcv e antibodies were incubated over night at • c with serum samples from patients positive for hcv rna, with different viral load, ranged - million copes/ml, then incubated min to hepg cells. rt-pcr and flow cytometry were used to study the inhibition binding and entry effect of e antibody. direct immunostaining of e antibody conjugated with fitic and flow cytometry analysis showed reduced the mean fluorescence intensity in the samples pre-incubated with e antibody compared with samples without e . of positive serum samples, ( %) samples showed completely inhibition of infectivity as detected by rt-pcr. conclusion: in house produced e antibody, blocks binding and entry of hcv virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. isolation of these antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his tnf␤ and n tnf␤ were expressed in e. coli. high solubility of n tnf␤ was expected, however, both analogs his tnf␤ and n tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with . % nls, while his tnf␤ and n tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. enzymatic modification of sphingomyelin long zhang, lars hellgren, xuebing xu biocentrum-dtu. e-mail: lz@biocentrum.dtu.dk (l. zhang) due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient, high yield production methods are of great interest. in the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin (sm) have been studied. sm is a ubiquitous membrane-lipid and rich in dairy products or by-products. in present study, we have optimized the production of ceramide from sm using phospholipase c from clostridium perfringens. water and enzyme amount had the biggest influence on sm hydrolysis in the system. botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. the toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. in this work we have reported isolation of the binding domain of type e neurotoxin by pcr and expressed in a proper expression vector. the output of this investigation can be used as a tool to study the mechanism of binding of holotoxins and also can be useful to study the antibody production against botulism syndrome. the synaptobrevin (vamp ) a protein which play a key role in the fusion and exocytosis of the vesicle of mammalian nerves terminals this protein is substrate different serotypes of botulinum neurotoxins. light chain of clostridium botulinum type b, d, f and g cleave end of neurons. due to intraction of clostridium botulinum neurotoxin with vamp , this protein can be used as one of the toolsin the detection of poisings cause by this bacteria in the clinical laboratory using the enzyme with proof reading activity the above gene amplified by pcr technique for the production of the recombinant protein, the prokaryotic expression vectors (pet system) was used. a recombinant protein developed on poly acrylamide gel analyzed by western blotting and eliza. most children with down syndrome (ds) are born to younger mothers (< years). recent reports linking down syndrome (ds) to maternal polymorphisms at the methylenetetrahydrofolate reductase (mthfr) gene locus have generated great interest among investigators in the field. in this study, forty mothers with their affected outcomes and control mothers were included. all mothers were subjected to complete medical and nutritional history with special emphasis on folate intake through food or oral supplementation. estimation of blood homocysteine level was done. also we examined the two polymorphisms in genes encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (mthfr), namely, c > t and a > c. folic acid intake from food and from vitamin supplements was significantly low (below the recommended daily allowance) in the group of mothers with ds children compared to control mothers (p < . ) using student t-test. blood homocysteine was normal in both control and ds mothers. the prevalence of the two polymorphisms, namely, c > t and a > c in mothers of ds children (case mothers) (n = ) was compared with controls (n = ). frequencies of mthfr genotypes (cc, ct, and tt) at position demonstrated no difference between the case and control groups. genotype frequencies of mthfr at position a (aa, ac, and cc) were different among the case and control mothers. we here report the first study on a possible relation between ds with mthfr a > c genotypes in egypt. our results showed that mthfr a > c polymorphism is a remarkable genetic entity among egyptian females with d.s. children. sufficient folate intake and supplementation is an important preventive strategy in overcoming the risk of nondisjunction. homologous recombination (hr) is the mechanism that permits the creation of genetically engineered strains through gene targeting. in order to further develop gene targeting techniques, notably for higher eukaryotes such as filamentous fungi, it is of crucial importance to fully understand the molecular mechanisms behind mitotic hr. in saccharomyces cerevisiae, a dna double strand break (dsb) is an essential intermediate in meiotic hr. however, as hr occurs at a low rate in mitotic cells, it has been difficult to determine the nature of the event(s) that triggers it. rad is a key protein involved in hr and is evolutionarily conserved from yeast to human. to shed light on the molecular events in hr, we have generated a large collection of defined rad mutant strains in the yeast s.cerevisiae. a screen of these mutants led to the identification of strains that fail to repair dna dsbs, yet are proficient for homologous recombination. this result strongly suggests that dsbs may not be the major cause of spontaneous mitotic hr and gives new perspectives in respect to novel potential gene targeting substrates. we have analyzed these separation of function mutants in a variety of new assays to obtain a more detailed understanding of their controversial phenotype. our latest results will be presented. the outcome of interferone plus ribavirine treatment of hepatitis c virus (hcv) genotype is unfortunately poor. development of alternative therapy for this genotype is of a paramount importance. inhibition of hcv gene expression in vitro by the use of antisense phosphorothioate oligodeoxynucleotides (s-odn) against internal ribosomal entry site (ires) elements were associated with favorable results. to assess s-odn activity, previous studies utilized viral subgenomic or full cdna fragments linked to reporter genes and transfected into adhered cells or in a cell free system. in the present study we utilized hepg cells infected with native hcv rna of genotype . the culture system presented herein was shown to support hcv replication on the following bases ( ) consistent detection of both plus and minus rna strands for weeks in cells and in fresh culture supernatent, ( ) ability of supernatent to infect naive hepg cells ( ) consistent expression of core and e envelope proteins in infected cells throughout the week culture. s-odns against aug translation start site (s-odn- , nt - ) of the viral polyprotein precursor and stem loop iiid within the ires region (s-odn , nt - ) were added to infected cells. intracellular viral replication was monitored by nested rt-pcr of plus and minus strands. the results of these experiments demonstrated that intracellular replication of hcv genotype was completely arrested after h in culture using either s-odn molecule (with more efficacy of s-odn than s-odn ) at concentrations as low as m. the inhibitory effect of s-odn appeared to be specific to hcv replication since equal levels of human glyceralehyde -phosphate dehydrogenase (gapdh) gene expression were noted pre and post supplementation of s-odns. in conclusion, the present study provides evidence antisense phosphorothioate oligonucleotides have potent inhibitory effect on genomic replication of hcv genotype , the most common type in egypt. a sensitive and specific pcr-elisa was developed to detect shigella dysentery in food. the assay was based on the incorporation of degoxigenin-labeled dutp and a biotin-labeled primer specific for shiga toxin genes during pcr amplification. the labeled pcr product were bound to streptoavidin-coated wells of a microtiter plat and detected by an elisa. the elisa detecting system was able to increase the sensitivity of the pcr assay by up to -fold, compared with a conventional gel electrophoresis. the detection limit of the pcr-elisa was . - cfu dependent upon shigella dysentery serotypes and genotypes of shigatoxin. the entire procedure took about h. isolation, cloning, expression and purification of snap- as a substrate of botulinum neurotoxin type a and e m.l. mossavi , f. ebrahimi , j. amani * , h. basiry , z. ahmadi : department of biology, faculty of science, imam hussein university, tehran, iran; department of biology, faculty of medicine, bageatallah university, tehran, iran. e-mail: kpjamani@ihu.ac.ir (j. amani) clostridial neurotoxin inhibit neurotransmitter relase by selective and specific intracellular proteolysis of synaptosomal associated protein of kda (snap- ); synaptobrevin/vamp- and syntaxin. snap- is one of the components that form docking complex in synaptic ends. this protein is substrate for botulinum neurotoxins type a and e. each of these toxin serotypes specifically cleave snap- in particular position and there by block docking and synaptic vesicle membrane fusion and finally prevent neurotransmitter exocytosis and transition of neurotic signals. in order to use the protein as a substrate for detection of different type of clostridium neurotoxin in vitro test, the protein was produced by recombinant technique. the dna encoding snap- was isolated from rat brain by pcr using the two primer the amplified fragment colonel into expression vector pet a.the expression protein was purified by affinity chromatography. confirm by different method the his tag fusion protein was digested with entrokinase. the present work deals with the preparation of pure alpha- antitrypsin (aat) protein from healthy subjects, which can be used in preparing its corresponding monospecific antibody in albino rabbits. this antibody was found very useful in the immuno-diagnosis of pulmonary emphysema. this study has been also concerned with the biochemical changes associated with aat deficiency in pulmonary diseases. to fulfill this work, a group of healthy blood donors was selected for separation of pure aat antigen from blood. the pure aat was used for the preparation of anti-aat, the purity and potency of antibody was checked by titration methods. the biochemical changes were studied in thirty subjects clinically divided into three groups including, control, heavy cigarette smokers with pulmonary emphysema, and non-smoking subjects with pulmonary emphysema. the activity of elastase and hydroxyproline (hp) level as a marker of elastin and collagen breakdown were assayed in bronchoalveolar lavage (bal) fluid. the activity of aat and to inhibit proteases as represented by tryptic inhibitory capacity (tic) was evaluated. serum ceruloplasmin, transferrin and iga level as well as thiobarbituric acid reactive substances (tbars) were estimated in all groups. our data revealed that aat and its tic showed very highly significant decreased levels in all patients with emphysema as compared to control, while the elastase activity and hp level in bal fluid were significantly increased in these patients. serum tbars was significantly increased in such patients associated with increasing level of both ceruloplasmin and transferrin. while, serum iga was significantly increased. furthermore, the biochemical changes were markedly changed in smokers with emphysema than non-smoking subjects. in conclusion, the preparation of anti-aat on the local level is very important where less expensive and less time consuming and can be useful in immuno-diagnosis and prognosis of pulmonary diseases. a new method combined with boosting and projective adaptive resonance theory for analysis of gene expression data from cancer patients hiro takahashi, yasuyuki murase, hiroyuki honda department of biotechnology, school of engineering, nagoya university, nagoya - , japan. e-mail: h d@mbox.nagoyau.ac.jp (h. takahashi) an optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. for this purpose, it is important to develop that a sophisticated algorithm that can manage large amount of data, such as gene expression data from dna microarray, for optimal and individualized diagnosis. in our previous study, we developed the projective adaptive resonance theory (part) as a gene filtering method and boosted fuzzy classifier with sweep operator (bfcs) as a modeling method. in the present study, we applied the combination of part and bfcs (part-bfcs method) to microarray data of brain tumor (central nervous system tumor) obtained from the website. the method enabled the selection of important genes related to the prognosis of the tumor, i.e., sensitivity for combined therapy with surgery, radiotherapy, and chemotherapy mainly with vincristine, cisplatin, and cytoxan. the constructed model showed about % higher accuracy than that of the conventional method. genomic signal analysis of hiv variability based on rt gene p.d. cristea, rodica tuduce d. otelea biomedical engineering center, university "politehnica" of bucharest, romania. e-mail: pcristea@dsp.pub.ro (p.d. cristea) dna sequences genotyped from clade f hiv- isolates from romanian patients in the laboratory of the national institute of infectious diseases "matei bals", bucharest, romania, have been studied. the symbolic sequences have been converted into digital genomic signals by using a complex quadrantal representation of the nucleotides described earlier. the cumulated phase and unwrapped phase of a complex genomic signal reflect the statistical distribution of bases and base-pairs, respectively. independent component analysis of the genomic signals has been used to characterize the variability of the f subtype hiv strains isolated in romania. the sequenced segment is of (about) base pairs, approximately aligning with the standard sequence of hiv- (accession nc in genbank) over the interval - bp. this segment, which is currently used for the standard identification and assessment of hiv- strains, comprises the protease (pr) gene and two thirds of the reverse transcriptase (rt) gene. only results referring to the analysis of the rt gene region are presented here and used for extracting features of virion isolates and for establishing phylogenetic trees of the studied strains. the analyzed rt encoding segment has the length bp and is located in the second interval ( - bp) of the analyzed dna segment, respectively along the - bp region of nc . taking into account the mutations identified in these sequences, the samples were classified in three groups from the point of view of their resistance to current antiretroviral compounds: sensitive, resistant and multiresistant. the paper presents results for the isolates in which mutations leading to multiple drug resistance have been identified. over expression of secb protein in e. coli enhances the periplasmic expression of human growth hormone m. ghafari , , a. zomorodipour : islamic azad university of jahrom, tehran, iran; national institute for genet eng and biotechnol., tehran, iran. email: maryamghafari @yahoo.com (m. ghafari) among several proteins involved in the secretion pathway of proteins in e. coli, secb plays a key-important role in solubilization of preproteins before processing. in order to increase processing of a human growth hormone precursor (pelb::hgh) which appeared to have problem in processing efficiency, as a possible solution a regulated co-expression of a secb gene was considered. in this regard, we designed an arabinose-regulated secb expressing plasmid compatible with an iptg/lactose-regulated pelb::hgh expressing plasmid. for the construction of the secb expressing plasmid the origin of replication and antibiotic resistant gene (amp) of a pbad vector was replaced by a p a-ori and a kanamycin resistant gene, respectively. the expression and processing of pelb::hgh preprotein in the two-plasmid containing bacteria in a secb over-expression state was compared to that of the pelb::hgh expression in normal bacteria. although a decline in total expression level of hgh during the overexpression of secb was observable, probably due to presence of two different expressing plasmids, but both the processing efficiency of pelb::hgh and the transport of mature protein into the periplasmic space was enhanced during prolonged arabinose induction. current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts. natural allergen extracts contain a mixture of allergenic and non-allergenic components that are difficult to standardize. recombinant allergens can improve diagnosis and de-sensitization against single component. major cat allergen is a heterodimer composed of disulfide linked . and . kda polypeptide chains. both chains of feldi protein were obtained in e.coli system. purification of recombinant proteins was performed in denaturing conditions using immobilized metal affinity chromatography specific for proteins with histidine tag. from ml culture approximately mg protein for feldi chain and mg of feldi chain were obtained. after purification histidine tag was removed by hydrolysis with thrombin. immunological activity of feldi against serum of patients allergic to cat was narrowed to subgroup of patients allergic to feldi protein by surface plasmon resonance. immunological activity of each chain and renatured heterodimer was also tested using immunoprecipitation techniques against serum of population group. subdoligranulum variabile -a novel member of the human gut micro flora with a high prevalence kim holmstrøm, trine møller bioneer a/s, hørsholm, dk- , denmark in we isolated and cultured for the first time a bacterium from a human fecal sample representing a hitherto unknown member of the clostridium leptum rrna supra generic cluster. the c. leptum rrna supra generic cluster represents one of the major phylogenetic lineages within the human gut microbiota, and is characterized by having only a small proportion of its members actually identified by cultivation compared to the estimated numbers of bacteria contained in this group from culture-independent gut flora analyses. s. variabile is an obligate anaerobe gram negative bacterium with a characteristic pleiomorphic coccoid-droplet-like cellular morphology. its closest previously cultivated relative based on a s rdna phylogenetic analysis is faecalibacterium prausnitzii, a rod-shaped and therefore easily distinguishable gram negative bacterium present in high numbers in the human fecal micro flora. based on s rdna sequence we designed a s. variabile specific oligonucleotide probe for use in fish analysis to estimate the prevalence of this "new" bacterium in fecal samples collected from healthy human beings. interestingly, we observed a high proportion of s. variabile present in all tested samples, and in some instances we observed a higher prevalence than the more well-known group of bifidobacteria equally estimated by fish analysis. documentation of these results will be presented. several species of sea cucumbers, long an incumbent of traditional medicines were selected as the source of animal based antibiotic compounds. swabs of the inner surface and coelomic fluid (inner fluid) samples from sea cucumber (holothuria atra jaeger) were taken. thirty strains of bacteria were isolated. these strains were grown in different antibiotic production media. nine human pathogenic bacterial species were used as test agents and they are, k. pneumoniae, mrsa, m. luteus, s. thyphimurium, s. epidermitis, s. saprophyticus, b. subtillis, p. aeruginosa and s. pyogenes; only four bacterial strains showed mild antibiotic activity against s. pyogenes and s. thyphimurium. similar testing on two other species, h. scabra and s. variegatus will be carried out. different media, especially antibiotic production enrichment media will also be used. characterization will be done upon obtaining an antibiotic compound, which shows moderate to high activity against at least one of the nine human pathogens used. for plant based medicines, three rhizomes, "cekor," "jerangau" and "bonglai" were analyzed. solvent extraction using ethanol, methanol and acetone was carried out, at a concentration of - mg per ml solvent. the filtrates were used for the antibiotic testing stage. all the three plant species showed moderate antibiotic activity against m. luteus, s. epidermitis, s. pyogenes and s. saprophyticus. interestingly, the antibiotic activity increased when combinations of the herbal extracts were used. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most % of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited % to % of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax and the musclespecific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. metabolic engineering design of an extracellular hgh synthesis system birgül Şentürk , pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre lab, department of chemical engng, ankara university, ankara, turkey; iblab, department of chemical engineering, metu, ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.Özdamar) metabolic engineering design of an extracellular human growth hormone (hgh) synthesis system is based on cloning the dna encoding human therapeutic protein together with the signal dna sequence of an extracellular enzyme gene into a host-vector system. in this context, extracellular protease (subc) signal dna sequence, i.e. pre-signal dna sequence, was fused into the frame in front of hgh mature dna sequence, by the use of four primers designed using pcr-based gene splicing by overlap extension method. b. licheniformis chromosomal dna and plasmid carrying hgh cdna were used as templates in pcrs, respectively, for the amplification of the subc signal dna sequence and hgh mature peptide sequence. for the fusion of two target genes, i.e. mature peptide sequence of hgh and, signal dna sequences were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their ends that are complementary to portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at end. extension of this overlap by dna polymerase has yielded the recombinant hybrid-gene; and hybrid-gene serve as template for the continuation of reactions for the increase of the concentration in the microreactors. the hybrid gene fragment was first cloned into puc , and then sub-cloned to pmk e.coli-bacillus shuttle plasmid. thus, a new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis (spo − ). the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the proposed metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, hgh, amino acids and organic acids concentrations as the constraints. on the basis of the intracellular bioreaction rates and the interactions with the bioreactor operation parameters, an in-depth insight will be provided for further metabolic engineering design for the extracellular hgh production in r-b.subtilis. medicines based on polypeptides consisting of and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin ␣ production impac system (intein mediated purification with affinity chitin-binding tag) has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin ␣ . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl and we have found that, in case of intein-thymosin ␣ , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of . - mm zinc chloride in buffers on all stages of thymosin ␣ isolation. the structure of recombinant thymosin ␣ of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and mg/l , -dichlorophenoxyacetic acid ( , -d) or mg/l naphtalenacetic acid (naa). the developed calli and regenerated plants were maintained on , -d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + , d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his tnf␤ and n tnf␤ were expressed in e. coli. high solubility of n tnf␤ was expected, however, both analogs his tnf␤ and n tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with . % nls, while his tnf␤ and n tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. high level expression of recombinant growth hormones in e.coli faces common problems such as protein aggregation and inclusion body formation. discussions are raised whether it is more beneficial to obtain soluble protein but to loose expression rates. here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. experiments were performed on bovine, chicken and mink growth hormones. expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. another approach to the problem was performed through genetic manipulation. we changed strong t promoter to e. coli promoter consensus sequence thus reducing expression rate. recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levelsseveral ( - ) percent of total intracellular protein. we developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation. to optimize the recombinant protein production in small-scale shake-flask system and high cell density fermentation, new tools have been developed at our laboratory which helps to get knowledge about the physiological state of the cell culture. these tools include (i) a quantitative monitoring system for cellular mrnas based on a sandwich hybridization technique, and (ii) a wireless online monitoring tool (senbit), applicable for standard sensors such as ph, po and temperature for the continuous data collection from shake flasks. the senbit system is a new tool supplying valuable information for the optimisation of the expression of recombinant genes in shake flasks and allowing conclusions towards the reproducibility of shake flask cultures. the presentation will focus on use of the sensitive sandwich hybridization technology for the quantitative analysis of process relevant marker genes in different kind of microbial cell cultures with a focus on the production of recombinant proteins. samples from shake flask cultures and high cell density fed-batch fermentations of the yeast pichia patoris, have been analysed. additionally the mrna analysis was combined with the application of the senbit wireless system to study the production of a recombinant protein in shake-flask cultures of p. pastoris. aside from p. pastoris, mrna sandwich hybridization also was used to monitor product expression in fed-batch fermentation of e. coli for the production of a protein with two subunits by sequential induction. quantitative rna analysis as a tool for optimization of tetrameric collagen prolyl -hydroxylase production in e. coli a. neubauer , m. bollok , j. myllyharju , p. collagen prolyl -hydroxylase (p h) involved in the biosynthesis of collagens is an ␣ ␤ tetramer. recombinant expression of p h in e. coli was described recently . the construct for cytoplasmic expression contains the genes of both subunits in one plasmid under control of different promoters. the ␣ subunit forms inactive aggregates, when expressed separately. in mammalian cells the ␤ subunit is available in large excess and keeps the ␣ subunit in a soluble active form. to mimic this in the bacterial system, we induced both genes sequentially. after induction of the ␤ subunit with iptg, expression of the ␣ subunit was initiated with anhydrotetracycline. here we use the analysis of the product mrnas with a bead based sandwich hybridisation assay (sha) (rautio et al., ) for optimization of the fermentation procedure. a high p h activity was obtained if a high mrna level of the ␣ subunit could be maintained over a longer time. the obtained results illustrate the importance of the second induction for a high level expression of the p h tetramer. the cells need to be in a "healthy state" with low metabolic load to react efficiently to the second induction. the data illustrate the optimization of a fermentation process by monitoring mrna levels which is of general interest for optimization of products which are difficult to detect. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most % of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited - % of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax and the muscle-specific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. collagen and its derived product gelatin are attractive mammalian proteins to be used as model for the production of complex heterologous proteins in plants. the availability of a recombinant product will provide a safer, more homogeneous product than the current animal-derived material. the aim of the project is to investigate the feasibility of a production system for the accumulation of recombinant collagen for conversion to gelatin using barley. the -end of the cocksfoot mottle virus (cfmv; genus sobemovirus) genomic rna sequence, called cfmv -element, has been shown to enhance recombinant protein synthesis in barley (wo / , mäkinen et al., ) . the -element will be used to study whether accumulation levels of complex mammalian proteins can be further increased, using collagen as a model that will serve as basis for exploring the expression of other complex proteins. this system can be study the production of barley-derived recombinant collagen for conversion to gelatin. classical swine fever virus (csfv) is an animal pestivirus which can be used as a surrogate model to elucidate the role of envelope glycoproteins of closely related human hepatitis c virus (hcv). the necessity to use the surrogate models for hcv is due to the fact that this virus cannot be grown in vitro cultures. csfv genome codes for three major antigenic glycoproteins which are located in the same cluster of genes; they are designated as e and e (e rns ) and e . glycoproteins form heterodimeric and homodimeric complexes on the external part of viral particles. it is generally accepted that envelope glycoproteins play a major role in the initial stages of viral infection both for csfv and hcv. formation of complexes is needed to effectively infect host cells. we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (western blotting). immunoblotting is a very useful technique in these studies because the complexes are formed via cysteine-cysteine disulphide bonds and they are retained during sds-page under non-reducing conditions. by modifying the glycoprotein genes and by arresting n-glycosylation of e and e we have investigated which factors influence the formation of complexes. it has been found that some glycosylation inhibitors which act at the early stages of glycan chain processing influence, not only glycosylation, but also the stability of e protein, effectively inhibiting the formation of glycoprotein complexes and the yield of the virus. these inhibitors are potential agents for arresting the multiplication and spread of csfv, and its relative -human hcv. recombinant proteins have been produced in a variety of heterologous protein expression systems. eukaryotic unicellular algae have distinct advantages, e.g. it can synthesize complex protein that requires post-translational modification. furthermore, microalgae can be grown in confined environment and thus prevents leakage of genes to the environment. our group has developed a platform technology for the production of recombinant proteins in red microalga porphyridium sp. we have constructed algal transformation plasmid vectors containing a camv s promoter and polya signal site. a streptoalloteichus hindustanus bleomycin-resistant gene was used as the selective marker. we have expressed ovalbumin and hepatitis-b surface antigen (hbsag) as model proteins. transformation was carried out by agitating algal cells and vector dna with glass bead. transgenic lines were selected by growing algal cells on agar plate containing g/ml zeocin. positive transgenic lines were selected by screening the colonies by pcr and confirmed by dna sequencing. expression of ovalbumin and hbsag protein was examined by western blot analysis. ovalbumin was found to be expressed inside the algal cells while small hbsag was secreted into the medium due to presence of signal peptide. these findings indicate that red microalgae are capable of producing heterologous proteins. life-material exhibition as ethical interpretation chang shih-lung biotechnology industry study center, taiwan institute of economic research, taipei , taiwan. email: schang@tier.org.tw in this thesis we aim to explore the medical-related life-material exhibitions within ntu hospital-the humanity building, and taipei mackay hospital-the historical showroom as abecedarian clues to understand the burgeoning phenomenon-medical museums in taiwan. following these clues, we treat the whole context of medical museums, which transform values through situational construction, as the background to interpret the ethical implications of group val-ues that are transformed in the medical profession. in this thesis, we see medical museums, as the social prescription transforming medical profession, format the ritual context of situation ethics with the cultural construction of life-material. multi-disciplinary interactions and visiting itinerary can be transferred to the exploring horizon of research approach through description and interpretation. among them, we observe that humanistic elements have become essential equipment (or mat'eriel) of medical profession. though humanistic equipment (or mat'eriel) has its bottleneck in the museum situation, it can also unblock new possibilities for museum exhibitions, ethical practice or life ethics. as part of a wide research program aimed at developing new antitumoral agents, we present herein a series of stereoisomeric derivatives of fused tetrahydrofuranes (fthf) substituted with diverse protecting groups either at the primary or secondary hydroxyl groups. unprotected derivatives were also synthesised to investigate the influence of substituents on the in vitro activity of fthf. data on the synthesis, chemosensitivity and apoptosis induction by this series of fthf will be correlated to substitution pattern, stereochemistry and protecting groups, as an aid to the rational design of novel antitumour drugs. establishment of in vitro test systems for pulmonary edema resorption by peptide drug candidates dominik geiger, aswin mangerich, rudolf lucas, klaus p. schäfer, inge mühldorfer department of biotechnology, altana pharma ag, konstanz, germany; imc university of applied sciences, krems, austria tnf-␣ was found to up-regulate the rate of lung liquid clearance (llc) in several animal models by the activity of its lectin-like tip domain. this activity can be mimicked by a circular peptide designated tip. tip was shown to induce llc and consequently pulmonary edema resorption in different animal models. in order to study the mechanism of action of tip, we established two in vitro test systems for edema resorption with the human lung epithelial cell line calu- : ( ) the ability of calu- cells for spontaneous dome formation within confluent monolayers was utilized for quantitative examination of tip's activity on active transepithelial fluid transport ("dome assay"). ( ) the effects of tip on bioelectrical properties of polarized cell monolayers were studied by using the transepithelial electrical resistance (teer) technology ("teer assay"). dome assay experiments confirmed that dome formation is a sodium dependent process and that tip is able to increase this process. teer assay experiments proofed that tip acts in a polarized and dose dependent manner. in conclusion, there is strong evidence that the dome and the teer assays are suitable systems for in vitro activity testing of tip and other anti-edema peptide drug candidates and are useful for studies on their mechanism of action. increasing safety concerns in gene therapy result in more stringent regulatory requirements. those cover the complete process chain of cell banking, fermentation, and purification. a wide range of applications for pdna requires gram to kilogram amounts for clinical trials and market supply. economic, productive and robust processes are a prerequisite for low cost of goods (cogs). therefore manufacturer of biopharmaceuticals need to address these considerations by developing new production processes meeting the new standards. boehringer ingelheim austria developed a novel pdna production process suitable for large-scale cgmp production. the process is based on e. coli fermentation. the process contains no components from animal origin. the optimized fermentation process yields up to g pdna/l fermentation volume. for isolation of pdna from e. coli alkaline lysis in glass bottles or stirred tanks is commonly used. cell wall structure is destroyed by a combination of alkaline ph and detergents. in our process alkaline lysis is operated in a closed, continuous system directly connected to clarification without using enzymes. we developed a scalable process for pdna purification based on different chromatographic principles. the capture step is carried out by hydrophobic interaction chromatography followed by anion exchange chromatography as intermediate step. final polishing is carried out by conventional size exclusion chromatography in a group separation mode providing also buffer exchange and desalting for the final formulation. the process results in a pdna drug substance of highest quality containing a low level of impurities (genomic dna, rna, proteins endotoxines) suitable for therapeutic applications. depending on the conditions during fermentation and the used host homogeneities of greater than % or even % are possible, while a high over all yield can be achieved (∼ %). during the development monolithic chromatography supports (cim ® ) were compared with conventional resins and evaluated as potential alternatives. the complete process is monitored by a set of analysis covering cell banking to final purification. new sensitive methods based on hpce, hpiex and fluorometric measurement were developed. whereas many natural amino acids are currently produced by very cost efficient biological processes, the manufacture of methionine is still performed by traditional chemical synthesis. this was mainly due to the poor performances of the currently available producing strains that inhibited the development and commercialization of a biological process to l-methionine. metabolic explorer has recently reinvestigated and developed an efficient biological process that employs an engineered microorganism and utilizes a renewable starting material (corn sugar) as its feedstock and converts glucose into l-methionine. we will describe (a) the general scheme for the engineering of the host organisms and (b) the general approach to maximize carbon and reducing equivalent throughput to l-methionine. to highlight this effort, we will present the global approach developed to improve the process combining metabolic flux analysis, traditional protein biochemistry, molecular biology and fermentation optimisation. saccharomyces cerevisiae is an established 'work horse' of the fermentation industry and modern biotechnology has led to a spectacular expansion of the range of products that can be produced by this yeast. however, for the large-scale sustainable production of chemicals, it is equally important that the range of carbohydrate feedstocks be expanded. especially relevant in this respect is the ability to consume the pentose sugars d-xylose and l-arabinose, which make up a substantial part of plant carbohydrates. wild-type s. cerevisiae strains cannot metabolise d-xylose, but are capable of slowly metabolising its keto-isomer, d-xylulose. therefore, efficient conversion of d-xylose into d-xylulose has long been a key issue in yeast metabolic engineering. non-saccharomyces yeasts that is capable of growing on d-xylose use two enzymes, xylose reductase and xylitol dehydrogenase for this purpose. while both enzymes have been successfully expressed in s. cerevisiae, this is not always compatible with efficient product formation. for example, in the case of ethanol production, the different cofactor specificities of these two oxidoreductases cause massive byproduct formation. theoretically, introduction of a xylose isomerase, which catalyses the interconversion of xylose and xylulose, might circumvent these problems. however, it is notoriously difficult to express bacterial and archaeal xylose isomerases in s. cerevisiae and, until recently, activities of heterologous xylose isomerases expressed in s. cerevisiae were vanishingly low, at least under physiological conditions. a breakthrough was reached when, in , a xylose isomerase gene from the anaerobic fungus piromyces was expressed in s. cerevisiae. while this led to high activities of xylose isomerase, these were not enough to enable fast growth or product (ethanol) formation. in this presentation, we will discuss how a combination of metabolic and evolutionary engineering led to fast and efficient xylose utilization by engineered saccharomyces cerevisiae strains under aerobic as well as anaerobic conditions. furthermore, we will illustrate how evolutionary approaches can be applied to facilitate the utilization of mixed substrates. hyaluronic acid (ha) is a natural and linear polymer composed of ␤- , -n-acetyl glucosamine and ␤- , -glucuronic acid repeating disaccharide units with a molecular weight (mw) up to mda. it is a major constituent of the extracellular matrices and the synovial fluid. in the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of ha and its unique physico-chemical properties. however, for some specific applications, the relatively high mw of ha is a limiting factor and the availability of low mw fractions would be highly desired. for food applications, low mw ha has been shown to penetrate the gastrointestinal barrier, thereby increasing the ha bioavailability. moreover, low mw ha fractions are able to re-establish the ha content in the skin and can thus be used in cosmetics as anti-aging and anti-wrinkle agents. finally, low mw ha has shown to prevent oxygen free radical damage in granu-lation tissue during wound healing. a range of methods has been described for the depolymerization of ha to low mw fractions. these techniques involve heat treatment, ultrasonication, uv/gamma irradiation, chemical and enzymatic degradation. we present results on a degradation process of ha originating from bacillus subtilis fermentation into well-defined low mw fractions. the process developed in lab scale is safe, well-controlled and produce low mw ha fractions with narrow polydispersity. moreover, the process is readily up-scalable. the low mw ha fractions are being evaluated in various cosmetic applications. metabolic pathway manipulation for improving the properties and productivity of microorganisms is becoming an established concept. metabolic engineering can be defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant dna technology. a detailed analysis of the physiological means of the different pathways is needed to be able to introduce modifications aimed to the production of not only important metabolites, but also to understand the fundamentals of cell biology. aimed to produce single compounds, metabolic engineering necessarily includes the modification of the cellular pathway(s) as well as the redirection of the energy toward the production itself. the existing metabolic engineering applications are the culmination of more than two decades of global experience developing processes for the production of fine chemicals, vitamins, nutraceuticals and animal nutritional aids such as amino acids. based on the relative low complexity, the first biotechnological applications have been developed from microorganisms. our laboratory has been engaged in this field since different years. yeasts like saccharomyces cerevisiae, kluyveromyces lactis and zygosaccharomyces bailii have been developed for the production of fine chemicals like lactic and ascorbic acids from d-glucose. in this contribution, we will present the last data obtained. since years amino acid production is in the focus of industrial microbiology. l-glutamate and l-lysine are produced with corynebacterium glutamicum, while escherichia coli is used for l-threonine production. the worldwide market of threonine drastically increases: in the amount of threonine produced worldwide was , t and raised to , t in and , t in . concerning predictions of experts the demand of threonine will rise with a two-digit rate of economic growth within the next few years. meanwhile the prices declined. these conditions enforce very efficient production processes. beside the strain development and an optimised downstream procedure the fermentation process is an important target for productivity improvement. strain development is dependent on detailed knowledge of the production strains. with innovative methods we are able to get a close look inside the cells under different culture conditions. these methods have been called 'omics' in recent literature. knowledge about genome, transcriptome, proteome, phosphoproteome, metabolome and fluxome leads to new ideas for strain improvements. data generated by these methods must be based on clearly defined culture conditions. therefore, highly parallel fermentations have to be performed to generate biological parallel samples. cutting-edge technical equipment is the basic requirement for experiments like this. other requirements are optimised sampling for different analysis, technical parallels of analytical steps and a detailed statistical analysis of data. these procedures guarantee distinction between real data and data noise. integration of all these data to a holistic model of the cell is the challenge for the future. by combination of a new strain, process and downstream improvements the plant productivity was increased drastically. we ended up in an optimized, fast and high yield process to scope challenges worldwide market comes up with. rieping, m., hermann, t. ( ); fermentation process for the preparation of l-threonine; wo/ . ) are potentially quite useful biocatalysts, as they allow for the regioselective and stereoselective hydroxylation of activated as well as non-activated carbon atoms. in addition, the large number of members of the p superfamily exhibits a wide diversity of specificities from which a useful biocatalyst may be selected. from a technical point of view, however, they have significant drawbacks. thus, they usually cannot be produced in large quantities nor recovered or stored without a severe loss in activity. their catalytic activity is mostly quite low, and their operational stability leaves much to be desired. most p enzymes require a complex protein/phospholipid machinery for activity, and the final electron donor in the reaction cascade, usually nadph, does require extensive recycling to arrive at a commercially satisfactory process. recently, the use of bacterial cytochromes as hydroxylation biocatalysts has received considerable attention. some of them are natural fusion proteins, which contain the heme and the reductase domains on a single polypeptide chain. they are catalytically much more active compared to, e.g. cytochromes occurring in human tissue. we thus have set out to further improve the technical applicability of these enzymes, and have centered our activities around several bacterial cytochromes. it proved very useful to apply rational mutagenesis and directed evolution to these enzymes, leading to a surprising compatibility of mutant enzymes with a wide variety of substrates. mechanisms for the limited stability of the enzymes were explored, leading to hybrid enzymes with enhanced stability, and the cofactor problem was alleviated using auxiliary enzymes or mediator-based technologies. as a result, a bioreactor based on microbial cytochromes was built and operated for several days. baeyer-villiger monooxygenases represent useful biocatalytic tools as they can catalyze reactions, which are difficult to achieve using chemical means. however, so far only a limited number of these monooxygenases were available in recombinant form kamerbeek et al. ( ) . using a recently described protein sequence motif fraaije et al. ( ) and the available genome sequence information, we were able to identify and overexpress a number of novel bacterial bvmos. one of the overexpressed bvmos was found to be relatively stable as it originates from thermobifida fusca, which grows at ∼ • c. the enzyme was shown to be active on a broad range of substrates, preferring aromatic ketones fraaije et al. ( ) . the best substrate discovered so far is phenylacetone, hence its name: phenylacetone monooxygenase. we have solved the crystal structure of phenylacetone monooxygenase, which represents the first structure of a bvmo malito et al. ( ) . the crystal structure provides insight into the complex mechanism of catalysis mediated by fadcontaining bvmos. by site-directed mutagenesis we have probed the role of several active-site residues. a crucial role is played by an arginine residue. as phenylacetone monooxygenase shares significant sequence identity (> %) with all known nadph-dependent bvmos, many of the observed structural features seem to be conserved within this class of atypical monooxygenases. by homology modeling using the phenylacetone monooxygenase structure, catalytic properties of other baeyer-villiger monooxygenases can be explained or predicted. screening for fungal baeyer-villiger monooxygenases l. butinar , j. friedrich , v. alphand : laboratory of biotechnology, national institute of chemistry, ljubljana si- , slovenia; groupe biocatalyse et chimie fine cnrs fre , université de la méditerranée, marseille, france the asymmetric form of the baeyer-villiger (bv) oxidation (transformation of ketones into lactones) is an important challenge for organic chemistry since the obtained lactones are valuable building blocks for synthesis of countless biologically active products. to date, enzymatic or microbial bv oxidations appears as more successful than their chemical counter-parts. (ten brink et al.) whereas most active bv monooxygenases are produced by bacteria (among which the well-studied enzyme of acinetobacter calcoaceticus), only a few fungal strains expressing bvmo were described (alphand et al., carnell and willetts) . in order to increase the number of available biocatalysts which perform such an asymmetric biotransformations, a screening of fungi belonging to major groups of zygo-, ascoand basidiomycetes was conducted using bicycloheptenone as testsubstrate. surprisingly, a large number of the tested fungi were able to transform the substrate into one to four different lactone isomers. the yields, the enantio-and regio-selectivity of the reaction depended on the fungal strain. alphand, v., furstoss, r., . j. mol. catal. b , - . carnell, a., willetts, a., . biotechnol. lett. , - . ten brink, g.j., et al., . pyranose oxidase (p ox) is a periplasmic enzyme that widely occurs in basidiomycetes. it catalyses the c- oxidation of several aldopyranoses to the respective -keto derivatives, transferring electrons to molecular oxygen to yield h o . p ox is of interest for carbohydrate conversions, as its reaction products ( -keto sugars) can be attractive intermediates in the production of food ingredients. we cloned the gene encoding p ox, and subsequently amplified a cdna clone by rt-pcr. the cdna was inserted into a bacterial expression vector and successfully expressed in e. coli. properties of the heterologous protein were compared to those of the native enzyme showing that they are essentially identical. both the native as well as the recombinant enzyme were used in biotransformations of sugars. recently, the d-structure of this tetrameric enzyme was elucidated. based on structural information, several enzyme variants containing point mutations were constructed and further characterised. two of these variants (e k and e r) displayed improvements in stability and certain kinetic properties thus making them attractive for biocatalytic applications. lactones are important compounds for the fragrance and flavour industry. right now the production of lactones is dependant on the import of crude materials from tropical countries. in this project, we want to tackle the manufacture of lactones via a biocatalytic route using p monooxygenases. cytochrome p monooxygenases catalyse the oxyfunctionalization of non-activated c-atoms. cyp a from bacillus megaterium, cyp a and cyp a from bacillus subtilis are soluble fusion proteins comprising p monooxygenase and fad/fmn reductase domains in one polypeptide chain. all three enzymes are highly homologous fatty acid hydroxylases. especially, cyp a also known as p bm- is well characterized and shows high activity compared to other p monooxygenases. the aim of the work is to change selectivity but conserve the high activity that is typical for those enzymes. using methods of structure modelling, rational protein design and directed evolution new mutants of these enzymes with changed regioselectivity are obtained. products of conversion with monooxygenases are intermediates in the production of lactones. the interface of biology and materials science has led to new materials with unique structural and functional properties, and new process technologies with the ability to produce, from "bottoms up", a wide range of biomimetic structures. these materials and their designs have broad application as catalysts, sensors, and devices for use in synthesis, cell and tissue engineering, bioanalysis and screening, and nanoelectronics. we have focused on the generation of sugar-based nanostructures, complete with tailored selectivities and biocatalytic activities at the molecular and nanoscales. these include biocatalytically-generated carbohydrate derivatives that selfassemble with high precision to give novel architectures with functional and responsive properties. izumoring: a strategy for total production of rare sugars ken izumori rare sugar research center, kagawa university, kagawa - , japan. e-mail: izumori@ag.kagawa-u.ac.jp we found a new enzyme, d-tagatose -epimerase (dte), that epimerize all ketohexoses at c- position. this epimerase catalyze not only between d-tagatose and d-sorbose, but also d-fructose = dpsicose, l-sorbose = l-tagatose, and l-psicose = l-fructose. this new enzyme offered us a useful key tool to connect all ketohexoses using hexitols as intermediates. the figure shows that all eight ketohexoses can be connected with dte and polyol dehydrogenases (pdh) in a ring. using this ring, we can easily find the pathway to transfer d-fructose to d-tagatose via d-psicose using dte and pdh. various aldose isomerases transform ketohexoses to the corresponding aldohexoses. so, we can connect all aldohexoses with ketohexoses using the enzyme. finally, all hexoses, ketohexoses, hexitols and aldohexoses are connected using enzyme reactions in a ring structure (not shown). this kind of strategy is effective also on transformation of tetroses and pentoses. now, we can produce all monosaccharides; tetroses, pentoses and hexoses by enzyme reac-tions. the bioproduction strategy of all rare sugars (monosaccharides that are rare in nature) is illustrated using ring form structures named as izumoring. we have already succeeded to produce d-psicose in large scale and are now in the progress of mass production of various rare sugars from natural and cheap sugars using izumoring. bioprocess development for chiral intermediates christian wandrey institute of biotechnology, forschungszentrum jülich gmbh, jülich d- , germany chiral alcohols, diols, amino alcohols and chiral acids (e.g. hydroxy acids and amino acids) play an important role in pharma and agro synthesis. in the past such chiral intermediates were obtained by racemic resolution via chiral reductions using prochiral precursors. here the problem of cofactor regeneration arises. this problem could be solved by enzyme-coupled or substrate-coupled cofactor regeneration using formate or isopropanol as reducing agent. alternatively, whole cell bioreductions were developed where glucose is used as the reducing agent. in recent years "designer microorganisms" were developed in which oxidoreductases (e.g. alcohol dehydrogenases) were over-expressed in escherichia coli. in such cases, cofactor regeneration was achieved intracellularly with isopropanol as the reducing agent or by coexpression of a formate dehydrogenase, so that once again format could be used for reduction. another route to obtain chiral intermediates is a fermentative approach using classical pathways (like the aromatic amino acid pathway). here, the pathway is interrupted after the intracellular production of chorismate. new chiral intermediates can be obtained by over expression of additional genes, which catalyzed the production of chorismate derivatives leading to cyclohexadiene-transdiols and the corresponding amino cyclitols. the last example can be regarded as an example of industrial biotechnology where glucose is used as starting material (white biotechnology). here bioprocess development is carried out in an integrated approach, in which molecular biochemical engi-neering cares for the optimal intracellular metabolic network and the classical biochemical engineering cares for the optimal environment of the cell in a fermenter. examples will be given which reach (in cooperation with industrial partners) up to kilogram scale. biotransformations are usually involved in just one or very few separate reactions in organic syntheses. the development of a cell-free "system of biotransformations" (sbt), in which a set of enzymes acts in a coordinated fashion in a one-pot synthesis, lead to increased catalytic complexity, selectivity and yield, as well as facilitated operation at reduced costs. the example chosen to prove the usefulness of the sbt-approach is the production of dihydroxyacetone phosphate (dhap). dhap, a c -compound from glycolysis, is an important precursor for asymmetric c-c-bond formation. so far, the production of dhap is difficult and expensive. for the construction of the dhap-producing sbt, e. coli's glycolysis is isolated from the metabolism to an as large as possible extent by the construction of a multi-ko-mutant. a culture is grown in an appropriate medium, homogenized in the production buffer, and used as the catalytic system. high production yields can be achieved since the production pathway is almost completely isolated from the metabolic network. the employed dhap-producing sbt provides not only a path from glucose to the product, but also an integrated atp-regeneration and nad-recycling system. in first experiments with a tpi-ko-mutant, a dhap-production yield on glucose of % could be achieved, without optimizing the system. the system remained active for more than h. up to now, atp cannot be applied in catalytic concentrations, but has to be present in equimolar amounts to glucose. the production yield could be increased by % through the addition of phosphate ions as substrate to the reaction, enabling the system to utilize atp more efficiently. these experiments indicate that the sbt-approach is viable and a large potential remains to improve the dhap-producing sbt. for some years novozymes have manufactured a pectate lyase for scouring of textile as an ecological alternative to the traditional harsh chemical treatment, and recently, we at novozymes discovered additional applications for pectate lyases. however, to be commercially attractive more robust pectate lyses had to be developed. in this paper, we will demonstrate how we for two different pectate lyases have improved their stability significantly. as the two enzymes are quite similar in sequence and structure, it was a new discovery for us to find that different concepts of protein engineering had to be used in our attempt to stabilize each individual pectate lyase. the stability of one enzyme was improved by substitutions in the internal of the structure whereas the stability of the other pectate lyase was primarily improved by changing surface residues. starting with knowledge from structural analysis, we have applied rational based protein engineering resulting in few selected variants. also random based protein engineering combined with screening of hundreds of thousands variants was used. in conclusion: the project team showed that by synergistic use of the two approaches, we were able to move faster towards a solution and eventually we succeeded finding new stabilized pectate lyase variants, applicable for new business areas. the importance energy independence as a national goal equals or exceeds that of the moon landing in . the development of a new industry to produce fuel ethanol from woody biomass would increase national security, improve employment and the environment, and provide substantial relief from the debt of imported petroleum. costs associated with the rapid development of this new industry (∼$ . billion per year) could be paid by re-assigning cent per gallon from existing federal gasoline taxes, a small price to pay for future energy independence. the corn-to-ethanol industry continues to make a remarkable contribution to our liquid fuel needs through expansion. today, one row of every six rows of corn is converted into fuel ethanol in the u.s. however, this expansion will be limited to - % of total automotive fuel by the economics of corn costs and production. corn can do more! corn stover is the single most abundant agricultural residue in the us and can be used as a feedstock to produce - gallons of ethanol per dry ton. further expansion with other biomass feedstocks such as agricultural and municipal residues (lignocellulose, woody biomass) could produce over billion gallons of fuel ethanol annually according at a recent joint report by the usda and doe (april, ) . current technology has been demonstrated at pilot scale for the production of fermentable sugars from hemicellulose by dilute acid hydrolysis and for the hydrolysis cellulose using fungal cellulose enzymes. biocatalysts such as recombinant escherichia coli have been developed and demonstrated for the efficient conversion of all sugar constituents of biomass to ethanol. a national goal for the full-scale deployment of current technology to produce biomass-based fuel ethanol will allow the us to reduce imported petroleum by %. together with increased efficiencies of hybrid vehicles, energy independence could be achieved within - years. similar gains could be realized by many nations around the world to provide new manufacturing and employment, redistributing wealth and ensuring a cleaner, healthier environment. bioethanol production using thermophilic bacteria marie just mikkelsen, birgitte k. ahring emab, biocentrum-dtu, lyngby, denmark the industry of bioethanol production is facing the challenge of redirecting the process from fermentation of relatively easily convertible but expensive starchy materials, to complex but inexpensive lignocellulosic biomass. on lignocellulosic hydrolysates, gram-positive thermophilic bacteria have unique advantages over the conventional ethanol production strains. the primary advantages are their natural broad substrate specificities, and in some strains, a high tolerance to lignocellulosic hydrolysates. moreover, ethanol fermentation at high temperatures also has the advantages of high productivities and substrate conversions, low risk of contamination and facilitated product recovery. some thermophilic bacteria naturally produce primarily ethanol from most sugar monomers present in lignocellulosics, but modifications are still necessary to increase ethanol yields. the release of useable sugars from lignocellulose biomass for industrial fuel-ethanol fermentation is often facilitated by a weak acid hydrolysis step. as a consequence, inhibitors such as furfural and -hydroxymethylfurfural (hmf) are formed as degradation products of xylose and glucose, respectively. moreover, the fermentative end-product of ethanol is also inhibitory. these, and other inhibitors present an environment, which elicits the expression of stress-related genes in saccharomyces cerevisiae. recently, s. cerevisiae genes have been identified as important in furfural stress tolerance. when furfural is present, yeast with these genes disrupted grows poorly compared to wild-type yeast. a sub-class of these genes suggests that yeast grown under furfural-induced stress may rely upon similar pathways as cells grown under various other stresses, including oxidative, heat, and sorbate. to investigate this link further, we analyzed stress-induced phenotypes such as ros activity, dna damage, and membrane damage in wild-type and mutant yeast exposed to furfural or hmf stress. moreover, we investigated whether overexpression of this sub-class of genes would provide protection from furfural-induced stress and oxidative damage. micro-organism to be used in fermentation of lignocellulose hydrolyzates should preferably have three characters: (a) high ethanol tolerance, (b) resistance to inhibitors found in the hydrolyzate, and (c) a broad substrate utilization range, since the hydrolyzate contains several sugars. in addition to the possibility of controlling the level of potential inhibitors, fed-batch fermentations also permit the parallel uptake of several different monomeric sugars. two strains of saccharomyces cerevisiae, cbs (a commonly used laboratory strain) and tmb (a strain isolated from a spent sulfite liquor fermentation plant), were characterized in batch and fed-batch fermentation of a dilute-acid hydrolyzate from spruce. the strains had different abilities to ferment spruce hydrolyzate. the study suggests that the furan reduction capacity of a yeast strain is a key factor for its performance in fermentation of lignocellulosic hydrolyzate. polyketides constitute a structurally highly diverse group of natural products that possess broad ranges of pharmacological properties and represent a major source for novel cancer therapeutics. however, these compounds may be sub-optimal in regard of activity, selectivity, availability and unwanted side effects. in addition, the sustainable production of these valuable metabolites can be a challenge. studying the molecular basis of the biosynthetic pathways may set the basis for improving the production and for rationally engineering derivatives with altered bioactivity profiles, e.g. through targeted knockouts, mutasynthesis ziehl et al. ( ) , and swapping of pathway genes. our results in elucidating and manipulating the biosynthesis of selected antitumoral polyketide metabolites from bacteria (aureothin, chartreusin) and fungi (cytochalasines, rhizoxin) are presented. analyses at the genetic and biochemical levels provided new insights into several unusual biosynthetic features, e.g. non-linear polyketide assembly for the nitroaryl-substituted polyketide aureothin he and hertweck ( , ) , an oxidative rearrangement cascade in the chartreusin pathway xu et al. ( ) , and a fungal iterative pks-nrps hybrid synthase schuemann and hertweck ( ) involved in cytochalasin biosynthesis. the most surprising result was obtained from elaborating the biogenesis of the antimitotic agent rhizoxin from rhizopus sp., which allowed for a significant improvement in large-scale production partida-martinez and hertweck ( ). he, j., hertweck, c., chem. biol. , , - chem. bio. chem. , , glycopeptides such as vancomycin and teicoplanin are the drugs of last resort for the treatment of severe infections caused by antibiotic resistant gram-positive bacteria. glycopeptides inhibit the peptidoglycan biosynthesis by binding as dimers to the d-ala-d-ala termini of the cell wall precursors. amycolatopsis balhimycina synthesizes the vancomycin-type glycopeptide balhimycin, whose structure and biological properties greatly resemble vancomycin and which only differs by its glycosylation pattern. using a "reverse genetics" approach we have identified the -kb gene cluster encoding the biosynthesis of balhimycin. by a combination of genetics, biochemistry and analytical organic chemistry, we were able to elucidate the biosynthetic pathway and to assign functions to almost all genes of the cluster. the biosynthesis starts with the pathway-specific provision of the non-proteinogenic amino acids ␤-hydroxytyrosine (␤-ht), hydroxyphenylglycine (hpg) and dihydroxyphenylglycine (dpg) which form together with (n-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. dpg is synthesized via a polyketide synthase mechanism (pksiii) similar to that known from plant chalcon/stilben synthases (pfeifer et al., ) . for the ␤-ht synthesis three genes are essential which form an operon (puk et al., ) : bpsd, an nrps binds a tyrosine molecule, which is then hydroxylated by the p monooxygenase oxyd. the perhydrolase bhp is required for the release of ␤-ht. subsequently bhaa, a nadh/fad-dependent halogenase catalyzes the chlorination of ␤-ht to form chloro-␤-hydroxytyrosine (puk et al., ) , which is needed to stabilize the dimerization. the amino acids are linked by non-ribosomal peptide synthetases (recktenwald et al., ) , and the aromatic side chains are interconnected by p monooxygenases; a series of reactions which lead to the first antibiotically active intermediate. inactivation of the oxygenase genes revealed the order of the cyclization steps (bischoff et al., ) : the oxygenases act in a stepwise fashion in the sequence oxyb, oxya and oxyc. the resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation. the biosynthesis is regulated by the strr-type regulator bbr, which was shown to bind in front of different operons of the balhimycin gene cluster. this ensures the coordinated expression of the biosynthetic genes. the non-producing mutants, defective in the supply of the non-proteinogenic amino acids, were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. thus, novel balhimycin derivatives were generated (weist et al., (weist et al., , . bischoff et al., . angew. chem. int. ed. , - . pfeifer et al., . j. biol. chem. , - . puk et al., . j. bacteriol. , - . puk et al., . chem. biol. , - . recktenwald et al., . microbiology , - . weist et al., . angew. chem. int. ed. , - . weist et al., spectroscopy guided discovery of novel bioactive microbial natural products thomas ostenfeld larsen, michael edberg hansen cmb biocentrum-dtu, technical university of denmark, lyngby, denmark the task of finding novel bioactive natural products is usually bioassay driven. often a certain type of compound (e.g. polyketide, alkaloid) turns out to be active in an assay. when having generated a promising hit in a bioassay the normal procedure in the drug discovery process usually is to produce a large number of structurally analogous compounds either by traditional chemical synthesis or by combinatorial chemistry in order to study structure activity relation-ships and to find even more active lead compounds. alternatively to chemical synthesis of analogues nature can be explored for structurally similar compounds by uv-spectroscopy guided screening. this work will present a new method for the systematic and automated computer assisted search of full uv spectra in large number of datafiles for both dereplication of known and discovery of new natural products based on the use of the new mathematical algorithm x-hitting. exploring the substrate spectrum of the antibiotic producing bacteria saccharopolyspora erythraea p. krabben, p. oliveira, f. baganz, j. ward department of biochemical engineering, university college london, london wc e je, uk knowledge of substrate utilisation capabilities play an important role in the development of genome scale metabolic models (borodina et al., ) and refinement of first generation annotations. furthermore, knowledge of the product formation during catabolism of different substrates provides valuable information about the distribution of metabolic fluxes and thereby forms a basis for rational strain improvement. we present here, data on the substrate utilisation capabilities and the corresponding product formation of s. erythraea. this analysis will help in improving the production of erythromycin and provide clues to the activation of the cryptic secondary metabolic pathways present in the s. erythraea genome. reference borodina, i., et al., ( ) . genome-scale analysis of streptomyces coelicolor a ( ) modeling of growth and product formation on complex media containing multiple substitutable substrates is a challenge. complex media offers the organism multiple choices of carbon and nitrogen substrates including free amino acids, peptides, soluble and insoluble proteins in addition to the defined sources such as glucose and ammonium sulfate. we present a structured model that accounts for growth and product formation kinetics of rifamycin b fermentation in a multi-substrate complex medium. the model considers the organism to be an optimal strategist with a mechanism to regulate the uptake of the substrate combinations. further, we assume that the uptake of a substrate depends on the level of a key enzyme, which may be inducible. the model also considers control parameters as fraction of flux through a given metabolic branch. the control parameters are obtained using a simple multi-variable constrained optimization. the model parameters were rigorously estimated via a specifically designed experimental plan. the model correctly predicts the experimentally observed growth and product formation kinetics and the regulated simultaneous uptake of the substitutable substrates under different fermentation conditions. the model and the model parameters provide useful insights into the growth and product formation strategy of this industrially important process. this presentation will describe the experimental results, the model development and the relevant model parameters for a. mediterranei s . the recent surge in oil price and the increasing concern on our environment have generated much interest in the production of chemicals from renewable resources. succinic acid, also called as amber acid, is a four-carbon dicarboxylic acid, which can be used as a precursor of numerous products including biodegradable polymers, green solvents, pharmaceuticals, and bulk and fine chemicals. a new capnophilic bacterium named mannheimia succiniciproducens mbel e was isolated from the rumen of korean cow. this bacterium can produce large amounts of succinic acid along with some other metabolites such as lactic, formic and acetic acids. we have completely sequenced the genome of m. succiniciproducens and charaterized its genome content in the context of metabolic pathways. we then constructed the genome scale in silico metabolic network for metabolic flux analyses, and carried out metabolic flux analysis under varying environmental conditions. based on the in silico analyses results, we selected several target genes to be manipulated for enhanced succinic acid production. detailed results of metabolic engineering based on genome-scale information will be reported. we have been developing tools for inverse metabolic engineering in order to identify gene targets that improve the phenotype of industrial strains and cells for medical applications. to this end, we create genomic fragment libraries from a source organism and use it to transform the host organism. cells are properly selected in environments that favor the phenotype of interest and genes enriched in these cells are sequenced and used in follow up transformations of cells with specific genetic backgrounds. this overall strategy is complemented with additional tools for modulating gene over-expression, gene deletion, and high throughput clone isolation. we will demonstrate applications of this strategy to the identification of gene targets for improved xylose assimilation in recombinant saccharomyces cerevisiae and improved lycopene production in escherichia coli. based on assumed reaction network structures, nadph availability has been proposed to be a key constraint in ␤-lactam production by penicillium chrysogenum. in this study, nadph metabolism was investigated in glucose-limited chemostat cultures of an industrial p. chrysogenum strain. enzyme assays confirmed the nadpspecificity of the dehydrogenases of the pentose-phosphate pathway and the presence of nadp-dependent isocitrate dehydrogenase. pyruvate decarboxylase/ nadp-linked acetaldehyde dehydrogenase and nadp-linked glyceraldehyde- -phosphate dehydrogenase were not detected. although the nadph requirement of penicillin-gproducing chemostat cultures was calculated to be . - . -fold higher than that of non-producing cultures, activities of the major nadph-providing enzymes were the same. isolated mitochondria showed high rates of antimycin a-sensitive respiration of nadph, thus indicating the presence of a mitochondrial nadph dehydrogenase that oxidizes cytosolic nadph. the presence of this enzyme in p. chrysogenum has important implications for stoichiometric modelling of central carbon metabolism and ␤-lactam production and may provide an interesting target for metabolic engineering. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. complete elucidation of the genetic control of a metabolic flux requires the availability of fine-grained expression levels of the gene(s) of interest. we developed a collection of promoters of varying strength for tuning gene expression in the yeast s. cerevisiae. engineered promoters were obtained through random mutagenesis of the constitutive tef promoter. eleven mutated promoters were selected by fluorescence-activated cell sorting (facs) spanning gradually increasing activities between about and % compared to the native tef promoter. data were also confirmed at the level of mrna via rt-pcr. by introducing selectable markers in front of the different tef promoter mutations, we provided plasmid collections, which can be directly used to amplify promoter replacement cassettes for genomic integration of the fine-grained promoter collection in front of any yeast gene. l-arabinose, widely distributed in plant kingdom, is a component of plant cell wall. l-arabinose does not abundantly exist in free state in plants, but usually in corn hull, sugar beet pulp, gum arabic, mesquite gum, as the polysaccharide such as arabinoxylan and arabinogalactan. to produce arabinose from agricultural wastes, we screened arabinogalactan degradable strain from compost. thereafter, putative arabinase gene from this strain was cloned (b ts - ). as a result of spectrometric assay using -nitrophenyl ␣l arabinofuranoside, recombinant showed - -fold higher activity than wild type e. coli strain. after enzymatic reaction with corn fiber, b ts - produced . g/l of l-arabinose, which was detected on hplc. however, the enzyme activity was very low. so, we are transferring the gene into expression vector system. further characterization study and enzyme engineering to enhance the activity toward corn fiber will be presented in poster. there are only a limited number of hypersaline areas all over the world, which include several locations in turkey such as van lake located in eastern region of turkey. isolation and identification of halophilic and hyperhalophilic microorganisms from such locations is essential for the determination of biodiversity in turkey. high-level production of extremozymes from these microorganisms has also many economical advantages due to their stability at extreme reaction conditions. proteolytic enzymes are the most important group of enzymes produced commercially. of these, proteases produced by alkalophilic microorganisms are investigated not only in scientific areas such as protein chemistry and protein engineering but also find wide application in food, pharmaceutical, leather and detergent industries. in this study, microorganisms isolated from van lake were screened for the presence of extracellular alkaline protease activity. the optimum screening temperature and ph were determined as • c and ph . , respectively. one of the isolates that could grow at - % salinity reached highest levels of extracellular alkaline protease activity. this best producer, which was identified as the halotolerant bacillus pumilus, was found to produce alkaline protease both in the presence and absence of nacl. to improve enzyme production yields, culture conditions such as medium composition, growth ph and temperature were optimized. the effect of different carbon sources, organic and inorganic nitrogen sources on the production of alkaline protease was studied. whereas a mixture of inorganic and organic nitrogen sources induced high protease production, use of only an organic nitrogen source supported poor enzyme production. halotolerant bacillus pumilus produced maximum alkaline protease activity when maltose, yeast extract and sodium nitrate were used as carbon source, organic and inorganic nitrogen sources, respectively. this project was supported by tubitak through project tbag - t . in the market of biochemical products a very important role is played by heterologous proteins production, and despite recent advances in mammalian cells exploitation, yeasts can still present advantages as host systems. among them, the spoilage yeasts belonging to the zygosaccharomyces genus have become, due to some peculiar properties, significantly attractive. in particular, z. bailii is characterized by acid resistance, osmotolerance to high sugar and ethanol concentration combined with high biomass yield. despite still little is known about its genetics and cellular biology, our group is working on its development and exploitation for recombinant productions with an integrated approach coupling physiological study with the creation of molecular tools for heterologous proteins production. we previously described and did a patent application regarding the first techniques necessary to transform this yeast and to express and secrete different proteins derived from different sources. here we present and discuss the last advances in optimization of heterol-ogous protein expression in particular, on one side we present a reproducible strategy for target gene deletion, leading to the first z. bailii auxotrophic mutant, and on the other we show the improvement of gene dosage and plasmid stability by building a set of multicopy expression vectors based on the sequences of the z. bailii -like endogenous plasmid psb and an integrative plasmid. all the known ␥-butyrolactone autoregulator receptors are highly conserved in the dna binding motif present in their n-terminal portions and have been proposed to play roles as transcriptional regulators in antibiotic production and/or morphological differentiation. previously, kim et al. reported that the cloned scar in streptomyces clavuligerus has several characteristics of the autoregulator receptors in the genus streptomyces. in this study, to clarify the in vivo function of scar, a scar-disrupted strain was constructed by means of homologous recombination after introducing a scar-disruption construct via transconjugation from e. coli. no difference in morphology was found between the wild-type strain and the scar disruptant. however, the scar disruptant showed a . -fold higher production of clavulanic acid than the wild-type strain. the phenotype was restored to the original wild-type phenotype by complementation with intact scar. therefore, the autoregulator receptor, scar, acts as a negative regulator of biosynthesis of clavulanic acid but plays no role in cytodifferentiation of s. clavuligerus. lactate dehydrogenase catalyses the production of lactate from pyruvate. it is the first target for many researches on lactic acid producer microorganisms like rhizopus oryzae. in the present study based on the known sequences of r. oryzae ldha and ldhb genes skory ( ), they were cloned and expressed in a citric acid producer fungus aspergillus niger. the aspergillus niger strains expressing ldha or ldhb gene resulted in increased production of lactate in aspergillus niger. among transformants tested ldha and ldhb expressing strains were found to have higher lactate dehydrogenase activity compared to wild type in the conditions tested. the highest specific activity obtained with ldha transformants was only . times of the wild type while this was times for one of the transformants expressing ldhb. in addition to increased lactate production citric acid production was also increased. however, gluconic acid production ceased in the ldha or ldhb expressing a. niger strains. the production of lactic acid in a. niger transformants and lactate dehydrogenase a and lactate dehydrogenase b enzymes are being investigated in the chosen strains. selection of n source suitable for production of rhodococcus sp. biomass for the purposes of microbial transformation of ␣h-epoxypregnanolone ( ␣h) and -epoxy-pregnenolone ( ) into their ␣-hydroxy-derivatives was carried out. three dehydrated and three non-dehydrated n sources were tested. the transformation reaction was carried out in phosphate buffered medium containing g l − of the steroid substrate and . g l − cells. the steroids were determined by hplc. the transformation resulted in formation of three derivatives appearing in the reaction medium in the sequence: - -keto-; ␣-hydroxy-and ␣, ␤-hydroxy-epoxy-pregnenolone. a strong influence of the n source on the hydroxylating activity of the biomass was observed. triptose (difco) gave a cell depot actively hydroxylating ␣h without any significant accumulation of the - -keto-derivative. the most effective accumulation of hydroxylated derivatives of was observed with biomass grown on freshly prepared meat extract while the commercial products triptose (difco), meat extract (difco) and lactalbumin (flika) gave valuable information about the dynamics of the transformation process. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene , nazif kolankaya : kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth ( % soytone, % d-glucose and . % cellulose pulp). maximal extracellular ligninase production was detected after day ( nkat). the optimum biobleaching conditions are • c and ph: . , with days. in this condition p. versicolor decreased the kapa number from . to . and increased brigthness from to . in day treatment. xylanase production, purification and characterization from a soil isolate, bacillus m- ayşegül ersayin , aytaç kocabaş , b. zümrüt ogel , ufuk bakir : biotechnology department, middle east technical university, ankara, turkey; food engineering department, middle east technical university, ankara, turkey; chemical engineering department, middle east technical university, ankara, turkey, . e-mail: ubakir@metu.edu.tr (u. bakir) xylan is a major component of the plant cell wall, representing up to % of the dry weight. xylan molecule is a complex polymer consisting of a ␤-d- , -linked xylanopyranoside backbone substituted with acetyl, arabinosyl and glucuronosyl side chains. hydrolysis of the xylan backbone is mainly catalysed by endo-␤- , -xylanases (ec . . . ). many bacterial and fungal species are able to utilize xylan as a carbon source. interest in the enzymology of xylan hyrdolysis has increased because use of xylanases in bioconversion of agricultural wastes to valuable products like single cell protein, xylo-oligosaccharides and fuel, in bio-bleaching processes, food and animal feed industries. in this study, xylanolytic nature of a soil isolate bacillus spp., bacillus m- , has been shown. bacillus m- produced multiple xylanases when grown on a liquid medium containing agricultural wastes as the sole carbon source. various agricultural wastes including corn-cobs and cotton waste, with and without pretreatments were used to maximize enzyme production. the major xylanase having molecular weight of kda upon sds-page and a pi of . upon ief was partially purified by liquid chromatographic techniques -fold with % recovery, including gel filtration, ion exchange and hydrophobic interaction chromatography. enzymes are important constituents in the laundry detergents due to their contribution to shortening washing times, reduction of energy and water consumption by lowering washing temperatures, provision of environmentally friendlier wash-water effluents and fabric care. however, they can loose a significant part of their activity in the chemically-hostile detergent matrix over a time period of several weeks. therefore, improving the storage stability of enzyme granulates is the main challenge in the development of a new product. the complexity of the detergent matrix implies the presence of a complicated mechanism involved in the inactivation of the enzymes. a combination of factors, such as oxidation by h o released by the bleaching agents, humidity, high temperature, autolysis of enzymes, high local ph in a granule, oxygen, and other detergent components, plays a role in the activity loss. an experimental investigation on the inactivation of the solid-state enzyme during storage has been initiated. the release rate of h o from the bleaching agent, sodium percarbonate, was determined using a simple and accurate method for measuring the gas phase h o concentration. the deactivation kinetics of pure enzyme was determined as a function of gas phase h o concentration and humidity. the preliminary results indicate that humidity plays a significant role in the inactivation mechanism of the detergent enzyme due to a possible increase in the mobility of the enzyme molecule and the surface area exposed to destructive agents. the effect of main granulate ingredients on the stability of the enzyme was investigated and the extent of the protection of each component was estimated. the study is important for the revealing of the phenomena occurring in the detergent matrix during storage. understanding the inactivation mechanism provides a valuable tool for the development of more effective protective coatings and stabilizers. the use of biosurfactants in cosmetic industry has attracted great attention of biotechnological researchers because they consist of two inexpensive, renewable and easily accessible starting agricultural materials: sugar and oil/fat. carbohydrate based products are non-toxic, biocompatible and biodegradable. in addition, the enzymatic processes present many advantages in comparison with the chemical methods, which employ high temperatures in the presense of alkalin catalysts, high-energy consuption and low selectivity of products. sugar esters present vast application, such as for antibiotics, biomaterials, surfactants, cosmetics and so on. we investigated the synthesis of sugar vinyl esters, using protease-catalysed transesterefication method applying protease from bacillus subtilis. sucrose . m and vinyl ester . m has been mixed in dimethylformamide at rpm of agitation. at first, we have studied the effects of protease from bacillus subtilis concentrations ( , , and mg/ml) as catalyst. afterwards, the influence of the temperature ( and • c). after that, the influence of the molar ratios ( : ; : ; : m/m) between vinyl laurate (ch (ch ) cooch ch ) and sucrose. subsequently, we investigated the effects of water amount, using , , and % of water in dmf. the conversion ratios of sucroseto-sucrose esters were determined decreasing sucrose measurement with hplc. the results showed that the best conditions to produce high activity on the enzymatic reaction was by using mg/ml of protease from bacillus subtilis at • c, molar ratio of : (vinyl laurate:sucrose) and adding % of water in dmf. finally, we succeeded in the characterization of vinyl sugar ester, which was produced after hours of reaction by c nmr. the results confirmed the c substituted sugar mono-ester ( -o-vinyl lauroyl sucrose). effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide Ç alık bre lab, department of chemical engng, ankara university, ankara, turkey ␣-amylase (e.c. . . . ) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣- , glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b- ), which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in . dm air-filtered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs ). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled . and . dm systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc s ultracentrifuge, ␣-amylase activity was measured by the dns method bernfeld ( ) . amino acid concentrations were determined with a hplc (waters), pro-tein and organic acid concentrations were measured with a hpce (waters, quanta e) Ç alik et al. ( ) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method rainer ( ) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources, i.e. glucose, fructose, maltose, lactose and soluble starch; n sources, i.e. (nh ) hpo , (nh ) so , and nh cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and by-product concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. bernfeld, p., . methods in enzymol. : - . Ç alık, p., Ç alık, g.,Özdamar, t.h., . enzyme microb. technol. , - . rainer, b.w., . geldanamycin is a benzoquinone ansamycin produced as a secondary metabolite by the actinomycete, streptomyces hygroscopicus var. geldanus, in submerged culture. it is a broad-spectrum antibiotic and exhibits an anti-tumour activity through its interaction with the heat shock protein family of chaperone proteins. the optimal recovery of geldanamycin from fermentation broths is the focus of the presented work. the application of adsorbent resins was assessed and the viability of developing a solid phase extraction process for geldanamycin was determined. it was found that recovery of geldanamycin from fermentation broth was possible using adsorbent resins and the use of resins facilitated the recovery of a product stream of high purity. the composition of the fermentation broth had an impact on the performance of the resins and it was found that assessing performance on the basis of experimentally derived data was more apt than studying the kinetics of adsorption alone. adsorptive processes are, by their nature, difficult to optimise and this was found to be the case when optimising the recovery of geldanamycin from partially clarified fermentation broth. considerable effort was required to optimise geldanamycin adsorption, via examining the effect of environmental conditions and process system configuration, and geldanamycin desorption, via examining the effect of environmental conditions and investigating selective elution patterns. it is well known that halophilic eubacteria synthesize compatible solutes in order to face the high ionic strength environment in which they proliferate. these biomolecules are gaining more and more importance as biotechnological tools in a wide array of applications, and the recently developed novel bioprocesses enabled large-scale production of these compounds and therefore commercial distribution. however, there is still interest in the optimization of the production process of sole hydroxyectoine that was demonstrated to have a superior stabilization capacity. in this research project, we optimized growth conditions of marinococcus m to obtain high yield of hydroxyectoine. their production proved faster in the batch experiments at a higher oxygen supply, even if the stationary phase was comparable in all cases. the mf experiments showed a final biomass which was -fold that obtained in the corresponding batch process. in addition the monitoring of compatible solutes production showed that in the last h experiment hydroxyectoine accounted for - % of the total content, accumulating up to - % of the cell dry weight. studies for improving downstream process for ectoine and hydroxyectoine recovery showed that short permeabilization cycles in water are effective in a temperature range between • c and • c using a ratio : /biomass:water. moreover, we evaluated the ability of ectoine to stabilize lactic acid bacteria during freeze-drying and to protect human cells from heat stress. in particular, the compatible solutes were added to the medium of confluent keratinocytes before subjecting the cells to heat stress, or lps insult. rt-pcr and western blot analysis demonstrated the hsp b' gene over-expression in heat stressed human keratinocytes treated with ectoine. finally, we demonstrated that even at low concentration ( mm) these compatible solutes are able to diminish cell death in lactic acid bacteria due to lyophilization procedure. among all existing alternative energy sources, biomass-derived bioethanol is especially advantageous since it is clean, sustainable and potentially inexpensive. the actual production of bioethanol is divided into a pre-treatment step, an enzymatic hydrolysis step and a fermentation step. while fermentation has been practiced by humans for centuries, our knowledge of enzymatic hydrolysis is still limited. nevertheless, it is well accepted that hydrolysis is a synergism among three classes of enzymes, ␤-glucosidase, endoglucanase and cellobiohydrolase. furthermore, a complete and efficient hydrolysis is only achieved when the enzymes are in the correct proportion. the common enzyme proportions have so far been based on the natural enzyme abundance as produced by the microorganism or mainly been determined by a trial-and-error approach. in this study, however, we used metabolic control analysis (mca) as a modelling tool to gain fundamental knowledge about enzymatic hydrolysis and to design an optimal enzyme mixture. using gepasi, a free software, the degree of control of each reaction step or each enzyme towards the overall hydrolysis can be calculated. our hypothesis is that the amount of each enzyme used for hydrolysis should be proportional to the degree of control of the enzyme. with mca, a significant amount of time, labour and reagents can be saved on developing hydrolysis enzyme mixture. furthermore, this study should demonstrate the usefulness of mca on understanding enzyme-catalyzed reactions outside the cell. process optimization for fed-batch fermentation of bacillus thuringiensis subsp. israelensis arindam chaudhury, gopinathan c department of biotechnology, university of calicut, calicut, kerala india. e-mail: g achaudhury@umassd.edu (a. chaudhury) bacillus thuringiensis (bt) is a desirable biopesticide because of its low cost and lack of toxicity. the use of bt in developing countries is limited due to process complications and economic non-feasibility of the fermentation process. in the present study, we have shown how regional production, using inexpensive alternatives for carbon and protein sources, can effectively reduce the cost of mass production of bt. while using alternative media supplements, the biomass production, nor the larvicidal activity was hampered. in addition, the positive effects of sparged aeration and the indispensable role of yeast extract were also proved. this work provides the first experimental proof of delineating the sporulation process and delta-endotoxin production. the role of various buffering agents and additives in increasing biomass production and early sporulation were also investigated. for the production of coenzyme q (coq ), an electron carrier in the respiration chain with antioxidant activity. with decrease of dissolved oxygen level from to %, the intracellular coq content increased about -fold, yielding mg per g-dry cell weight at % dissolved oxygen level. azide significantly increased the intracellular coq content, with the highest value of . mg per g dry cell weight in the presence of . mm of sodium azide. however, dnp (up to m) and h o (up to m) did not affect the intracellular coq content, indicating proton gradient release and oxidative stress do not affect the synthesis of coq . these results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular coq content. fourier transform infrared spectroscopy (ft-ir), combined with in situ heat sterilizable attenuated total reflection (atr) probes, constitutes a promising and versatile technique for on-line monitoring of bioprocesses. the ft-ir enables rapid determinations of the medium composition without the requirement of sample withdrawal and preparation. in this work the concentration levels of the substrates glycerol and methanol were monitored on-line in pichia pastoris cultivation. partial least squares (pls) models were used for obtaining the concentration readings. the glycerol concentration measurement proved to be very reliable and reproducible in the glycerol batch phase. however, the on-line information regarding the glycerol concentration was not utilized for any process control purposes. on the other hand, the availability of on-line information about the methanol concentration proved to be crucial for the successful implementation of the cultivations. the temperature strategy in the methanol fed-batch phase utilized temperatures as low as • c. in order to keep the metabolic activity at a reasonable level the culture was therefore pushed towards the maximal substrate consumption rate, rather than being a conventional substrate limited fed-batch. as a consequence methanol accumulation occurred on occasions. without on-line information about the concentration this accumulation, if sustained, would have resulted in a poisoning of the culture, either from methanol itself, or perhaps more importantly from formaldehyde. therefore, it can be concluded that the ft-ir/atr instrument was very useful in this application. jørgensen department of chemical engineering, denmark technical university, building , dk- lyngby, denamrk. e-mail: fpd@kt.dtu.dk (f.p. davidescu) modeling biochemical reaction network in microorganisms still represents a challenge due to the very large number of enzyme catalyzed biochemical reactions, to the very complex system and to the many feed-forward and feedback regulation mechanisms. the presented approach to model such a system is based on the stochastic grey-box modeling framework proposed by kristensen et al. ( ) . this methodology consists of parameters estimation based on a prediction error method followed by different statistical tests for parameter significance and for model (in-) validity. the methodology furthermore allows estimation of unknown functional relations, e.g. kinetic rates. a set of experimental data zangirolami ( ) obtained during continuous cultures of a high enzyme producing aspergillus oryzae strain. the oxygen concentration was decreased stepwise and the substrate concentration was modified from one experiment to other. a model proposed by agger et al. ( ) is investigated on these data. the primary interest is to develop a physiologically feasible model, also at the low oxygen concentrations often found in industrial practice. microbially produced secondary metabolites such as antibiotics have tremendous economic importance. streptomyces spp. have long been identified as sources of antibiotics and chemotherapeutic compounds, synthesising over bioactive compounds. geldanamycin is a novel chemotherapeutic agent produced by streptomyces hygroscopicus var. geldanus in submerged fermentation. initial studies have focused on optimisation of media design through understanding and controlling metabolic routes of biosynthesis within the cell. geldanamycin is a by-product of the shikimate or aromatic amino acid biosynthesis pathway. stimulation of this pathway and concomitant production of geldanamycin is achievable through amino acid control. increasing concentrations of primary carbon source greatly influence biomass generation and product formation, as does the inclusion of cations such as magnesium and calcium to the fermentation media. optimisation of production media through balancing minerals, nitrogen, and carbon sources has significantly improved antibiotic yields in shake flask cultures and the development process will be extended into pilot scale through the use of bioreactors. microbiology and biotechnology research group, school of life sciences, napier university, edinburgh, eh dt, scotland. email: m.el-mansi@napier.ac.uk (m. el-mansi) synopsis: during growth of corynebacterium glutamicum on glucose or other glycolytic intermediates, pep carboxylase fulfils an anaplerotic function as it replenishes intermediary metabolism with biosynthetic precursors that are essential for growth and glutamate production. under these conditions, pep carboxylase plays a central role and this in turn is characterised by a high flux control coefficient thus rendering this enzyme an ideal target for metabolic interventions. further analysis in silico revealed that any increases in the concentration of the enzyme was accompanied by increases in flux through the enzyme itself as well as glutamate formation, presumably as a consequence of sustaining a high intracellular level of ␣-ketoglutarate; the immediate precursor for glutamate biosynthesis. a combined approach to enhance periplasmic expression of human growth hormone in escherichia coli, using a modified signal peptide from alpha amylase gene of bacillus licheniformis s.k. falsafi , , a. zomorodipour : islamic azad university of jahrom, iran; department of mol genet. national inst for genet eng & biotechnol., tehran, iran. e-mail: soheil falsafi@yahoo.com (s.k. falsafi) the alpha amylase gene signal peptide, originated from a strain of bacillus licheniformis, was shown to be able to transport its native protein, when expressed in e. coli the competence of the fusion protein being processed and translocated through the inner membrane is highly dependent on the amino acid sequences in the signal peptide. therefore, in order to increase the expression efficiency of bla signal peptide, we reconstructed the bla signal peptide coding fragment with the following modifications. two rare codons of arg (cgg) and arg (cga) and codons for leu (tta) and pro (cct), in the signal peptide were substituted with their corresponding e. coli major codons. two other changes, including phe (ttc) → leu (ctg) and ala (gcg) → met (atg), were also introduced to increase the processing efficiency. the hgh-expressing plasmid equipped with the modified bla (blaf ) was subjected for further expression analysis in a t -based expression system. the results obtained from the protein patterns of the induced bacteria indicates in high expression level of hgh preprotein (hgh::blaf ) followed by efficient transfer of the mature hgh to e. coli periplasm. (ip ) has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip ) and inositol tetrakisphosphate (ip ) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia converted ip into ip (myoinositol , , , , pentakisphosphates) and another isomer, which is yet to be elucidated. characterization of the novel ␤-peptidyl aminopeptidase (bapa) from sphingomonas sp. - w that cleaves synthetic ␤peptides birgit geueke, hans-peter e. kohler environmental microbiology, eawag, duebendorf, switzerland. e-mail: birgit.geueke@eawag.ch (b. geueke) non-natural peptides, which are capable of evoking a specific biological response, are currently receiving much attention. oligomers of ␤-amino acids (␤-peptides) are representing a group of pharmaceutically interesting peptides because of their very high stability towards enzymatic degradation and their ability to mimic the structure of naturally occurring biologically active peptides. the pharmaceutical potential on the one hand and the high stability on the other hand aroused interest for studies on the environmental fate and the degradation behaviour of this class of compounds. a novel bacterial strain (sphingomonas sp. - w ) that was capable of degrading short ␤-peptides was isolated from an enrichment culture. the ␤peptide degrading enzyme was purified and its gene sequence was determined (bapa). the gene encodes a ␤-peptidyl aminopeptidase (bapa) of amino acids that is synthesized as preprotein with a signal sequence of amino acids. it belongs to the n-terminal nucleophile (ntn) hydrolase superfamily and is the first peptidase that is capable of cleaving amide bonds in ␤-peptides composed of synthetic ␤-amino acids. the biochemical properties of recombinant bapa were investigated regarding its substrate specificity and possible application in the synthesis of ␤-peptides. to produce efficient strains of agaricus bitorquis (quel.) saccardo, which are resistant to high temperatures p. guler, a. ergene, s. tan kirikkale university, faculty of science and literature, department of biology, yahsihan-kirikkale in this study, the culture mushroom agaricus bitorquis (quel.) sacc. the growth of the mycelium and the fructifications under high temperature is examined. the spores taken from the mushrooms that were collected from nature were grouped as a, b, c, d, e. the spores were inoculated into malt extract agar and incubated at • c and primer mycelium was produced. the mycelium discus taken from primer mycelium in mm diameter were inoculated into the center of malt extract agar and incubated at , , , and • c separately. during the incubation period the growth of the mycelium were measured. during the growing period the radial growth speed of the mycelium were taken as criteria. the best mycelium growth for all groups was seen at • c. at • c the e group mycelium and at • c other group's mycelium did not grow. these temperatures were determined as thermal lethal point for the groups. from all the mycelium produced from all temperatures spawn was prepared and with the results taken from these, spawn calendar is prepared. in this research, the spawn was inoculated to compost with mixing system and separately put in culture rooms, temperatures as and • c. at this level the culture mushroom production techniques were used. the harvested mushrooms were inspected morphologically. at this morphological inspections the cap width, cap tissue thickness, stalk thickness and stalk long ness was taken as criteria. in the study the best growth was seen at d group mushrooms and this group mushrooms tyrosinase's activities were measured and graphics were made. introduction: viral contamination of biological products; cause many problems in viral diagnostic laboratories, blood transfusion organizations, and biological producers. bovine viral diarrhea virus (bvdv), from the pestivirus genus, is the most common viral contamination in (fetal) bovine serums (fbs). also, bvdv used as a module, for study hepatitis c virus inactivation due to its similarity in structure and genome. pulsed uv lights (puvls) have this potential to inactivate known and unknown or reemerging viruses as well as prions. two puvl with the wavelengths of and nm, were produced by q-switched nd + :yag laser in its third and forth harmonic, respectively. the energy of each pulse for nm was . mj/cm and for nm was . mj/cm . bvdv were produced and titrated in mdbk cell line. mdbk and fbs were already checked for non-cytopathic or cytopathic pestiviruses, using related ag-elisa kit. bvdv suspended in solution with the dilution of : before exposure. the quartz tube with the minimum uv-absorption in compare with air, used as a container for exposed solutions. calculation of the virus titer, . tcid /ml, was done based on the reed and muench method. bvdv suspended in pbs was exposed into the . - j/cm of puvls with the wavelength of nm and also, was exposed into the . - . j/cm of puvls with the wavelength of nm. furthermore, bvdv suspended in fbs was exposed into the , , and j/cm of puvls with the wavelength of nm and also, was exposed into the . , . , . , and . j/cm of puvls with the wavelength of nm. results: the minimum dose for inactivation of bvdv suspended in pbs with the and nm wavelengths of puvls, were and . j/cm , respectively. also, the minimum dose for inactivation of bvdv suspended in fbs with the and nm wavelengths of puvls, were and j/cm , respectively. to evaluate the fbs quality to support cell culture, treated fbs with the dose of . j/cm of nm puvls was used to grow vero cell line in successive passages. the viability of cells in two study groups was identical. the statistical evaluation of two treated groups showed no significant difference, in passages. conclusion: because inexpensive equipment can be used to produce puvls capable of handling different volumes of biologics with operational ease, this viral inactivation technique is cost effective for relevant industries. the procedure has the potential to be combined synergically with other inactivation method. puvls offer a new, nonadditive and chemically safe alternative for the treatment of fbs to inactivate adventitious viruses and to preserve the biological activity necessary for the propagation of cell culture. characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid y. iwasaki , y. ishii , k. kino , k. kirimura : dept. appl. chem., sch. sci. eng., waseda univ., tokyo, japan; bme, asmew, waseda univ., tokyo, japan. e-mail: iwasaki@moegi.waseda.jp (y. iwasaki) for selective production of ␥-resorcylic acid (␥-ra, , -dihydroxy-benzoic acid) from resorcinol (re, , dihydroxybenzene) under mild conditions, we screened various microorganisms and found the reversible ␥-ra decarboxylase (rdc) as a novel enzyme applicable to carboxylation of re to form ␥-ra, in a bacterial strain rhizobium radiobacter wu- ) . rdc catalyzed the decarboxylation of ␥-ra, and regio-selective carboxylation of re to form ␥-ra, without formation of ␣-ra and ␤-ra. the molecular weight of rdc was estimated to be kda by gel-filtration, and that of the subunit was determined to be kda by sds-page, suggesting that rdc is a homotetrameric structure. the gene encoding rdc was sequenced, and a site-directed mutagenesis study revealed that the two histidine residues at positions of and in rdc are essential for the catalytic activity of rdc. through the reactions using e. coli cells highly expressing rdc, . mm ␥-ra was produced from mm re at • c for h, with a yield of %. ishii, y. et al., . biochem. biophys. res. commun., , - . laccase biosynthesis in stirred fermenters teresa jamroz, stanislaw ledakowicz, barbara sencio department of bioprocess engineering, technical university, lodz pl - , poland industrial applicability of enzymes is closely related to development of efficient methods of their production. currently, significant interest in lignolytic enzymes, including laccase, has been observed. laccase is an enzyme applied in various industrial branches and environmental processes. broad laccase applicability induces researchers to develop urgently efficient methods for its commercial production. laccase (ec. . . . . p-diphenol oxidase) is produced by cap mushrooms from the class basidomycetes. this is the so-called white rot fungi which in natural conditions appears on both living and dead wood. as shown in the practice of biotechnological processes, high-efficient strains have low resistance to destructive factors in bioreactors. hence, to preserve a proper morphology and physiological state of an organism, strictly determined culture conditions must be obeyed. this is very important in the case of basidomycetes for which submerged culture in the liquid phase is not a natural habitat. results of studies on laccase production from cerrena unicolor family are discussed. cultivation of active biomass was carried out in stirred tank and rotating disc bioreactors of different volume (b. braun of working volume dm ; fas- of working volume . dm ). experiments in both fermenters were made at impeller revolutions and min − , on a modified lindberg substrate. significant differences in the rate and yield of laccase production were reported. an almost three times higher values of laccase activity were obtained in the b. braun fermenter, at rotational speed min − . to retain suspended cells in bioreactor a filtration process can be used. the biomass is concentrated by withdrawing cell-free culture broth. if the desired product is dissolved in the broth (extracellular production), the procedure enables the continuous harvest in the cellfree permeate. an application test of filtration system for suspended biomass of aspergillus niger in submerged single stage continuous culture was presented in this report. the system is easy to construct and there is a possibility of its sterile exchange during cultivation. the culture medium contained the following substances (g/dm ): white sugar, . ; nh no , . ; mgso · h o, . ; kh po , . ; feso · h o, . . fermentations were carried out in the lab bioreactor biomer . the bioreactor was a standard cstr (continuous stirred tank bioreactor) with working volume of dm . high citric acid concentration in culture medium (p = . g/dm ), high yield of citric acid (y p/s = . %) and high efficiency coefficient (k ef = . ) were observed in single stage continuous culture with biomass retention. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar Ç alık, iblab, department of chemical engineering, metu, ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc ::bal carrying recombinant escherichia coli on a defined medium with . kg/m glucose were investigated in order to fine-tune the bioreactor performance, in v = dm batch bioreactors at five different conditions with the parameters at, i.e. q o /v r = . vvm and n = , , , min − and; q o /v r = . vvm and n = min − . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph o = . are optimum for maximum bal activity, i.e. u/cm at h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains metabolites and reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e. coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. saprophytic mycobacterium strains belong to the best known microorganisms which have been applied to the pharmaceutical industry for the production of steroid drugs. the mycobacterial cell wall is the permeation barrier to chemical compounds, including lipophiles. using isoniazid (inh), the inhibitor of the mycolic acids biosynthesis, we were able to demonstrate increased ad production and susceptibility to antimicrobial agents. the process of sterol transformation and products accumulation was monitored using gas chromatography. isoniazid was shown to intensify ␤-sitosterol side-chain degradation by mycobacterium sp., and accumulation of -androstene- , -dione (ad) and , -androstadien- , -dione (add), which are the starting materials in the biotechnology of medically important steroids. to confirm these results, the sensitivity of the bacteria to antimycobacterial drugs was performed. the minimum inhibitory concentration mic of rifampicin and erythromycin decreased markedly in the presence of inh. this work was supported by grant nr p c of the committee for scientific research. for the purpose of high-throughput screening and to reduce experiments with animals in pharma biotechnology biosensor systems gain importance. the principle of a biosensor is the combination of cultured cells and a sensorchip device, which allows the monitoring of cellular activity. in contrast to traditional analytics with a biosensor you can measure on-line cellular activity change caused by an effector as well as the restored activity after privation of the effector (re-native activity). cmos technology can be used for the realisation of various biological sensorchips such as adhesion sensorchips, metabolical sensorchips and electrophysiological sensorchips. the standard cmos technology allows a high reproducibility of the chips, the integration of electronic components on the chip, which reduces the amount of external devices and the combination of different sensors on one chip. in cooperation with the semiconductor company micronas and the biotech company bionas we have realised different types of cmos sensorchips to measure adhesion of a cellular monolayer with interdigitated electrodes (ides), metabolical activity via acidification with ion-sensitive field effect transistors (isfet) and sponteneous neuronal network activity with passive palladium electrodes. microbial agents have been applied to the different stages of pulp and paper processing. the work presented describes a study on the effect of applying ligninolytic enzymes, such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and subsequently subjecting these to different ageing processes. industrial pulps were obtained from different portuguese pulp and paper companies. the pulps used were ( ) unbleached pine pulp from portucel tejo; ( ) unbleached eucalyptus pulp from portucel setúbal; ( ) bleached eucalyptus pulp from portucel setúbal; and ( ) pulp made from recycled paper from renova s.a. several types of handsheets were produced with different grammage namely, and g/m . the prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet radiation (wavelength of nm), temperature ( • c) and moisture ( %); and thick saline fog at a concentration of % and temperature of • c. in order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each type of handsheet. in the first, the moisture varied ( , and %), while the temperature was held constant ( • c); in the second the temperature varied ( , and • c) and the moisture was held constant ( %). following the aging phase, the handsheets were subject to several chemical (viscosity and index kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in order to characterize the effect of the aging conditions. results will be presented describing the effect of application of the laccase-mediator system on the optical and mechanical properties of the prepared handsheets. fundação para a ciência e a tecnologia, project pocti/ agr/ / . aspergillus niger is a filamentous fungi widely used in industry. its growth as freely dispersed hyphae leads to an increase in the medium viscosity and to problem of mass transfer, especially oxygen transfer. oxygen acts both as final electron acceptor in the mitochondrial chain and as nutrient for the biosynthesis of unsaturated fatty acids and sterols. thereby, a lack of oxygen affects the nadh/nad ratio, the atp production, the growth and has a strong influence on the physiology of the microrganism. in the present study, the metabolic changes of a. niger in response to a lack of oxygen was investigated using oxygen limited chemostats combined with nitrogen pulse. under these conditions, the main consequence of a sudden decrease of oxygen availability is an increase in the mannitol production. this work showed that the mannitol biosynthesis, involving the enzyme mannitol- -p dehydrogenase, helps the reoxidation of nadh when the final electron transport acceptor, oxygen, is limiting. investigation of the lipase activity of the bacteria isolated from olive mill wastewater sevgi ertugrul , nur koçberber , gönül dönmez , serpil takaç department of biology faculty of science ankara university beşevler ankara turkey; department of chemical engineering faculty of engineering ankara university tandogan, ankara, turkey the bacteria that could grow on media containing olive mill wastewater (omw) were isolated and their lipase production capacities were investigated. the strain possesing the highest lipase activity among strains grown on tributryin agar medium was identified as bacillus sp. the effect of ph on the lipase activity of the strain was investigated in tributryin medium and ph was found to be the optimal. the liquid medium composition was improved by adding different carbon sources and fatty acids into tributryin medium -omitted tributryin -to increase the enzyme activity. the cultivations were performed at • c and ph . lipase activity of the bacillus sp. was measured spectrophotometrically through the hydrolysis of p-nitrophenol palmitate. among the media containing different compositions of tricapryn, trimyristin, tributyrin, triacetin, tween , glycerol-trioleate, glycerol-trioctanoate, glycerol-tridodecanoate, omw, glucose, and whey; the medium consisted of % whey + % glycerol-trioleate was found to give the highest lipase activity. cultivation of bacillus sp. in the optimum medium at ph = and • c for h was resulted in the extracellular and intracelluar lipase activities of and u/ml, respectively. this study was supported by ankara university biotechnology institute (project no: - and - ) . under different abiotic stresses, cell growth and metabolic activity are highly influenced in all types of living organisms medium osmolality is usually one of those factors affecting different types of biological systems in different ways. however, even in the same organ of higher eukaryotes the degree of osmoregulation mechanism is highly variable in different types of cells. therefore, studying the effect of osmotic stress on mammalian cell is very important subject for particular cell line. the effect of hyperosmotic pressure on the kinetics of cell growth of and metabolic activity of mesenchymal stem cells (mscs) and two industrially important cell lines, hybridoma cells and human embryonic kidney cell (hek) were investigated in batch cultures at different osmotic pressures in the range from to mosm kg − . in case of mscs cells, the maximal specific growth rate [] of . [h − ] associated with the highest specific glucose consumption rate [-q gluc ] of . × − [mol cells − h − ] was obtained in medium of mosm kg − . in case of hybridoma cells, osmotic pressure showed not only influence on the kinetics of cell growth and metabolism but also on the monoclonal antibody production. the maximal mab production was obtained in case of cells cultivated under osmotic pressure of mosm kg − . further increase in osmotic pressure resulted in significant reduction in growth rate as well as mab production. on the other hand, hek cells were more sensitive to osmotic pressure in industrially used serum free medium and the addition of serum decreased the inhibitory effect of high osmotic pressure on the cells. gustavo g. fonseca , , andreas k. gombert , elmar heinzle , christoph wittmann : biochemical engineering, saarland university, saarbrücken, germany; chemical engineering, são paulo university, brazil kluyveromyces marxianus cbs is a potentially interesting yeast strain characterized by a high capacity of conversion of substrate into biomass. however, this yeast has been only marginally studied so far. therefore, we performed a metabolic characterization in batch and chemostat cultures at dilution rates of . , . and . h − . the specific rate of o consumption (qo ) increased with dilution rate from . to . mmol (g dw) − h − . the respiratory coefficient remained almost stable around . for all metabolic states investigated. even at the dilution rate of . h − , which is close to the maximum growth rate of the strain of . h − , no significant overflow metabolism was observed. the concentration of extracellular metabolites increased with the dilution rate, but remained below % of the carbon consumed as glucose. all carbon balances closed near % underlining the consistency of the data. in contrast to s. cerevisiae the respiratory capacity of k. marxianus cbs is not strongly influenced by the dilution rate in aerobic chemostat or batch cultures, indicating its high potential for biomass-directed applications. a thermostable l-arabinose isomerase for enzymatic production of d-tagatose o. hansen, f. jørgensen, p. stougaard department of enzyme technology, bioneer a/s, kogle allé , dk- hørsholm, denmark. e-mail: och@bioneer.dk (o. hansen) d-tagatose, an isomer of d-fructose, is a low-calorie bulk sweetener with a sweetness equivalent to sucrose. d-tagatose has obtained gras approval for use as a food ingredient, and is currently produced by chemical isomerization of d-galactose, which may readily be obtained by hydrolysis of lactose. structurally, d-galactose is closely related to l-arabinose, and it has previously been shown that some variants of l-arabinose isomerase (araa) may catalyze the conversion of d-galactose to d-tagatose, in addition to the metabolic conversion of l-arabinose to l-ribulose. we have screened a number of bacterial araa enzymes for their ability to catalyze the isomerization of d-galactose to d-tagatose. the best enzyme was found in the thermophilic bacterium thermoanaerobacter mathranii (dsm ). the araa gene of t. mathranii was cloned, sequenced and expressed in e. coli. amino acid sequence comparisons of the t. mathranii sequence and other known araa sequences showed a relatively low sequence identity of about %, indicating a distant phylogenetic relationship to the other members of the l-arabinose isomerase group. the t. mathranii enzyme was thermostable with optimal activity at • c and it required manganese ions. unlike other araa variants, the t. mathranii enzyme showed k m values in the same order of magnitude for l-arabinose and d-galactose, suggesting that this enzyme is a versatile isomerase capable of isomerizing structurally related aldoses. the enzyme was immobilized by chemical cross-linking of a crude e. coli cell homogenate, and the immobilized enzyme efficiently converted d-galactose into d-tagatose. currently, we are developing an enzymatic method for industrial production of d-tagatose using the immobilized enzyme. the agricultural production can be negatively affected by different pest insects (pi) and the use of chemical insecticides (chi) has been the traditional method for controlling pi during decades. nevertheless, there are various ecological implications due to the extensive application of chi. a viable alternative for the use of chi in certain agro-systems, is the use of entomopathogenic nematodes (epn) of the genera steinernema and heterorhabditis that are natural pathogens for different pi; besides, the presence of a symbiotic bacterium is necessary for an effective entomopathogenic activity can take place. the nematode/bacterium complex does not represent a risk for the environment. different authors propose that the best alternative for the massive production of epn is the submerged culture within bioreactors; nevertheless, more research is required to have really robust processes. particularly, information regarding the actual hydrodynamics during epn production and its relation with the epn productivities are scarce, among other aspects. the present study deals with the hydrodynamic characterisation during the production of the epn steinernema carpocapsae and its symbiont bacterium xenorhabdus nematophilus, in submerged monoxenic culture in an internal-loop air-lift bioreactor (v l = . l) using two culture media: one of them containing whey and the other one, agave juice, aguamiel, (agave spp.). process viscosity of the culture broth was determined along the time, exhibiting a maximum value of mpa s. moreover, it was determined that the hydrodynamic conditions were always located within the laminar region (re < ). at the experimental conditions tested, it can be inferred that the epn productivity are more sensitive to changes in the culture medium composition than on the prevailing hydrodynamic conditions during the fermentations. bacillus thuringiensis is a gram-positive bacterium used as a biological pest control agent. moreover, it is able to produce, several biologically active molecules such as bacteriocins and hydrolytic enzymes among which chitinases that play double roles, fungicide and improving the insecticidal effect of b. thuringiensis deltaendotoxins. a newly isolated b. thuringiensis subsp. kurstaki strain bupm , was shown to produce a novel bacteriocin named bacthuricin f . the highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. upon purification of bacthuricin f , the specific activity was increased -fold. this bacteriocin was heat-stable up to • c and resisted up to ph . . its molecular mass, determined by mass spectrometry was . da. direct n-terminal sequencing of bacthuricin f revealed the following sequence: dwtxwsxl. the latter was unique in the databases. bacthuricin f was active against bacillus species while it had little or no effect on gram-negative bacteria. the bacteriocin produced by the b. thuringiensis strain bupm respond to both criteria of thermostability and stability to low phs. thus, it could be used as a source of bacteriocin active against related species of bacillus harmful for agricultural products and as food preservative. the other example of antimicrobial compound produced by b. thuringiensis is a chitinase. we describe the selection of b. thuringiensis high chitinase-producing strain bupm , and the characterization and the heterologous expression of a novel chitinase encoding gene. the cloning and sequencing of the corresponding gene named chi showed an open reading frame of bp, encoding a amino acid residue protein. both nucleotide and amino acid sequences similarity analyses revealed that the chi is a new chitinase gene, presenting several differences from the published chi genes of b. thuringiensis. the identification of chitin hydrolysis products resulting from the activity, exhibited by chi through heterologous expression in e. coli revealed that this enzyme is a chitobiosidase. the addition of the sequence of chi to the few sequenced b. thuringiensis chi genes might contribute to a better investigation of the chitinase "structure-function" relation. cloning and characterization of s-adenosyl-l-methionine synthetase from pichia ciferrii dscc - kwon-hye ko , gee-sun yoon , gi-sub choi , joo-won suh , yeon-woo ryu s-adenosyl-l-methionine(sam) has an important role for dna methylation and cell signaling. sam was synthesized from methionine and atp by sam synthetase and play an pivotal function in the primary and secondary metabolism of cells. recent studies have revealed in the effect of sam in case of morphological differentiation in both eukaryotes and prokaryotes. the p. ciferrii produces large quantities of sphingoid base. tetraacetylphytosphingosine(taps), which is a precursor of sphingolipid, could be used for the production of pharmaceuticals and cosmetics. we isolated sam gene from p. ciferrii and cloned it into expression vector for e. coli and p. pastoris, respectively. an . kb sam-s gene fragment was isolated by low-strigency pcr using degenerated primer. by the analysed primary sequence deduced from dna sequence, this gene included conserved domains similar with other well-known sam synthetase. first of all, sam synthetase gene cloned pgem-t vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. typical characteristic analysis of this enzyme is underway. metabolic networks offer a large variety of different synthesis pathways starting from cheap substrates and leading to interesting high-value compounds, i.e. metabolites. in case an interesting pathway can be disconnected from the remaining metabolic network, the perforated cell or the crude extract could be used for a one-pot multi-step synthesis of the desired compound. pathway isolation, achieved by deletion of genes encoding gene products enabling side reactions, interferes with the viability of the organism, which is a requisite for the production of the system of biotransformation (sbt). in this work, a rational systems biology-derived approach is presented for the design of a sbt. it is illustrated for a sbt allowing for production of dihydroxyacetone phosphate. the design procedure comprises three steps: (i) a production pathway is identified in the metabolic network of e. coli. the e. coli pathway is complemented by two additional enzymes in order to obtain a fully energy and redox balanced production pathway. (ii) an optimal combination of gene knockouts is designed and a suitable growth medium composition is identified, both by a model-based approach: flux balance analysis of a genome-scale metabolic network is used to predict enzyme expression within the wild-type organism on different media, while a mixed-integer optimisation is employed to identify viable mutants as this approach strongly depends on the quality of the fba prediction, available regulatory information on the usage of metabolic pathways and thermodynamic constraints were taken into account. (iii) thermodynamic analysis of the obtained, partially branched sbt reaction cascade revealed the extent of loss in yield by the remaining side reactions. in summary, this systems biology-driven approach potentially enables the substitution of a elaborative multi-step synthesis process by a one-pot enzyme reaction cascade. institute of industrial biotechnology, inha university, incheon - , korea. e-mail: leecg@inha.ac.kr (c.-g. lee) algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. currently, its application is being extended to the areas of wastewater treatment and agriculture. however, lack of suitable photobioreactors (pbrs) makes the cost of algally-derived compounds higher than those derived by chemical synthesis and thus has prevented widespread use of algal cultures. the culture of algae prior to the late s was apparently restricted to laboratory scale operations. experiments on outdoor algal mass production began in the late s with nearly concurrent development of experimental culture facilities in germany and the united states. for the next two decades, outdoor mass culture of algae was undoubtedly the hottest topic in the algal biotechnology area. recent developments of high-density pbrs enable the production of valuable biologically active compounds by algal mass cultures. however, light is almost always the limiting factor in high-density photobioreactors. key factors for successful photobioreactors will be discussed and various photobioreactors will be analyzed and compared for their advantages and disadvantages. the new techniques, such as pigment redcution and application of flashing light and lumostatic operation, will be discussed for possible solutions to overcome the light limitation in high-density microalgal cultures. a quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host. southern analysis is laborious and time consuming. several colony hybridisation methods have been developed for the analysis of a large number of transformants. unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (van zeijl et al., ) . recently, methods for pcr-based analysis of fungal transformants have been described. most of these methods require high quality dna. many methods for dna extraction from fungi have been described in the past few years. these methods often are tedious, time consuming, costly or limited to a small number of samples each time. most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (cassago et al., ) . lately a few methods have been described to isolate dna from fungi suitable exclusively for pcr and appropriate for the simultaneous treatment of a large number of samples. some methods also describe the direct use of mycelium (cooke et al., ) in the pcr-reaction mixture. we compared the applicability of a few rapid dna extraction methods for myrothecium gramineum and tested the resulting dna samples on there suitability for pcr-applications. myrothecium gramineum is a filamentous ascomycete used in ongoing research as a new cloning and expression host. five methods were tested. in four of these methods dna was extracted from mycelium (goodwin et al., and aljanabi et al., ) or spores (ferreira et al., and xu et al., ) prior to pcr. a fifth assay used mycelium straight in the pcr-reaction mixture. only this last method seemed useful for myrothecium to isolate dna suitable for pcr. fragments up to bp were amplified. cheese whey is a liquid effluent from cheese-making processes. there is an increased interest in the economic utilization of whey produced by the dairy industries, because the whey is a pollutant, due mainly to its lactose content. the goal of this work was to find the most suitable values of some fermentation parameters for lactic acid production from whey by a lactic acid bacterium, lactobacillus helveticus (atcc ). the effects of lactose content, temperature, ph and the supplementation with yeast extract were investigated using surface response methodology. a composite central design was used with three center points, making a total of operational conditions. the region of maximum production is outlined by the following intervals: temperature around • c; lactose concentration between and g/l; concentration of yeast extract between and g/l; ph between and . . fermentation studies on a continuous fermentative process coupled to a vacuum flash evaporator were carried out in lab scale equipment. the phases of this work consisted in an assembly and instrumentation of the prototype and elaboration of a supervisory system coded in labview . , which allows the data acquisition and control through personal computers. the experiments in continuous fermentation used saccharomyces cerevisiae and sugar cane molasses as substrate. the analytical follow up was done through analysis of total reducing sugars, ethanol, glycerol, dry mass and viable cells. the system worked for months uninterruptedly, producing an alcoholic solution at the condenser with • gl. the fermentation operated with concentrations of ethanol at • gl, which is a weakly inhibitory value for the yeast of the process, even when fed with concentrated cane molasses, containing up to g/l of sugar. the result meets the initial goal, which was to operate the system with low level of ethanol and to guarantee high productivity, even in high concentrations of sugar in the feeding. the results showed that system productivity was superior to that of the conventional continuous process. lactic acid (la) is a versatile chemical, used as an acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, and for production of biodegradable poly lactic acid (pla). l(+)lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. in this research, lactic acid production was improved on l fermentor. in our experience, among six strains of lactobacillus were examined for the production of l(+) lactic acid, lactobacillus casei ssp. casei atcc was selected as a highest l(+) lactic acid producer. optimized medium used for lactic acid production contained (per l) g glucose, g whey powder and g corn steep powder. for a homofermentative process, ph . was found to be optimal. in order to avoid product inhibition, the produced lactic acid was neutralized using calcium hydroxide. maximum production and productivity of lactic acid in batch system, were g and . g/lh, but in fed batch system, after feeds of glucose, production and productivity increased up to g and g/lh. saleh a. mohamed molecular biology dept., national research centre, cairo, egypt an extracellular polygalacturonase (pgii) from trichoderma harzianum was purified to homogeneity by two chromatography steps using deae-sepharose and sephacryl s- . the molecular weight of t. harzianum pgii was , da by gel filtration and sds-page. pgii had isoelectric point of . and optimum ph of . . pgii was very stable at the ph . . the extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (de). pgii had very low activity toward nonpectic polysaccharides. the apparent km value and kcat value for hydrolyzing polygalacturonic acid (pga) were . mg/ml and s − , respectively. pgii was found to have temperature optimum at • c and was approximately stable up to • c for min of incubation. all the examined metal cations showed inhibitory effects on the enzyme activity. , phenanthroline, tween , tween , triton x- and sds had no effect on the enzyme activity. the rate of enzyme catalyzed reduction of viscosity of solutions of pga or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. the storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to one year. these properties of t. harzianum pgii with appreciable activity would be potentially novel source of enzyme for food processing. tarek m. mohamed, biochemistry division, chemistry department, tanta university, tanta, egypt preparation of peroxidase from horseradish, which could be used for commercial applications such as diagnostic kits, was occurred through a simple reproducible method consisting of extraction, ammonium sulphate precipitation, filtration through non-binding protein filter and lyophilization. the purification method was developed allow the preparation of mg of enzyme from kg of horseradish roots. the one mg of enzyme contains units of peroxidase. this value is similar to that produced by sigma ( - unit mg − powder). the final preparation is salt free reddish brown powder with free ammonia content less than . g − units. the rz value (a /a ) of the enzyme, which is a good criterion of purity and heme content, was . . the lyophilized enzyme was stable at − • c for at least one year. the liquid form of the enzyme in presence of . % sodium azide was stable up to days at • c, while it lost most of activity at room temperature in the same period. the properties of horseradish peroxidase including km, optimal ph and temperature, activation energy, thermal stability and effect of different compounds were studied. the applicability of this enzyme in determination of serum glucose was performed. the analysis of glucose in human sera gave results using the kit containing the prepared peroxidase similar to those obtained with a commercial glucose kit. lactobionic acid production using lactose oxidase: from laboratory to l scale mikkel nordkvist , per munk nielsen , peter budtz , john villadsen : center for microbial biotechnology, technical university of denmark, dk- lyngby, denmark; novozymes a/s, dk- bagsvaerd, denmark; chr. hansen a/s, dk- hørsholm, denmark. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) currently, lactobionic acid is mainly a high-price specialty product used, e.g. in solutions for organ stabilization. however, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology. with lower production costs it has the potential to become a bulk chemical. the kinetics for the oxidation of lactose to lactobionic acid by a new carbohydrate oxidase was studied in a l bio-reactor with control of ph, temperature, and dissolved oxygen. the byproduct hydrogen peroxide has a negative influence on the lactose oxidase enzyme, and hence additional experiments were made with addition of catalase to remove hydrogen peroxide, thereby also providing extra oxygen. on the basis of the experiments in l scale, experiments were performed in a l reactor equipped with a new system for dispersion of air to supply the necessary oxygen for the oxidation. the aeration system in the large scale reactor was able to supply oxygen sufficiently fast to give the same production rate, at low values of the air flow rate and the energy input, as was obtained in the high-performance laboratory reactor. the non-characterized gene previously proposed as d-tagatose -epimerase from agrobacterium tumefaciens was cloned and expressed in escherichia coli. the expressed enzyme was purified by affinity chromatography on histrap hp, desalting chromatography on hiprep / , and gel filtration chromatography on sephacryl s- hr with a final specific activity of . u/mg. using maldi-tof-ms, the native protein was estimated to have a molecular mass of , da and a monomeric structure. the purified enzyme exhibited maximal activity at • c and ph . without the addition of metal ions and at • c and ph . with . mm mn + . among various metal ions, mn + was the most effective divalent cation for d-fructose epimerization activity. the addition of mn + significantly increased the thermal stability and the epimerization activity with other ketoses such as d-psicose, d-tagatose, d-ribulose, d-sorbose, and d-xylulose. the activity, substrate affinity, maximum velocity, and catalytic efficiency (k cat /k m ) of the enzyme for d-psicose were higher than those for d-tagatose, which suggests that the enzyme is not d-tagatose -epimerase but d-psicose -epimerase. the equilibrium ratio between d-psicose and d-fructose was : at • c with . mm mn + . when the enzyme was used at u/ml, dpsicose was produced at g/l from g/l d-fructose containing mm mn + after min, corresponding to a conversion yield of . %. the role of ammonium ions in glucosamine formation during the citric acid fermentation process by aspergillus niger m. papagianni , f. wayman , m. mattey : department of hygiene and technology of food of animal origin, school of veterinary medicine, university of thessaloniki, thessaloniki , greece; department of bioscience, university of strathclyde, glasgow, g xw, uk. e-mail: mp @vet.auth.gr (m. papagianni) stoichiometric modeling of the citric acid fermentation process by aspergillus niger, in -l stirred tank reactor, indicates that nh + ions combine with a c-containing metabolite inside the cell to form a nitrogen compound which is then excreted by the mycelium. the close correlation between calculated and experimental profiles indicates that this metabolic process is rapid and takes place before the c-structure of the glucose has been greatly altered by glycolysis or the pentose phosphate pathway. hplc analysis identified glucosamine as the product of this relationship. a clear effect of medium concentration of nh + on glucosamine formation was observed when fermentations carried out with optimal and sub-optimal ammonium concentrations. (nh ) so addition when medium nitrogen was depleted, enhanced the formation of new cells from the tips of fragmented hyphae and led to glucosamine accumulation in amounts depending to pulse concentration. the fungus reacts in excess ammonium by converting it to glucosamine, to be utilized later when a regeneration process takes place with fragmentation of vacuolated hyphae and subsequent regrowth, depending always on the culturing conditions. however, depending on carbon and ammonium concentration in medium, glucosamine can be secreted in concentrations as high as g/l. about microorganisms originated from traditional korean food origin were screened for efficient palatinose production. an isolate designated fmb was exceptionally efficient in sucrose-palatinose conversion activity. conversion of sucrose into palatinose by fmb was much faster than a reference strain of erwinia rhapontici. fmb is a gram negative, facultatively anaerobic, motile, noncapsulate, and straight rod-shaped bacterium producing acid from glucose. based on api and s rdna analyses, fmb was determined to be enterobacter sp. the maximum conversion of % sucrose to palatinose and trehalulose by enterobacter sp. fmb was achieved within h. the preliminary dna sequencing result of the gene corresponding to sucrose isomerase of enterobacter sp. fmb revealed that it showed % similarity to that of klebsiella sp. (??). within the scope of an r&d project developed in collaboration with leather tanning portuguese industrial partners a screening of new proteases to be used in the industrial process was performed. a bacillus subtilis strain isolated from alkaline spent purge liquor was shown to be a promising protease producer. microorganism growth was studied for optimisation of temperature, agitation, ph and medium composition either for biomass or proteases production. optimal growth temperature is different for maximum biomass growth ( • c) and optimal proteolytic activity ( • c) yielding biomass specific growth rate of . and . h − , respectively. the achieved proteolytic activities were . and . u/ml of protease, respectively. optimised medium composition ( g/l beef extract, g/l yeast extract, g/l peptone and . g/l cacl ) yielded a specific growth rate of . h − and . ku/l of protease, in shake flask experiments. bioreactor experiments (from to l) with the selected medium were performed at • c in order to test aeration rate ( and vvm), stirring ( - rpm) and ph (uncontrolled, controlled at and ). best protease activity was u/ml in l bioreactor without ph control at rpm and vvm. the proteolytic extract was characterized and compared to commercial bates. results indicate that these proteases can be employed in the purge phase of the leather tanning process in industry. the gram positive bacterium bacillus megaterium is known for its capacity to produce exoenzymes including amylases, proteinases, and penicillin amidase at industrial scale. here, we describe the development of various vectors for the production and export of recombinant heterologous proteins employing b. megaterium signal peptides. the target gene can be cloned directly adjacent to the signal peptide coding sequence (bart et al., ) . this arrangement allows for a correct n-terminal sequence of the mature protein after processing by the signal peptidase sipm. using this newly developed protein production and export system lactobacillus reuteri levansucrase (van hijum et al., ) was secreted in significant amounts (∼ mg/l) into the growth medium. fusion of the recombinant levansucrase to affinity tags allowed one-step purification of the recombinant protein from the growth medium. however, fused peptide tags resulted in a decreased secretion of the fusion protein. . mg his -tagged levan-sucrase were purified per litre of culture. the system was further enhanced via coexpression of a gene for the signal peptidase sipm (malten et al., a) and deletion of the gene for the extracellular protease nprm. developed new tools allow for various strategies of integrated high level production, export and purification of heterologous proteins in b. megaterium. methods for high-throughput screening of secreted enzymes are under development. the determined sequence of the b. megaterium genome, studies using high-cell density cultivations (malten et al., b) and proteome data from batch fermentations implicate new targets for directed genetic optimization of b. megaterium production and secretion strains. novel strong and inducible promoters are currently under investigation. toru matsui , naoya shinzato , hisashi saeki , hitoshi matsuda : center of molecular biosciences, university of the ryukyus, okinawa - , japan; japan energy co., japan. e-mail: tmatsui@comb.u-ryukyu.ac.jp (t. matsui) optically active epoxides are considered as the potential intermediate for chiral drugs synthesis. although s-styrene oxide (so) have been extensively investigated using styrene monooxygenase from pseudomonas sp., microbial production of r-so with high enantiomeric excess was hardly examined. in this study, r-so producing bacteria from styrene was screened using various alkene assimilating bacteria. r-so with the highest ee (ca. %ee) was obtained using ethene utilizing bacteria, identified as mycobacteirum sp., while produced relatively lower at around %ee when using propene utilizing bacteria. the alkene monooxygenase gene homologue sequence amplified from the genomic dna revealed a significant similarity to that of etnabc. these bacteria also showed stereoselective degradation of racemic so, suggesting that the produced so might be further stereoselectively degraded to increase the ee. the ethene utilizing bacteira produced not only r-so but also s-epichrolhydrin at high ee.when using arylchloride as the substrate. this research was supported by nagase science and technology foundation. the secretion efficiency of the escherichia coli sec pathway is dependent on the growth phase but not on protein size f.j.m. mergulhão, g.a. monteiro centro de engenharia biológica e química, instituto superior técnico, av. rovisco pais, - lisbon, portugal. e-mail: filipem@alfa.ist.utl.pt (f. mergulhão) the secretion efficiency of the escherichia coli sec pathway was evaluated through the expression of green fluorescent protein and human proinsulin fusion proteins. translocation to the periplasm is dependent on the growth phase of the bacterial culture and the highest secretion efficiency is attained in mid-exponential phase. secretion performance is independent of protein size ( - kda) and even when the amino acid composition of the secreted proteins is very similar, the amino acid distribution within the protein can affect translocation. in silico prediction analysis suggests that proteins that are prone to form ␣-helix structures are more efficiently translocated. culture medium composition plays an important role on secretion performance with the highest secretion results being obtained in minimal medium. streptokinase is a common fibrinolytic drug. that is used in thrombolytic therapy for long time. to compare with another thermbolytic drugs like tpa, it has lot of advantages. in this present research dna was extracted from s. equisimilis h a for the first time in iran. streptokinase gene was amplified by using two forward primers and one reverse primer. a common restriction enzyme, bamh-i, was used for cloning. both ends of the pcr products (full length: bp and mature section: bp) and the restriction site on mcs of pqe- vector were digested. in this study, pqe- expression vector was used with high level expression ability for production of recombinant fusion streptokinase with simplifying the purification by employing affinity-metal chromatography method. in addition, the cloning results were controlled by double digestion and sequencing. takesono oxidation of short-chain iso-alkanes was studied with propanegrown resting mycelia of scedsporium sp. a- . isobutane was oxidized to tert-butanol, but not to isobutanol. isobutanol was used for growth, but both isobutene and tert-butanol were not used for growth. isopentane was oxidized to -methyl- -butanol, -methyl- -butanol, and -methyl- -butanol but not to -methyl- -butanol. -methylpentane was oxidized to -methyl- -pentanol, -methyl- pentanol, and -methyl- -pentanol but not to -methyl- -pentanol or -methyl- -pentanol. -methylpentane was not oxidized. oxidation of branched alcohols was also studied. application of nadph-dependent . -diketo-gluconic acid reductase for production of l-ascorbic acid claudia pacher , : division of food biotechnology, department of food sciences and technology, boku, university of natural resources and applied life sciences, muthgasse , a- vienna, austria; research centre applied biocatalysis, petersgasse , a- graz, austria. e-mail: claudia.pacher@boku.ac.at ascorbic acid is an organic acid with various applications in the food and pharmaceutical industries. at present, the majority of commercially manufactured vitamin c is synthesized via the reichstein process, which is highly energy-consuming, involves considerable quantities of organic solvents and gives an overall yield of about %. therefore, during the past decades a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic or fermentative means, which show some advantages regarding costs and environmental friendliness. one of the fermentation routes runs via . -diketo-d-gluconic acid ( . -kdg), produced by pectobacter (erwinia) cypripedii. this compound has been reduced by a nadph-dependent . -diketogluconic acid reductase (dkr) from corynebacterium glutamicum to the key intermediate -keto-l-gulonic acid ( -klg) before chemical rearrangement leads to the final product. for economical reasons we wanted to express dkr heterologously. based on our long term experience with coenzyme regeneration we wanted also to perform the reaction in homogeneous solution. the spent coenzyme of nadph-dependent dkr has been regenerated by a second isolated nadp-dependent enzyme like glucose dehydrogenase. we describe here the recombinant production, purification and characterization of dkr and the results of enzymatic -klg formation by using the recombinant enzyme in a homogeneous system with conjugated coenzyme regeneration. grp residing in endoplasmic reticulum (er) functions as a molecular chaperon by associating transiently with incipient proteins as they traverse the er and aiding in their folding and transport. furthermore, the protein can also be induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, blockage of vesicular trafficking by brefeldin a and er-calcium-atpase pump inhibition by thapsigargin. thus, substances that directly down and up-regulate grp transcription are expected to be useful for treatment of cancer and alzheimer's disease, respectively. in the course of our screening program to obtain substances, which regulate grp expression, we first constructed an assay system monitored by the expression of a reporter gene. hela cells, which are transformed with luciferase gene under the control of grp promoter designated as hela c cells, respond sensitively to luciferase grp induction by er stress such as treatment of tunicamycin. by using this screening system, we isolated pyrisulfoxin as an up-regulator of grp , and valinomycin, citreoviridin and alternariol as down-regulators. detailed studies on other biological activities were now under way. pdh is an enzyme that was described only several years ago in a number of ecologically related litter-decomposing fungi (agaricales, gasteromycetales). it catalyzes the c- and/or c- oxidation of several aldopyranoses to the respective keto sugar derivates. pdh shows a very broad substrate range, oxidizing almost all major sugar components of wood polysaccharides, and is implicated to play a role in lignocellulose degradation. agaricus bisporus, the white button mushroom, is an economically significant agricultural crop. the cultivation, which is done by solid-substrate fermentation on straw-and hay-based composted substrate, is sometimes seen as one of only few economically feasible methods for bioconversion of lignocellulosic agricultural waste material. deeper insight in the physiological role of pdh may provide help for mushroom growers to increase yield, improve quality or make new sources of raw materials utilizable. we amplified a fragment of the pdh gene with degenerated primers derived from internal peptide sequences. the screening of a genomic library led to the isolation of the pdh gene. subsequently we amplified a cdna clone by rt-pcr and investigated the transcriptional regulation by different carbon sources on a defined minimal medium. optimization of monoclonal antibody production processes with simulation and scheduling tools demetri petrides, charles siletti intelligen inc., scotch plains, nj , usa. e-mail: dpetrides@intelligen.com (d. petrides) this presentation will review the state of the art in batch process simulation and scheduling tools and their applications in the design and debottlenecking of integrated biopharmaceutical processes. a systematic methodology will be presented for identifying and eliminating size, time, and throughput bottlenecks that limit production in single and multi-product facilities. the methodology will be illustrated with an industrial case study dealing with the optimization of a multi-product facility that produces therapeutic monoclonal antibodies (mabs). mab processes are characterized by a long bio-reaction time (e.g., . - weeks for fed-batch operation and - months for perfusion operation). the cycle time for processing a lot in the recovery and purification train typically takes - days. consequently, one way of increasing throughput is by installing extra bioreactors that operate in staggered mode and utilize the same recovery train. the result is that multiple batches may be at different stages of completion at any given time. since cleaning equipment (e.g., cip skids) and buffer preparation and holding tanks are shared by multiple steps across many batches, this type of operation leads to time/scheduling bottlenecks that constrain the cycle time and the throughput of a process. the problem becomes more challenging in the context of multi-product facilities and when constraints imposed by the limited availability of resources are considered. our methodology and its computer implementation will illustrate how to systematically identify and eliminate such bottlenecks. the industrial case study will provide a real world example of the methodology. application of two stage continuous cultures of aspergillus niger for citric acid biosynthesis jerzy j. pietkiewicz, malgorzata janczar, wladyslaw lesniak food biotechnology department, university of economics, wroclaw - , poland. e-mail: jerzy.pietkiewicz@ae.wroc.pl (j.j. pietkiewicz) the aim of the work was application test of submerged two stage single stream continuous cultures of aspergillus niger for citric acid production from sucrose. studies were carried out in lab fermenters with working volume of dm . the bioreactors were standard cstrs. in two stage continuous cultures (tscc) high mycelium growth and high citric acid production were observed in the first bioreactor. there was almost four times lower growth of biomass rate and about three times lower citric acid production rate in the second bioreactor. studies on influence of dilution rate in race from . to . dm /(dm h) on course and efficiency of tscc showed, that the highest citric acid yield (y p/s = . %), high volumetric rate of its production (r pc = . g/(dm h)) and the highest biosynthesis efficiency coefficient (k ef = . ) were obtained with dilution rate d = . dm /(dm h). there was also high citric acid concentration (p = . g/dm ) and low residual sugar concentration (s k = . g/dm ) in the medium flowing out the second bioreactor in those cultures. the beta-galactosidases in commercial use are of different origins and yeast and fungal lactases present the greatest interest. the yeast lactases present neutral optima ph and are suitable for the hydrolysis of lactose in milk. in this work, the aim was to study the influence of aeration in the production of beta-galactosidase in batch fermentations with kluyveromyces marxianus atcc . the medium composition for culture was as follows (in g/l): lactose pa , yeast extract , (nh ) so and kh po . the fermentations was carried out at • c, ph . , at rpm starting with an initial cellular concentration of × cels/ml, with different aeration rates. the cells were disruped with chloroform % (v/v) as solvent. the enzymatic activity was determined as initial rate of lactose hydrolysis at defined conditions. the studies have revealed the importance of aeration on kluyveromyces marxianus in the growth and beta-galactosidase synthesis. the enzymatic activity of fermented medium with . vvm was % higher than one without aeration. furthermore, the cellular growth was faster in the aerobic fermentation than in the anaerobic one. the aeration has taken an important place in the enzymatic synthesis and in the cellular growth, however the results have shown that the aeration rate increase of . - . vvm has not implied a increase in the cellular growth neither in the enzymatic reached activity. the lactose presents in the milk has a solubility of only % at • c, and a high percentage of the world population presents intolerance to this sugar, due to the low or absence of the activity of the lactase enzyme in the organism. to minimize such problems, the most viable alternative for nourishing dairy products is the enzymatic hydrolysis of milk, although it is an expensive process due to the high cost of the beta-galactosidase enzyme. an alternative that has been greatly studied is the immobilization of this enzyme, originated from many different sources. there are several immobilization procedures for this enzyme, however, a procedure considered ideal was not obtained yet. the objective of this work was to study the immobilization process of beta-galactosidase from aspegillus oryzae in sodium alginate with commercial gelatin. in the immobilization process was studied the glutaraldehyde influence for , and %, in the presence of commercial gelatin at the concentration of % at the immobilization medium. the activities of the immobilized enzymes were obtained in a stirred micro-reactor, at the temperature • c, ph . with a gl − lactose solution in acetate buffer. the experimental results showed that the immobilized biocatalyst that presented the larger initial activity was the one obtained at the immobiliza-tion medium that contained % of glutaraldehyde. after daily determinations of enzymatic activities, a fall of , and % was verified in the enzymatic activities for the immobilized biocatalysts using glutaraldehyde at , and %, respectively, however, in all cases, the enzymatic activity reached the half of their initial activity after determinations. hydrolysis of sucrose by immobilized beta-fructofuranosidase in silica eloízio júlio ribeiro, ubirajara coutinho filho faculdade de engenharia química, universidade federal de uberlândia, uberlândia - , brazil. e-mail: ejribeiro@ufu.br (e.j. invertase, known as beta-fructofuranosidase (ec . . . ), plays a catalytic role in the conversion of sucrose into glucose and fructose. it is largely used in the food industry to prevent the crystallization of sucrose in sugar mixtures and can be used in enzyme reactors for hydrolysis of sucrose. the objective of this work was to study the kinetic of sucrose hydrolysis by immobilized betafructofuranosidase in a continuous recirculation reactor, evaluated the enzyme stability and determine the effective half-life of immobilized enzyme. invertase was covalently immobilized on sillanized controlled pore silica. nonlinear fitting were used to determine the kinetic parameters for substrate and product inhibition observed in the enzymatic hydrolysis of sucrose. the kinetics studies of immobilized invertase were carried out in a continuous recirculating reactor. the half-time of enzyme inactivation (t / ) was calculated from the initial rates of the remaining enzyme activity. the model of inhibition by substrate and product adequately represented the enzymatic hydrolysis. the fructose effect was competitive inhibition (k f = . . − mol/ml) and the glucose effect was noncompetitive inhibition (k g = . . − mol/ml). the effective diffusivity of sucrose into the support was shown to be the same as for sucrose in dilute solution ( . × − cm /s at • c). the half-time of enzyme inactivation (t / ) was h. controlled pore silica showed to be an excellent immobilizing support. the immobilized invertase was very stable at temperatures lower than • c. the intrinsic parameters (k i , k f , k g and v m ) were shown to be similar to the apparent values. the low permeability of mycobacterial cell wall envelopes is a result of the unique composition and organization of the cell wall lipids. the permeability of mycobacterial cell wall can be changed by means of partial disintegration of its compounds. the aim of present work was to characterize the changes in the cell wall skeleton (cws) and non covalently bound free lipids under the influence of isoniazid, the inhibitor of mycolic acids biosynthesis. fatty acid (fames) and mycolic acid methyl esters (mames) obtained from all tested preparations were analyzed by gc/ms analysis. the analysis of free lipids and cws revealed distinct changes in the composition of the frac-tions obtained from the cells exposed to action of the isoniazid. the changes in the quantity of fatty acids in the inh-treated cells indicates that inh interferes with the synthesis of lipidic compounds of the mycobacterial cell wall also. the decreased amount of covalently bound mycolic acids in the cws is responsible for the enhanced penetration of hydrophobic compounds through the cell wall. this work was supported by grant nr p c of the committee for scientific research. barbara sencio, teresa jamroz, stanislaw ledakowicz department of bioprocess engineering, technical university, lodz, poland the enzyme laccase (ec. . . . . p-diphenol oxidase) is a subject of research in many centres dealing with improvement of bioprocesses with the use of different white rot fungi species. most strains that produce this enzyme in vitro require inductors initiating its biosynthesis. when cerrena unicolor was applied, it was found that the strain produced laccase very efficiently without additional toxic compounds. to specify optimum conditions for laccase production in a submerged culture, research was undertaken to obtain the most efficient inoculum c. unicolor. the goal of this research was to determine the effect of form and incubation time of inoculum on enzymatic activity of the laccase producing strain. the experimental inoculum was the mycelium prepared on a solid and liquid substrate. basing on results obtained, it was found that the laccase yield was the highest in the cultures where the mycelium was grown on a solid substrate. maximum activity of the c. unicolor strain was achieved on the th day of culture, and the amount of laccase produced was higher by, ca. % as compared to the mycelium obtained from the liquid substrate. results of these experiments were used to continue studies on the impact of inoculum age. experiments were carried out using an inoculum incubated for - weeks at the temperature • c in a certomat bs shaker at rpm. the best results in the c. unicolor strain culture were achieved using a -day-old inoculum. effect of alcohol treatment on hydrolytic activity of candida rugosa lipase serpil takaç, a. ezgiÜnlü department of chemical engineering, institute of biotechnology, ankara university, tandogan, ankara, turkey candida rugosa lipase (crl) was treated with , , and % concentrations of methanol (m), ethanol (e), -propanol ( p) and -butanol ( b) to investigate the changes in its hydrolytic activity toward p-nitrophenylacetate. the treatment included the following steps at + • c: (i) stirring crl with phosphate buffer for h; (ii) treating the solutions with alcohols; (iii) stirring treated-crl for h; (iv) centrifugation at , rpm for min; (iv) dialysis the supernatant against bidistilled water for h. the activity of crls was followed for h at • c in the presence and absence of isooctane. the enzyme activity was measured spectrophotometrically and the protein concentration was measured by lowry's method. it was found that the recovered protein did not change considerably with the type of alcohol; however, decreased with alcohol concentration. in the presence of isooctane, specific activities of the untreated and treated-crls increased compared with those obtained in the absence of isooctane. b-crls and e-crls showed higher activities than m-crls and p-crls whereas untreated-crl exhibited higher activity than m-crls, e-crls and p-crls. the highest and the lowest activities were obtained with % b-crl and % p-crl, respectively. the changes occur in the structure of crl after treatments were investigated by electrophoretic analysis. this study was supported by ankara university biotechnology institute (project no: ) . different genera, species and strains of microorganisms were found to posses different cryoresistance. optimal ways for cryopreservation of microorganisms-producers of antibiotics, microorganisms, used in food industry, agriculture and veterinary have been developed. it was demonstrated, that non-lethal damages could occur in cryopreserved microorganisms after their returning to physiological culture conditions, which were manifested in streptomyces' hypha fragmentation, that of cyanobacteria's, streptococci's chains as a result there was an increase in a number of colony-forming units, a reversible inhibition of microorganisms' proliferative activity in bacillus thuringiensis and lactic streptococci, stimulation of the enzyme processes and antibiotic production. non-lethal damages are repaired during microorganism culturing in the first passage. the cause of non-lethal damages is a reversible inhibition of biosyntheses of protein and nucleic acids respiratory activity. the repairing of non-lethal damages is accompanied by the production of stressproteins, different from heat shock proteins. effect of ph in the -propanol treatment of candida rugosa lipase on its enantioselectivity in the hydrolysis of racemic naproxen methyl ester serpil takaç, a. ezgiÜnlü department of chemical engineering, ankara university, tandogan, ankara, turkey candida rugosa lipase (crl) was treated with -propanol ( p) at the ph values of . , , , . , and to investigate the changes in its enantioselectivity in the hydrolysis of racemic naproxen methyl ester. the treatment included the following steps at + • c: (i) stirring crl with different buffer solutions to maintain the desired ph values for h; (ii) treating the solutions with % p; (iii) stirring treated-crls for h; (iv) centrifugation at , rpm for min; (iv) dialysis the supernatant against bidistilled water for h. hydrolyses of racemic naproxen methyl ester to form s-naproxen were performed in shaking flasks at rpm and • c for h in isooctane-phosphate buffer solution biphasic system using treated-crls with the activity of . u. the concentrations of the enantiomers of naproxen methyl ester and naproxen were determined with hplc. it was found that the treatment ph has an important role on the enantioselectivity and conversion. the highest enantiomeric excess for the substrate, for the product, enantiomeric ratio, and con-version were obtained with crl treated at ph . as , , and %, respectively. these values were followed with p treated crl at ph as , , and %. however, lower enantiomeric excesses, conversions and enantiomeric ratios were obtained at the treatment ph values between . and . the effects of fatty acids, nitrogen (as nh no ), phosphorus (as kh po ), ph value, manganese (mn + ), iron (fe + ) and methanol concentration on growth and production of oxalic acid from post refining fatty acids by a mutant of aspergillus niger xp in submerged fermentation experiments was studied. of the a. niger strains screened, a. niger xp was identified as the best oxalate producer on lipids. the influence of the ph on oxalic acid formation shows that the maximum production rate and higher concentration of product are observed at the ph ranging from to . with a medium containing g fatty acids/l, the production reached a maximum of g oxalic acid/l after days. the addition of . % (w/v) methanol to seed culture increased the product yield and concentration of oxalic acid but decreased the amount of an undesired by-product (citric acid). under this condition, the maximum oxalate productivity ( - g/l days) was maintained for - days of fermentation. other results of the experiments show that supplementation of the production medium with manganese and iron enhances oxalate production. fatty acids proved to be a very good substrate for oxalic acid production by a. niger xp giving excellent yields and productivity at low ph. the results are very promising as they may lead to cheap alternative processes for oxalic acid production from renewable lipid resources. department of food engineering, middle east technical university, ankara , turkey. e-mail: banuy@metu.edu.tr (b. yalcindag) laccase (e.c. . . . , p-benzenediol:oxygen oxidoreductase), which is an enzyme belonging to the multi-copper oxidase family, catalyzes the oxidation of a broad variety of polyphenols with a preference for p-isomers, which are converted to p-quinones. fungi generally contain several laccases which have been found to be involved in delignification, melanin synthesis and pathogenesis. laccase has also important potential application areas especially in food and chemical industries. after aspergillus fumigatus genome data were released, research on functional analysis of laccases has been initiated in our laboratory. laccase genes of aspergillus nidulans, ya and tila, and laccase and multi-copper oxidase genes of aspergillus fumigatus, abr and abr , were used to analyze a. fumigatus genome for laccases. this sequence analysis resulted in probable laccase genes, one of which was the previously cloned abr gene. in this study, one of these genes (aflac ) was further characterized. after sequence alignment and characterization studies, aflac was predicted to have bp having six introns, which makes the protein amino acid long, and the predicted protein sequence showed % homology with the dihydrogeodin oxidase of aspergillus terreus and % homology to the laccase of botryotinia fuckeliana. aflac gene is found within an uncharacterized gene cluster containing genes with homology to glutathione-s-transferase, polyketide synthase, o-methyl transferase, and others. the information obtained from sequence analysis was employed in designing pcr-primers to amplify the aflac gene, followed by cloning onto pan - and pan - vectors for heterelogous expression in aspergillus sojae. in addition, by the use of rt-pcr, aflac cdna will be cloned and expressed in escherichia coli. furthermore, gene silencing studies will be performed to enlighten the function of aflac and associated gene cluster. stability of growth rate of photosynthetic cells is an important factor in designing of effective photobioreactors especially in long term operations. in our experiments, in order to keep operational parameters almost constant, a semi-continuous culture method was developed. in this method, a part of culture broth containing grown cells was repeatedly replaced by fresh medium at a predetermined time interval. the replacement of broth with fresh media could keep the cell concentration, volume of broth and distribution of light intensity constant at initial values throughout the cultivation. it was shown that in one side illumination with a halogen lamp, if the ratio of the light intensity at the front side of a flat plate photobioreactor to that at the rear side was kept lower than , the growth rates was sustained in constant levels. however, at higher ratios the growth was followed by rapid decrease after - h. supplemental illumination with a fluorescent lamp from the rear side of the flat plate photobioreactor could sustain almost stable growth rate. beside of the illumination conditions, increased ferrous ion concentrations in medium could keep the stability of growth rate even in unstable illumination conditions, while consumed ferrous ion was slight. glutathione (gsh) plays a pivotal role in protecting cells from by-products generated by oxidative metabolism. these characteristics make this active tripeptide an important drug for the treatment of liver diseases and is of interest in the food additive industry, therapeutics and sport nutrition. in the first part of the research, a screening was carried out among yeast strains, to find out those able to accumulate higher gsh intracellular levels. two saccharomyces cerevisiae strains proved to be the best gsh producers ( . %dw), in every samples the presence of s-adenosyl-methionine in traces ( . % dw) was also evidenced. s-adenosyl-methionine (sam) plays a role in the immune system, maintains cell membranes, participates in detoxification reactions and in the manufacture of brain chemicals and cartilage. the second part of the research was aimed at increasing, in a post fermentative procedure, gsh levels present inside the cells at the end of the growth phase. moreover, time course of sam intracellular levels, to be related with accumulated gsh, was also monitored. cells were then suspended in an appropriate solution containing mineral salts, glucose and the aminoacids, precursors of the two studied molecules. according to this procedure, gsh intracellular levels reached . % dw after h incubation. moreover, gsh levels can be related to sam production (up to . % dw). the presence, in several samples, of intermediate metabolites, such as cystathionine and omocysteine, proved the establishement of an intracellular equilibrium between gsh and sam; this behaviour represents a promising starting point for the set-up of a microbial process for the simultaneous production of the two studied molecules. three acetate mutants of y. lipolytica yeasts, which varied in colony morphology (rough and smooth), were employed for continuous citric acid production from glucose and fructose syrup in a membrane reactor with cell recycle. the strains were compared for their product yields, specific acid production rates and ratios of citric acid to isocitric acid. experiments shoved that glucose syrup was a better substrate for citric acid production by y. lipolytica. citric acid concentration in the effluent ranged from to g/l, depending on the yeast strain used. all y. lipolytica strains produced very low amounts of isocitric acid. its concentration did not exceed g/l. based on the results of these experiments, smooth strain awg- was found to be the most suitable for citrate production both from glucose and fructose syrup during long time continuous processes ( h). in the steady state, the highest citrate productivity ( . g/lh) was obtained with this strain, when the feed medium contained g/l of glucose and dilution rate (d) was d = . /h. supplementation of the feed medium with bacto-pepton improved the productivity, citric acid yield and stability of the continuous process in the cell recycle fermentation system. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. glycine oxidase is the product of the yjbr gene of bacillus subtilis that was predicted by sequence homology to be a flavoprotein similar to sarcosine oxidase. glycine oxidase catalyzes the oxidative deamination of various primary and secondary amino acids (e.g. sarcosine, n-ethylglycine, and glycine) and d-amino acids to form the corresponding ␣-keto acids and hydrogen peroxide. previous investigations reported on the cloning and production of the glycine oxidase gene in escherichia coli was up to u/g cell. the present works has improved the expression of the recombinant his-tagged glycine oxidase by -fold by using pet a and rosetta cells under the optimal iptg, temperature and time of induction. the protein obtained represented % of total soluble proteins in crude extract. the enzyme was purified to near homogeneity using imac with a % recovery and with and specific activity of . u/mg protein. the enzyme was active towards glycine, sarcosine and different d-amino acids, having in general, a basic ph optimum. the kinetic parameters were also studied, showing a km range from . to mm. the enzyme was immobilized, and used to obtain pyruvic acid (␣-keto acid) from d-alanine with a good yield. the enzymatic synthesis of lipophilic derivatives of various natural antioxidants including flavonoid glycosides, as well as derivatives of cinnamic acid, was performed using various immobilized lipases in ionic liquids such as -butyl- -methylimidazolium tetrafluoroborate (bmim-bf ) and -butyl- -methylimidazolium hexafluorophosphate (bmim-pf ). the influence of various reaction parameters on the catalytic behavior and the selectivity of lipases was pointed out. a response surface methodology was applied for the optimization of the yield and the productivity of the biocatalytic process. the antioxidant activity of the biocatalytically prepared lipophilic derivatives of natural antioxidants, as expressed on cu + -induced oxidation of low-density lipoprotein (ldl) and total serum, was investigated. process strategy for reduction of proteolysis in pichia pastoris fermentations jan weegar , john dahlbacka , noora sirén , niklas von weymarn , kaj fagervik : faculty of chemical engineering,Åbo akademi university, finland; laboratory of bioprocess engineering, helsinki university of technology, finland. e-mail: jan.weegar@abo.fi (j. weegar) the yeast pichia pastoris is a popular host organism for production of recombinant proteins. it is, however, common that the products are degraded by proteases towards the end of the fermentation, resulting in productivity and purity decreases. proteolysis of recombinant proteins in p. pastoris fermentations is affected by the temperature and ph of the growth medium. in this study, it was shown that decreasing the temperature from to • c during the induction phase effectively prohibited proteolysis. on the other hand, the temperature decrease resulted in a reduced maximal methanol consumption rate, which subsequently resulted in a culture highly sensitive to residual methanol. the temperature was slowly decreased according to a predetermined trajectory. as the temperature reached values below • c, the methanol concentration had to be closely monitored and the substrate feed rate adjusted in order to prohibit methanol poisoning as well as to maintain the culture as a substrate limited fed-batch. measurement of protease concentrations revealed that proteases were present at • c, but at this temperature the proteolysis rate was evidently effectively reduced. the recombinant protein produced could almost totally be recovered (i.e. high purity) with this process strategy compared to a constant high temperature culture ( • c) where severe breakdown of the product was observed. with the growing of an ecological conscience in the public opinion, more and more industrial processes are analyzed for a possible ecologically beneficial alternative. for the production of ascorbic acid, which is nowadays done by the reichstein process, a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic means, which show some advantages regarding costs and environmentalfriendliness. besides of two-stage fermentation, our approach is to design a tailor-made organism that produces -keto-l-gulonic acid, which is the direct precursor of ascorbic acid from glucose or gluconic acid and which can easily be converted to the final product by conventional methods. erwinia (pectobacter) cypripedii, which is a natural producer of , -diketo-d-gluconic acid, was selected as a suitable host for a , -diketo-d-gluconate reductase from corynebacterium glutamicum. to increase the yield of the desired compound we investigate two -keto-reductases in the host organism that diminish the yield of -keto-l-gulonate by reducing the compound to l-idonic acid, or by metabolisation of intermediates. these two enzymes were investigated with vari-ous biochemical and molecular biological methods, which will be presented. overproduction of bioinsecticides by heat and salt stress and control of dissolved oxygen in cheap media of bacillus thuringiensis nabil zouari, dhouha ghribi, samir jaoua laboratoire des biopesticides, centre of biotechnology of sfax, tunisia, bp:k, sfax, tunisia bioinsecticides based on preparations of spores and insecticidal crystal-proteins (icps) produced by the bacterium bacillus thuringiensis (bt) proved to be a high tool for fighting some agricultural pests and vectors of diseases. however, the use of bt preparations as commercial insecticides would be prohibitively expensive because it is not easy to reach cheap overproduction of icps during large-scale fermentation. here, we report possibilities to improve delta-endotoxins production as a consequence of responses of bt strains to low levels of heat and salt stress. each stressor results differently in the improvement of delta-endotoxins production, but both were shown to be most efficient at the beginning or the midexponential phase of the cultures which become resistant at the stationary or the sporulation steps. heat stress caused increase of % of synthesis yields of the sporulating cells, in contrast, salt caused increase of % of spores counts, corresponding to % toxins production improvement. combined effects of both stressors lead to toxins production improvement of %, yield improvement of %. we focused on the overcome of carbon repression catabolite, closely related to oxidative metabolism, by an adequate control of dissolved oxygen in the cheap media we formulated for bt insecticides production. we showed that an equilibrium between the high density of vegetative cells and their ability to synthesize toxins during their sporulation was necessary to take into account. % increase of icps production was reached into l fermenter combination of mutagenesis, heat and salt stress and oxidative metabolism control allowed more than % improvement of delta-endotoxins. these results are of great importance in practical point of view, since high bioinsecticides concentrations could be produced without decrease of the yields of their production. mechanism and function of the intramembrane-cleaving protease rhomboid marius lemberg , javier menendez , christopher koth , matthew freeman : mrc laboratory of molecular biology, cambridge, uk; ontario center for structural proteomics, toronto, canada rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. oligosaccharides and -keto-glycosides. availability of the enzyme is, however, hampered by the very slow growth and low production rates of the fungus. cloning of the encoding gene and production of the protein by heterologous expression are therefore a prerequisite not only for any biotechnological application, but further scientific investigations as well. on the basis of peptide sequences degenerated primers were designed, and the resulting pcr fragment was used as a probe to isolate the gene from a genomic library. two very similar genes encoding previously uncharacterized proteins were also found, and flanking regions were amplified using rage-pcr. furthermore cdna clones of all genes were isolated by rt-pcr. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. the behavior of phytate degrading enzymes isolated from malaysian zea mays root in rice bran media anis shobirin meor hussin , abd-elaziem farouk , hamzah mohd salleh , ralf greiner : biomolecular engineering research group, department of biotechnology engineering, kulliyyah of engineering, international islamic university malaysia, jalan gombak, kuala lumpur, malaysia; centre for molecular biology federal research centre for nutrition and foods, haid-und-neu-straße , d- karlsruhe, germany phytate degrading enzymes catalyze the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen. they are widespread in nature, occurring in plants and microorganisms, as well as in some animal tissues. phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for phytate degradation and their application for animal feed and human health. from over isolate screened isolates of phytate degrading enzymes, three isolates from the root malaysian maize plantation have shown phytate degrading activity. the production of phytate degrading enzyme was studied using different concentrations [%, w/v] of rice bran media during different stages of cultivation. the dephosphorylation of phosphate from rice bran phytate has shown regulatory effect on the secretion of bacterial phytases. in this conference, we will present data for the characterization of the enzymes. in this paper we present the properties of a phytase purified from a bacterium isolated from malaysian wastewater, which might find application as an animal feed supplement. the phytase described herein is a periplasmic enzyme. the phytase was purified about fold to apparent homogeneity using ion-exchange chromatography and gel-filtration with a recovery of % referred to the phytatedegrading activity in the crude extract. the enzyme exhibits an activity of about u mg − . gel filtration of the native enzyme on a calibrated sephacryl s- column gave a molecular mass of the phytase of , ± da with elution position being measured by determination of enzyme activity. lower molecular mass species or higher molecular mass aggregates were not observed. the phytase appeared homogeneous by polyacrylamide gel electrophoresis under non-denaturing conditions at ph . and . and gave a single protein band upon sds gel electrophoresis after coomassie staining of the gels. these results indicate that the phytase could be regarded as homogeneous. the estimated molecular mass after sds-page indicated that the phytase having a molecular mass of , ± da. consequently, this enzyme is a monomeric protein. the purified enzyme had a single ph optimum at ph . and was virtually inactive above ph . . at ph . , % and at ph . , % of the activity at optimal ph was observed. the effect on enzyme stability was studied in the ph range . - . at • c. within days the phytase did not lose any activity in the ph range from . to . , but at ph values below . a rapid decline in activity was observed. at ph . , % and at ph . , % of the initial activity was lost during h. in the range of temperatures studied, - • c, the optimum temperature for the enzyme was found to be • c. the apparent activation energy was estimated at ph . from the slope of log v max versus /t. the data showed excellent linearity from to • c. the arrhenius activation energy for the hydrolysis of phytate was calculated to be . kj/mol. in order to check thermal stability, the purified enzyme was incubated at different temperatures, cooled to • c and assayed using the standard phytase assay. the enzyme lost no activity in min at temperatures up to • c. when exposed for min at • c, it retained % and at • c % of the initial activity. in order to determine the substrate selectivity of the purified phytase, several phosphorylated compounds in addition to phytate, were used for k m and v max estimation by detecting the release of the phosphate ion during hydrolysis using formation of a soluble phospho-molybdate complex in an acidic water-acetone mixture. only phytate was identified as a substrate. the kinetic parameters for the hydrolysis of phytate were determined to be k m = . mmol l − and k cat = s − at ph . and • c. like other bacterial phytatedegrading enzymes, the purified enzyme showed substrate inhibition. the activity of the purified enzyme was inhibited at substrate concentrations > mm. the study of the effect of metal ions on enzyme activity showed that none of them had an activating effect when used at a concentration between − and − m. mg + , ca + , mn + , co + , ag + , hg + , and cu + had little or no effect on enzyme activity, while zn + , fe + , and fe + showed strong inhibitory effects. the reduced phytate-degrading activity in the presence of fe + and fe + is attributed to a lower phytate concentration in the enzyme assay because of the appearance of a fe-phytate precipitate. when compounds which tend to chelate metal ions, such as o-phenanthroline, edta, oxalate, citrate or tartrate, were tested for their effect on enzyme activity, it was noted that none of them was inhibitory at a concentration from − to − m. fluoride, a known inhibitor of different phytate-degrading enzymes from bacteria and the hydrolysis product phosphate as well as its structural analogs molybdate, wolframate and vanadate were found to be strong inhibitors of the purified enzyme. flouride inhibited the hydrolysis of phytate with a k i value of mol l − . several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (amps) in recombinant bacterial expression systems. in the present work, we investigated the use of the baculoviral polyhedrin (polh) protein as a novel fusion partner for production of a model amp (halocidin subunit; hal ) in escherichia coli. the recombinant hal amp could then be hydroxylamine cleaved from the fusion protein and easily recovered by simple dialysis and centrifugation. this was facilitated by the fact that polh was soluble in the alkaline cleavage reaction but became insoluble during dialysis at a neutral ph. importantly, recombinant and synthetic hal peptides showed nearly identical antimicrobial activities against e. coli and staphylococcus aureus, which were used as representative gram-negative and -positive bacteria, respectively. these results demonstrated that baculoviral polh can provide an efficient and facile platform for production or functional study of target amps. extensive industrial and food additive applications of succinate have attracted much effort towards finding an environment-friendly alternative to the petrochemical production processes. it is very attractive to engineer s. cerevisiae for succinate production because of its generally regarded as safe (gras) status, ease of genetic manipulation and fermentation. we approached this metabolic engineering problem with a two-step methodology combining modern computational as well as molecular biology tools. in the first step we identified potential metabolic engineering targets leading to high succinate yield and productivity, with the aid of genome scale metabolic model and a bi-level optimization framework using flux balance analysis and quadratic programming. in the next step, various deletion mutants are being constructed and characterized for physiology and succinate production. results from these experiments then will be used to improve the predictions in computational models. so far, we have constructed a saccharomyces cerevisiae mutant deleted in sdh , which encodes a major subunit of sdh-complex converting succinate to fumarate in mitochondria. the physiology of sdh mutant has been characterized in aerobic and anaerobic batch cultivations and in glucose limited chemostat at dilution rate as low as . h − . in aerobic batch fermentations, the mutant showed reduced maximum specific growth rate as compared to the wild-type, and it was incapable of growing on ethanol as sole carbon source, as predicted from the model. interestingly, the mutant showed much higher specific growth rate in anaerobic conditions, close to the wildtype strain. moreover, the chemostat cultivations indicate that the critical dilution rate of the mutant is below . h − . this opens further opportunities to investigate interesting behavior of the mutant and the underlying regulatory processes to improve our understanding of yeast mitochondrial metabolism. department of chemical engineering, yıldız technical university, davutpaşa campus, esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) lactose is the dominant carbohydrate in milks which are, in turn, the only significant natural sources of lactose. a large number of people do not digest lactose properly due to a lack or inactivity of the intestinal beta-galactosidase and they suffer from intestinal dysfunctions -gas, abdominal pain and diarrhea -if their diet contains lactose. moreover, lactose is a sugar with a high bod, low sweetness, low solubility, when compared to the products of its hydrolysis (glucose and galactose) and being a hygroscopic sugar has a strong tendency to adsorb flavours and odours. the hydrolysis of this sugar is very attractive towards the improvement of processes for the production of ice cream and other refrigerated dairy products and it could be very interesting for the development of additives for animal and human alimentation. the enzymatic hydrolysis of lactose is carried out by beta-galactosidases, enzymes that are widely distributed in nature, appearing in micro-organisms, plants and animal tissues. the present investigation describes the effects of the sonication process parameters on enzymatic hydrolysis of milk lactose and enzyme stability. bandelin sonopuls sonicator was used for the lactose hydrolysis experiments. ␤-galactosidase enzyme used is produced from kluyveromyces marxianus. the reactions were carried out in ml of milk. the process variables for the sonicator are duty cycle, acoustic power and enzyme concentration. the amount of residual lactose concentration (g/l) and residual enzyme activity (%) against time were investigated versus process variables. beside of this; the mathematical models depending on the operating conditions were also derived by using the experimental data of lactose concentration and enzyme activity. kinÖzbek department of chemical engineering, yıldız technical university, davutpaşa campus, esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) over the last decade, the use of plant protein hydrolysates alternative to animal protein hydrolysates in human nutrition has broadly expanded. protein hydrolysates are often used in different nutritional formulations, such as supplementation of drinks to enhance their nutritional and functional properties, or special medical diets. there are many processes which employ protein hydrolysis and hydrolytic products. among these processes, the use of enzymes allows selective hydrolysis of protein and produces a potentially safer and more defined material. in the present study, the effect of the temperature, ph and viscosity on the hydrolysis of corn gluten was investigated using a stirred batch reactor system. the corn gluten was hydrolysed by using neutrase enzyme, a bacterial protease produced by a selected strain of bacillus amyloliquefacien. the reactions were carried out in . l of aqueous solutions containing % (w/v) corn gluten and . % (v/v) enzyme. the degree of hydrolysis (%) and soluble protein concentration depending on the time were investigated at the temperatures , , , , and • c; and at the ph values . , , . , and . to investigate the effect of viscosity, the various amounts of glycerol was added to the reaction solutions to produce viscosities in the range of . - . cp. the degree of hydrolysis (dh) was computed by using ph-stat method. for the soluble protein determination in the hydrolysates samples, the folin-lowry method ( ) was used. polyhydroxyalkanoates (pha), one of the most promising bioplastics for the partial replacement of synthetic polymers like polypropylene, are polyesters produced by bacteria as intracellular storage reserves of carbon and energy. the industrial production of pha is achieved by pure cultures in its natural state or using genetically engineered organisms. the main obstacle to the replacement of synthetic plastics by biopolymers is their great cost difference. research on the field of biopolymers synthesis using mixed cultures and waste organic carbon sources as substrates prove to decrease substantially the production costs of pha. the optimization of pha production under aerobic feeding conditions (adf) was achieved recently in our group, obtaining the highest value of pha content stored by mixed cultures ( . % of cell dry weight). in this work only a homopolymer of polyhydroxybutyrate (phb) was obtained and since it is a highly crystalline and brittle material its application field is limited. the mechanical and thermal properties of pha can be varied to a great extend by adjusting the monomer composition. the incorporation of different monomeric units, other than hb, in the polymer chain, originates copolymers with improved mechanical properties. optimization of pha production from propionate by a mixed culture was studied varying the carbon and ammonia concentrations. propionate only, acetate alone or a mixture of acetate and propionate were tested. copolymers of hydroxybutyrate and hydroxyvalerate, p(hb/hv), with different compositions were obtained. consequently polymer properties could be manipulated by feeding the selected volatile fatty acid composition. the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of weeks. mean transformation frequency ranged from % (for up to % (for ). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba tl-dna and the s gus gene showed an average of more than %. these obtained root cultures were additionly elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g.uralensis were obtained by infection of a. rhizogenes have produced gl at an yield of . % dry weight on the period of culture as a days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels ( . g/l) of the total flavonoids production have been identificated on the strains which transformed by lba . this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. production, purification and characterization of scytalidium thermophilum phenol oxidases didem sutay , ufuk bakir , zumrut b. ogel : chemical engineering department, middle east technical university, inonu bulvari, ankara, turkey; food engineering department, middle east technical university, inonu bulvari, ankara, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) phenol oxidases (pos) are a group of enzymes which are responsible for oxidation of various phenolic compounds in the presence of molecular oxygen. there are different types of pos present in nature and three major groups of these enzymes are laccases (e.c. . . . , p-benzenediol: oxygen oxidoreductase), catechol oxidases (e.c. . . . , o-diphenol oxidoreductase) and tyrosinases (e.c. . . . , monophenol monooxygenase). another group of enzymes, peroxidases (e.c. . . . ), can also be considered as a member of po family. pos have very wide substrate range and final oxidation products of these substrates are quinones, which are highly reactive molecules and polymerize into brown, red or black waterinsoluble compounds. pos are very common in nature, they can be found in almost all plants, animals and microorganisms. pigmentation and protection from the environment are main functions of these enzymes. pos have different applications in food, pharmaceutical, textile industries and waste-water treatment systems. the objective of this study was po production by the thermophilic fungus, scytalidium thermophilum, purification and characterization of the enzyme. for this purpose, enzyme production was performed either in a shaker-incubator or a temperature, ph and dissolved oxygen controlled l bioreactor (probiotem) to optimize enzyme production medium composition and bioreactor parameters. as the carbon and nitrogen sources, % glucose and . % yeast extract were determined as the optimal concentrations, respectively. copper, gallic acid and tannic acid were determined to increase enzyme production. purification was performed by using membrane and chromatographic techniques. hydrophobic, ion exchange and gel filtration columns were used by using a fplc system;Äkta prime (amersham biosciences). especially the phenyl sepharose tm high performance column appeared to be very efficient for po purification from scytalidium thermophilum. purified po have been characterized by electrophoretic techniques and kinetic studies. isolation of lipolytic microorganisms from subtropical soils. cloning, purification and characterization of a novel esterase from strain pseudomonas sp. cr- núria prim, cristian ruiz, cristina bofill, f.i. javier pastor, pilar diaz department microbiology, university of barcelona. av. diagonal , microorganisms or their enzymes are used in a wide range of biotechnological activities such as polymer hydrolysis, synthesis of added-value compounds, sample decontamination, etc. thus, there is an increasing interest for isolating new enzymes and new enzymeproducing organisms for their use in industrial conversions (cherry and fidantsef, ) . among these enzymes, lipases, esterases, cellulases, xylanases and pectinases play an important role in many biotechnological processes like those related to pulp and paper processing. three samples of subtropical forest soil from puerto iguazú (argentina) were used for the isolation of autoctonous microorganisms growing in an organic matter-rich environment. a total of pure cultures of bacteria and fungi were obtained and their hydrolytic activities on polysaccharide and lipidic substrates were assayed using olive oil, tributyrin, cholesterol esters, xylan, cellulose and pectin as substrates. among the isolates analysed, were active on one or more of the substrates evaluated, and of them degraded all substrates. nearly half of the strains displayed lipolytic activity, whereas the number of strains active on xylan, cellulose, pectin and cholesterol esters, was much lower. the strains bearing the highest hydrolytic activities were selected and stored for further characterization (ruiz et al., ) . among them, strain cr- , one of the most active isolates on tributyrin, was selected for identification and characterization of its lipolytic system. lipolytic strain cr- was identified by morphological, physiological and phylogenetic tests, as a pseudomonas sp., closely related to p. fluorescens. sds-page and zymogram analysis (diaz et al., ) of cell extracts and supernatants from the strain revealed a complex lipolytic system consisting of at least two lipolytic enzymes. sequence alignment and clustering of previously described pseudomonas lipases and esterases allowed the design of different sets of primers for the isolation of the lipase/esterase coding genes. a gene coding for an esterase with homology to family vi bacterial lipases (arpigny and jaeger, ) was isolated and cloned in escherichia coli. the cloned enzyme was further purified and characterized, showing preference for short fatty acid esters and displaying a typical michaelis-menten kinetics, with no interfacial activation. the substrate profile, together with the kinetic behaviour and sequence similarity of the cloned enzyme to family vi bacterial esterases, allowed to identify this enzyme as an esterase and was named esta . maximum activity was achieved on muf-butyrate at • c and ph . , suggesting that it could be of interest for biotechnological purposes. microbial xylitol production from agricultural wastes has recently attracted much attention from industries because it has potentials to realize the cheaper production of xylitol with low environmental impact (tada et al., ) . in order to realize the effective xylitol production by a xylose utilizing yeast, the oxygen supply is a key for maximizing xylitol yield over consumed xylose (y xl ) because the intracellular xylitol metabolism is strongly influenced by the amount of available oxygen. in the present work, we tried to apply a metabolic reaction model in order to determine the optimal oxygen transfer rate (otr) in a fermentor for maximizing xylitol yield. corn cob hydrolysates containing g-xylose/l was employed as medium for xylitol production by computer-controlled batch cultures using candida magnoliae (ferm p- , aist). a metabolic reaction model considering main xylitol metabolisms including glycolysis, pentose-phosphate pathway, tca cycle and cell synthesis was developed. the model allows to estimate various intracellular metabolic flux distributions including a xylitol production rate. the oxygen uptake rate to maximize the ratio of xylitol production rate over xylose consumption rate corresponds to the otr condition to maximize a xylitol yield over xylose consumed. based on the metabolic reaction model, the otr was optimized by a linear programming, the optimal otr and the maximum xylitol yield were estimated as . mmol o /l h and . g-xylitol/g-xylose, respectively. the experimental verification using the optimal otr demonstrated that the xylitol yield was greatly improved to . g-xylitol/g-xylose from . g-xylitol/g-xylose in our previous study. expression of a bacterial sugar phosphate transporter in s. cerevisiae to release l-glycerol -phosphate accumulated by metabolic engineering almut popp, huyen thi thanh nguyen, ulf stahl, elke nevoigt department of microbiology and genetics, university of technology, berlin, germany. e-mail: a.popp@lb.tu-berlin. de (a. popp) in contrast to glycerol, its phosphorylated precursor l-glycerol- phosphate (l-g p) is retained by the plasma membrane. therefore, engineered yeast strains accumulate l-g p in the cytosol resulting in low overall yield of the desired product and laborious downstream processing. a suitable sugar phosphate transporter in the yeast plasma membrane would overcome these limitations. the glycerol- -phosphate transporter (glpt) of e.coli is an antiporter and naturally mediates the uptake of l-g p in exchange with inorganic phosphate. we assume that this transporter would also mediate the excretion of accumulated l-g p into a phosphate-rich medium if it was present in the plasma membrane of engineered yeast. despite many inconsistencies in codon usage, we were able to express the bacterial glpt gene in yeast. expression was monitored by a c-myc tag added to the c-or n-terminal hydrophilic tail, respectively. the quantity of the construct with n-terminal tag clearly exceeds the quantity of the construct with c-terminal tag. both gene products are located in the endoplasmic reticulum, as shown by immunofluorescence microscopy. obviously, yeast's transmembrane protein sorting machinery does not recognise it as a substrate for the secretory pathway. metabolic flux analysis of c-and p-limited shikimic acid producing e. coli gaspard lequeux , louise johansson , jo maertens , peter vanrolleghem , gunnar lidén : biomath, ghent university, coupure links , gent, belgium; department of chemical engineering, lund university, p.o. box , lund, sweden. e-mail: gaspard.lequeux@biomath.ugent.be (g. lequeux) metabolic flux analysis (mfa) was applied to decipher why plimited e. coli fermentations are more optimal for shikimic acid production in comparison with glucose-limited fermentations. as mfa allows obtaining insight in the intracellular flux distribution over different pathways by only measuring net production and consumption rates of metabolites, under the condition that parallell pathways are removed. to this end a detailed metabolic network model was created and checked for consistency, dead-ends, and parallell pathways. several fermentations were performed at different dilution rates (ranging from . to . h − ) and different limitations (phosphate and glucose). the e. coli strain used was w with genetic modifications in the aromatic amino acid pathway to enhance shikimic acid production. for each dilution rate, a metabolic model was solved. this way, the evolution of each flux can be followed with respect to the dilution rate. the p-limited cultures showed a better yield which can be explained by the diminished excretion of dehydro-shikimic acid (as is known from literature) and a reduction of the hydrolysis of atp. takasumi hattori, kuniki kino, kohtaro kirimura department of applied chemistry, school of science and engineering, waseda university, tokyo, japan. e-mail: takasumi@suou.waseda.jp (t. hattori) alternative oxidase is a terminal oxidase in respiration chain, which is a branched chain of cytochrome pathway, and inhibited by salicylhydroxamic acid (sham), but not by cyanide. the citric acid-producing fungus aspergillus niger wu- l has a cyanide-insensitive and sham-sensitive respiration catalyzed by the alternative oxidase (kirimure et al., ) and did not produce citric acid when cultivated with sham (kirimure et al., ) . in this study, the transcript levels of alternative oxidase gene (aox ) (kirimure et al., ) and activities of alternative oxidase under the conditions of citric acid production were examined during days-cultivation. the amount of aox mrna was determined by northern blot analysis, and the specific activity of alternative oxidase as that of duroquinol oxidase. the transcript level and the activity were highest at days at log-phase, decreased during and days, and thereafter maintained at low levels. however, the transcript and alternative oxidase activity was constitutively detected during whole the cultivation periods under the conditions of citric acid production. the sequence analysis of aox chromosomal dna revealed the presence of potential binding site of cyclic amp responsive element (cre), stress responsive element (stre) and heat shock factor (hsf) in its upstream region. these results indicated that the expression of alternative oxidase was regulated in the transcription level and alternative oxidase contributes as the main respiration chain during citric acid production. kirimura, k., et al., . curr. genet. , - . kirimura, k., et al., . biosci. biotechnol. biochem. , - . trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. metabolic versatility displayed by these fungi makes it a very amenable source of new gene products. functional analysis of candidate genes goes by two complementary ways: gene overexpression and loss-of-fuction mutants generation. a few expression systems are available mainly to direct constitutive gene expression in catabolite repression conditions. following a genomic approach, we have recently cloned some gene promoters with high expression in glucose in trichoderma harzianum cect . a main goal in a gene functional analysis is the generation of knock-out mutants. up to date, there is no reference about successful gene disruptions in t. harzianum mainly due to a very low homolog recombination frequency. rna-mediated gene silencing has been shown as an efficient tool to diminish or totally abolish specific gene expression. especially those strategies based on the use of hairpin constructs allow rapid and easy generation of strains with reduced levels of mrna from genes of interest. we have used the t. harzianum cect tss promoter to direct the expression of a hairpin dna construct that induced the appearance of small-interfering rnas (sirnas) and the silencing of the uida reporter gene in a previous uida overexpressing strain. reduced levels of mrna and gus activity correlated with the presence of sirnas. this is the first report on rna-mediated gene silencing in trichoderma and constitutes a useful and promising tool for functional genomic studies in fungal systems. analysis of the metabolic response of escherichia coli to quantitative modulations of the glucose- -phosphate dehydrogenase based on c-labelling experiments cécile nicolas, fabien létisse, stéphane massou, philippe soucaille, jean-charles portais. e-mail: fabien.letisse@insa-toulouse.fr (f. létisse) microorganisms have an efficient capacity for adapting their metabolism in response to genetic or environmental changes, and understanding metabolic robustness has become an emergent issue. part of the robustness originates from the network organization of metabolic systems, where the interplay between all available biochemical reactions provides alternative mechanisms for compensating the perturbations. recently, c-metabolic flux analysis ( c-mfa) has been applied to escherichia coli knock-out mutants lacking key enzymes to determine the phenotypic effects of structural changes in the metabolic network, providing further evidences for compensatory phenomena. the aim of our on-going work is to understand how the central metabolism in e. coli responds to quantitative alterations at a specific key-point of the metabolic network. the glucose- -phosphate dehydrogenase (g pdh), a key enzyme in the central metabolism for which the effects of deleting the gene (zwf) has been already described (zhao et al., ) , was chosen as the target. to this aim we have generated a set of expression mutants, i.e. mutants having each a fixed level of expression of the zwf gene. four different levels of expression, leading respectively to g pdh activity ; . ; . and times higher than in the wt strain, have been obtained. for each mutant, transcriptomics analysis will be carried out and compared to both the zwf-and wt strains to detect changes in the network structure, and the distribution of fluxes will be measured using c-mfa. the flux maps obtained for the various strains will be compared to evaluate the quantitative response of the central metabolic network to imposed and increased g pdh activity. metabolic control analysis will be applied to provide insights onto the sensitivity of the measurable metabolic fluxes to g pdh activity. combination of transcriptomics and fluxomics approaches will provide information on the nature and extent of the compensatory mechanisms. because the activity of a single enzyme is tuned at different levels in knock-out and expression mutants, this investigation provides a situation that mimics gene-level regulation of metabolism. , j., et al., , metab. eng., , . with the depletion of the world's petroleum supply, there has been an increasing worldwide interest in ethanol as an alternative, nonpetroleum source of energy. this fact caused increased interest in the new ethanol technology fermentation process research. as reported before, bacteria zymomonas mobilis possesses more advantages than saccharomyces cerevisiae, microorganism used for ethanol production in industrial scale. for that reason, we have focused on the fermentation studies of this facultative bacterium in free and immobilized form. the immobilization of the cells into polyvinylalcohol (pva) hydrogel lens-shaped capsules lentikats, improved the batch fermentation process efficiency nine times. starch, the substrate considered as one of the best of renewable energy source is considered as fuel ethanol feedstock. due to z. mobilis disability of maltose, maltotriose and dextrin utilization, the starch has to be converted into glucose monomers. this pre-fermentation step can overcharged whole ethanol production process. this ineffective part of the process was resolved with immobilization of glucoamylase into lentikats. the system with immobilized enzyme and cell was stabile in continuous mode for days without any significant change in the system efficiency. the combination of cell and enzyme immobilization can significantly improve the efficiency and the cost of ethanol production in industrial scale. fermentation of an inhibitory dilute acid-hydrolysate from spruce using a fed-batch procedure combined with cell-reuse andreas rudolf, gunnar lidén department of chemical engineering, box , lund university, se- lund, sweden. e-mail: andreas.rudolf@chemeng.lth.se (a. rudolf) a well-controlled addition of hydrolysate to the fermentation has proved very efficient in reducing yeast inhibition due to compounds formed during lignocellulose hydrolysis. furthermore, using high cell mass concentrations has been another way of avoiding the negative impact of the inhibitory compounds. if possible, the yeast should therefore be re-used in the process. in the present work a dilute-acid hydrolysate from spruce was fermented using a fed-batch procedure with reutilization of yeast. the fermentation procedure worked satisfactorily, with more than % of fermentable sugars consumed in each of the four consecutive fed-batch fermentations performed. the ethanol yields on fermentable sugars reached . g/g. there was continued cell growth in the repeated fed-batch experiments, with an average cell yield on fermentable sugars of . g/g. in contrast, only about % of the fermentable sugars were consumed within h, when the fermentation of the hydrolysate was run in a batch process. the work shows the potential to re-use the yeast in a suitably designed process. metabolic engineering is defined by bailey in his seminal paper as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant dna technology". the manipulation of these functions ultimately results in the manipulation of metabolism, which is the purpose of many biotechnological processes. metabolic engineering has sought its methods and tools in mathematics and the physical sciences, and later in information technology (it) leading to the proliferation of bioinformatics. in this work we propose a novel approach to metabolic engineering that regards it as a business process reengineering (bpr) endeavour. hammer and champy in their celebrated book define bpr as "the fundamental rethinking and radical redesign of business processes to achieve dramatic improvements in critical contemporary measures of performance, such as cost, quality, service and speed". our thesis is that metabolic engineering with its goal of reengineering the metabolism of the microorganism, is equivalent to business process reengineering (bpr) in business and management. indeed this is essentially what metabolic engineering does to the cell through the use of recombinant dna technology, which can be viewed as a radical redesign of the metabolic process. after all, it causes changes that cannot be attained otherwise, and whose purpose is to achieve dramatic improvements in cellular activities such as the increase in production of some metabolites by orders of magnitude. the cost incurred by the process, which is metabolism in this case, can be, for example, energy requirements or change to a cheaper substrate. in this study we elucidate this parallelism between the two with emphasis on modelling of metabolism and how the concepts of business process modelling can be applied to it. the purpose is not to produce a model, but rather to introduce the modelling methodology and demonstrate its utilisation and benefits and outline its limitations and challenges. we believe that the novelty of our work lies in applying a new paradigm in approaching metabolic engineering that has not been considered previously. thermophilic ethanol production from wheat straw hydrolysate in continuous culture tania i. georgieva, birgitte k. ahring biocentrum-dtu, technical university of denmark, building , dk- lyngby, denmark. e-mail: tig@biocentrum.dtu.dk (t. georgieva) ethanol production from lignocellulosic biomass has attracted widespread attention as an unlimited low cost renewable source of energy to transportation fuels due to increasing petroleum use and air pollution towards greenhouse gases. wheat straw available as agricultural residue has been considered as a potential lignocellulosic substrate for industrial bioethanol production. a major technical obstacle to commercialize bioethanol production form lignocellulose (such as wheat straw) is associated with a lack of microorganism able to rapidly and efficiently ferment both hexose and pentose sugars into ethanol and to tolerate the inhibitors present in undetoxified hydrolysates. currently used industrial mesophilic microorganisms (saccharomyces cerevisiae and zymomonas mobilis) are excellent ethanol producer from glucose, however, they are not able to ferment other sugars such as xylose, which is the second most abundant sugar in lignocellulose. thermophilic anaerobic bacteria have been considered for ethanol production from lignocellulosic biomass as an alternative to mesophilic ethanol producing strains, predominantly because of their abilities naturally to ferment the whole diversity of sugars found in lignocellulosic biomass. an increase attention to thermophilies for ethanol production have also arise from broad spectrum of advantages regarding industrial scale ethanol fermentation such as high growth and metabolic rates, low oxygen solubility, reduced risk of reactor contamination, and cost savings via mixing, cooling and facilitated product recovery. in addition, simultaneous co-fermentation of glucose and xylose in a single operation unit could substantially reduced the ethanol production cost. research has been attempted to study the potential of using a thermophilic anaerobic bacterium for continuous ethanol fermentation of lignocellulosic biomass, with particular emphasis on effectiveness of our strain to ferment undetoxified wet oxidized wheat straw hydrolysate with respect to sugar (glucose and xylose) conversion and ethanol yield. the experiment was carried out in a lab-scale reactor operated at • c with wheat straw hydrolysate as a substrate in concentrations from to wt.% equivalent to total sugar mixture of - g/l. wheat straw hydrolysate (woh) [ g/l wheat straw, . % dry matter (dm)] was prepared using wet oxidation pretreatment process followed by enzymatic saccharification with commercial enzymes mixture of celluclast . , and novozym (novozymes, denmark). both xylose and glucose sugars were simultaneously converted to ethanol. the sugar utilization was higher than %, and high ethanol yields were achieved. reactor shows good long-term performance ( days) in terms of operation stability and reactor contamination. maltotriose is the second most abundant fermentable sugar in wort and due to incomplete fermentation, residual maltotriose in beer may cause problems in the brewing industry. to study genes that might improve utilization of maltotriose we used a library with dna from brewer's strains and a laboratory strain and identified a new transporter encoded by mtt . mtt gave lager strain a the ability to grow on yp/ % maltotriose in the presence of mg/l of the respiratory inhibitor antimycin a. this transporter gene shares % similarity with mph and mph , % similarity with agt and % similarity with mal and mal . moreover, mtt shares even higher similarity ( %) with the s. pastorianus mty gene (m. salema-oom, unpublished, ncbi accession number aj ). purified radiolabeled maltotriose and radiolabeled maltose were used to study sugar uptake of lager strains a and ws / , and of a containing mtt or mtt alt, a more efficient, altered version of this gene lacking the basepairs from the end and containing base-pairs of vector sequences. these transport studies show that mtt and, especially, mtt alt encode maltose transporters with relatively high activity towards maltotriose compared to maltose. this study is part of a multi-disciplinary project, funded by the european union (contract no. qlk -ct- - ) focusing on the development of high-gravity resistant brewer's yeast strains. metabolic engineering of l-phenylalanine pathway in bacillus subtilis yasemin demirci , pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre laboratory, department of chemical engineering, ankara university, ankara, turkey; iblab, department of chemical engineering, metu, ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.Özdamar) metabolic engineering design of a recombinant l-phenylalanine (phe) production system is based on coordinated overexpression of the flux-controlling genes in the aromatic-amino acid pathway. based on the insights gained by the work carried out in our laboratories (Özçelik et al., ) , aroh for the reaction r at the branch-point chorismate that connects the preceding reactions of the aromatic group amino acid pathway to the proceeding reactions towards phe, was predicted as the first-, and aroa for dahp synthase (r ) predicted as second-metabolic engineering sites. aroa gene was cloned next to aroh, by the use of four primers designed using pcr-based gene splicing by overlap extension method. the genes were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their ends that are complementary to the portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at end. extension of this overlap by dna polymerase has yielded the recombinant two-gene product; and the two-gene product serve as template for the continuation of reactions for the increase of the concentration in the micro-reactors. the two-gene fragment was first cloned into puc , and then sub-cloned to pmk e. coli -bacillus shuttle plasmid. the new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis. the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, phe and other amino acids, and organic acids concentrations as the constraints. on the bases of calculated intracellular fluxes of recombinant b. subtilis carrying pmk ::aroa::aroh, an in-depth analyses of the metabolic engineering design will be presented. ozçelik,İ., Ç alık, p., Ç alık, g.,Özdamar,t.h., . metabolic engineering of aromatic group amino acid pathway in bacillus subtilis for l-phenylalanine production. chem. eng. sci. ( - ), - . the % respiratory-deficient nuclear petite amylolytic saccharomyces cerevisiae npb-g strain capable of excreting a hybrid protein possessing both ␣-amylase and glucoamylase enzyme activities was generated and its employment for direct fermentation of starch into ethanol was investigated under both shake flask and controlled bioreactor cultivation conditions. when compared with a standard host strain, higher ethanol concentrations and yields were achieved with the nuclear petite strain under both cultivation conditions. further improvement in ethanol production was achieved by the use of an initial glucose supplement. comparison of the ethanol fermentation performances of the respiratory-deficient npb-g and the parental respiratory-sufficient wtpb-g strain showed an increase of, ca. % in both ethanol production yields and ethanol productivities with the respiratory-deficient strain. response surface methodology (rsm) was used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both strains with concomitant improvement in their amylolytic potentials. high ethanol yields on substrate values of the bioreactor cultures, that were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient strain developed in this study was very effective in the direct fermentation of starch into ethanol. establishing a biotechnology educational framework to support a knowledge-based economy in puerto rico rosa buxeda, lorenzo saliceti-piazza industrial biotechnology program, university of puerto rico, mayagüez campus, mayaguez , puerto rico. e-mail: rbuxeda@uprm.edu (r. buxeda) industrial biotechnology has been identified as a major thrust area of economic development within the past five years for the island of puerto rico. the portfolio of biotechnology manufacturing investments in the island has passed the two billion dollar mark. world known companies like amgen, abbott and eli-lilly lead these investments, which have catalyzed a strong technology transfer to the island. a strong collaboration between industry and academia was needed to provide a well-trained workforce for these company startups. as a result, the university of puerto rico, mayagüez campus (upr-m) developed four initiatives which are part of the educational pipeline in biotechnology. these are: (i) an industrial biotechnology program, a -year bs degree which contains a novel curriculum with courses from science and engineering with undergraduate research and industrial internships as part of the degree requirements; (ii) an industrial biotechnology learning center, which provides customized biotechnology and bioprocessing training modules, including lectures and hands-on experiences to train and develop the workforce needed in the biotechnology manufacturing plants; (iii) a biotechnology summer camp, which addresses the high school student population and its main purpose is to educate and advise on the different career paths that can be followed in the field of biotechnology; and (iv) a biotechnology center for research and training in bioprocessing, that will address the development of corporatesponsored research projects to strengthen links between industry and academia in order to build up a knowledge-based economy. our paper will describe each initiative in detail as well as its outcomes and impact on puerto rico's knowledge-based economy goals. isolation of acid phosphatase from sweet potato and immobilization using different adsorbent d. omay, y. güvenilir, n. deveci istanbul technical university, department of chemical engineering, maslak , istanbul, phosphatase enzymes occur in a wide range of plant and animal tissues. they catalyze the hydrolysis of phosphate bonds in organic phosphates, between the phosphate group and rest of the molecule. immobilization of enzymes and biological compounds is currently gaining importance due to its wide variety of applications in the food and pharmaceutical industries and also its biomedical applications. it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan. the aim of this experimental study was determined as partial purification and isolation of acid phosphatase enzyme and its immobilization. the purification was realized by applying centrifugation, ammonium sulfate precipitation and dialysis respectively. the specific activity of the supernatant was . u/mg and after % saturation this value increased . u/mg. furthermore, acid phosphatase was investigated using different adsorbent (chitin, chitosan, synthetic zeolite and raw zeolite) and evaluated the storage stability and re-usability of the immobilized acid phosphatase. it was estimated that, acid phosphatase activity was shielded the ratio of , , , and % by using raw zeolite, synthetic zeolite, chitin and chitosan respectively under h operation condition. Øyvind m. jakobsen , , michael c. flickinger , svein valla , trond e. ellingsen , trygve brautaset : department of biotechnology, norwegian university of science and technology, norway; sintef applied chemistry, sintef, norway; biotechnol. institute, department of biochemistry, molecular biology and biophysics, university of minnesota, usa aerobic methylotrophs can utilize one-carbon (c ) compounds such as methane and methanol as a sole c-source for growth and energy. the majority of research on these organisms has focused on their biochemical novelity and commercial viability. for the industrial production of bulk products such as the amino acids lysine and glutamate raw material costs and abundance are important, and c sources are thus attractive compared to sugars. bacillus methanolicus can secrete up to g/l of glutamate upon methanol growth at • c (thermotolerant and methylotroph) and mutants producing - g/l of l-lysine have been selected. we study the genetics for conversion of methanol into biosynthesis of glutamate and lysine, and in the present report we unravel the regulation of genes impor-tant for the consumption and tolerance level for c compounds by b. methanolicus. b. methanolicus has a methanol dehydrogenase gene (mdh) for oxidation of methanol into formaldehyde and a ribulose monophosphate (rump) pathway for assimilation of formaldehyde. we recently discovered that mdh and five rump genes are carried by natural plasmid pbm in this bacterium and this represented the first documentation of plasmid-dependent methylotrophy in any microorganism. we here use real-time pcr to analyse the regulation of plasmid-and chromosomally located rump genes, in cells upon methylotrophic and non-methylotrophic growth. high methanol concentrations in the growth medium is cell toxic and the mechanisms for this sensitivity of b. methanolicus is poorly understood. our results indicate that plasmid pbm plays a fundamental role for this trait as well and the impact of our results on the biotechnological applications of this bacterium is discusssed. department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, . (t ), . (t ) and . (t ) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period ( days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t showed more less firm after days of storage being . g/ g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t and t of and mpa s respectively, compared with control of mpa s at days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t gained the highest scores ( points) followed by the control ( . points) after days of storage, while yogurt of t showed a low scoring being . from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of . units of plant proteinases/ml milk. the penicillium chrysogenum oat gene encoding a class iii omega-aminotransferase has been cloned and characterized. this enzyme that converts lysine into -aminoadipic semialdehyde is important in providing -aminoadipic acid, a precursor of penicillin and other ␤-lactam antibiotics. the enzyme is related to ornithine- -aminotransferases and to lysine- -aminotransferases encoded by the lat gene located in the bacterial cephamycin gene clusters. expression of oat is induced by lysine, ornithine and arginine and repressed by ammonium ions. area-binding consensus sequences and an -bp direct repeat associated with arginine induction in emericella (aspergillus nidulans) have been found in the oat promoter region. deletion of the oat gene resulted in the loss of omegaaminotransferase activity. the deletion mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed a reduced growth on lysine. complementation of the deleted mutant with the oat gene restored growth on ornithine, arginine and lysine to the levels of the parental strain and omega-aminotransferase activity. the strong expression of oat gene after induction by the basic amino acids may provide additional -aminoadipic acid for the formation of the -aminoadipyl-cysteinyl-valine tripeptide for ␤-lactam biosynthesis. morphological characterisation of two high producing strains of penicillium chrysogenum carrying a disruption in the nadph-dependent glutamate dehydrogenase k. rueksomtawin, j. thykaer, h. noorman, j. nielsen center for microbial biotechnology, biocentrum-dtu, technical university of denmark, dk- lyngby, denmark. e-mail: kr@biocentrum.dtu.dk (k. rueksomtawin) metabolic engineering has proven to be useful in optimisation of ␤-lactam production, e.g. constructing superior strains with multiple copies of the ␤-lactam gene cluster. it is however, of equal importance to gain insight into other aspects of the metabolism to establish a general overview in order to apply metabolic engineering for further improvement of the production strains. in that context, the redox metabolism is essential, as it functions as a tightly controlled connection between the different parts of the metabolism. in order to investigate this role of the redox metabolism in more detail, the gdha-gene, encoding the nadph-dependent glutamate dehydrogenase, was disrupted in two industrial strains of penicillium chrysogenum. during physiological characterisation of the two strains it became apparent that considerable changes in the morphology had occurred due to the genetic alteration. since the morphology is an important parameter in process optimisation, an examination of the morphology of the two strains was undertaken. in this work, the morphological differences between the gdha-disrupted strains and the reference strains were comprehensively investigated both during growth on solid media and submerged growth in a flow-through growth chamber. with the advance development of computerized image analysis techniques, the key morphological properties of the individual hyphal elements were quantified. in comparison to the reference strains, the disruption of the gdha gene resulted in a morphological change from short hyperbranched hyphal elements to long elongated hyphal elements with less branches. in the parallel studies with aspergillus nidulans and its corresponding gdha-deleted strain, no difference in the morphology was observed. polyketides (pk) represent one of the largest groups of natural products and are found in fungi, bacteria and plants. since many useful polyketides either originate from sources that are difficult or even impossible to cultivate or are produced in inadequate amounts, we are interested in expressing polyketide synthases (pkss) in heterologous hosts. saccharomyces cerevisiae, aspergillus niger and aspergillus nidulans were chosen as initial hosts, because the techniques necessary to cultivate and manipulate these strains genetically are well established. -methylsalicylic acid synthase ( -msas) was chosen as a model pks. replicative plasmids carrying the genes encoding -msas from penicillium patulum and phosphopantetheinyl transferase (pptase), respectively, were transformed into s. cerevisiae. in addition, an integrative vector was designed and the gene encoding -msas was integrated in the yeast chromosome. batch cultivations on galactose minimal media were performed. the results are presented and in particular the effect of expression mode and type of pptase (bacterial versus fungal) is discussed. furthermore, the progress of the work on expressing -msas in a. niger and a. nidulans using an integrative vector system is presented and discussed. the valorisation of functionalized chemicals from biomass resources compared to the conventional fossil production route ben brehmer, wageningen ur agrotechnology & food sciences, workgroup: valorisation of plant production chains, p.o. box , aa wageningen, the netherlands. e-mail: ben.brehmer@wur.nl at some undisclosed point in the foreseeable future, cheap fossil fuels resources will become depleted and the industry will be forced to pursue more difficult reserves with increasingly high extraction costs. most alternatives available and under research do not consider price as the main motivation for replacement, but focus solely on sustainability and environmental benefits. sustainability is an interesting word as there are enough fossil resources scattered around the world to be sustainable in quantity but not sustainable in price. seeing that fossil fuels are derived from prehistoric biomass it is not at all presumptuous to assume that every application and product can be replaced by the biomass of today. in fact, many highly specialised pharmaceutical chemicals already have a biomass origin. yet, not all of the uses of fossil fuels need to be replaced by a comparable carbon based source, such as biomass. energy and transportation in particular do not necessary need to rely on carbon cleavage, whereas practically all of the petrochemicals contain a carbon backbone. the main stipulation in substituting fossil-based chemicals with bio-based chemicals is availability and cost. it is proposed that already today, by utilising existing, recently developed and developing technology, it is economically advantageous for many chemicals to derive from biomass, in particular the functionalized chemicals. the only way to validate this conjecture is to develop a complete comparative life cycle analysis. as opposed to a traditional lca, the "multicriterion" developed here will revolve around energy flows and process efficiency in terms of exergy. the aim is to assess the optimum route with the best production options along the whole production chain while determining any possible limiting factors. using this tool, a systematic production matrix relating several logical source crops and a few key chemicals of varying derivative levels can be created and compared to the conventional fossil routes. combined with economic considerations and some unambiguous environmental factors, the investigation will provide all the information relevant to the industry. the goal is to create an objective and reliable simulation system ratifying the economic and environmental feasibility of exploiting biobased chemicals today and indicate the steps necessary for further improvement. biosynthesis of multi-enzymatic preparation from aspergillus niger ibt- useful in textile fabric treatment rita pyc , jadwiga sojka-ledakowicz , tadeusz antczak , joanna lichawska : institute of technical biochemistry, the technical university of lodz, lodz - , poland; textile research institute, lodz - , poland among many methods of producing enzymatic preparations, i.e. by liquid surface fermentation -lsf, submerged fermentation -smf or solid state fermentation -ssf, this last is most advantageous. cultivation in solid state means fermentation on a matrix formed by industrial and agricultural wastes. most often filamentous fungi -due to the lack of available water in the foundation -are the efficient, competitive microorganisms applied in solid state bio-conversion. the aim of research works carried out at the institute of technical biochemistry of the technical university of lodz and at textile research institute, lodz was defining optimal conditions for biosynthesis and testing the possibility of applying multi-enzymatic preparation from aspergillus niger ibt- in the treatment of woven fabrics made of natural cellulose fibres. as the result of biosynthesis optimization process, malt sprouts, wheat barn and beet pulp were selected as the best media to obtain enzymes of high pectinolytic activity maintaining at the same time high activity of cellulolytic enzymes and xylanase. the highest obtained activities reached: pm for total pectinolytic activity, j/ml for endoglucanase and j/ml for endoxylanase and they were . - . times higher than those achieved before optimizing process. performed research works demonstrated that the optimum activity of applied enzymatic system is obtained in the range of ph . - . . woven fabrics made of flax and cotton fibre blends, subjected to bio-pre-treatment, were evaluated with reference to their sorption properties. comparative evaluation of liquid sorption by woven fabrics allowed to notice efficient enhancement of fibres' sorption capabilities after pre-treatment using enzymes system from aspergillus niger ibt- . this offers the possibility of substitution of alkali scouring of linen-cotton woven fabrics before their bleaching by bio-treatment. the phylum actinobacter includes many bacteria of industrial importance both for accumulation of primary and secondary metabolites. both primary and secondary metabolites are dependent on precursors and cofactors that are provided by the central carbon metabolism of microorganisms. there are alternative pathways for catabolism of carbon either via the embden meyerhof parnas (emp) and pentose phosphate (pp) pathway or through the entner doudoroff (ed) pathway. the emp pathway is energetically more favorable and has therefore been presumed to be the dominating route for carbon metabolism in bacteria producing secondary metabolites. however, primary metabolism is poorly studied for most actinobacter species as focus traditionally has been on secondary metabolism. with the aim to gain more knowledge about the diversity of central carbon metabolism within the phylum actinobacter, different strains were collected from various sources and strain collections. the strains were grown in minimal medium with supplement of standard vitamin solution and [ - c] glucose as carbon source. the c-labeling patterns of proteinogenic amino acids were determined by gc-ms analysis. through this method, the fluxes in the central carbon metabolism during balanced growth were estimated and pathways for carbon metabolism were determined. in particular the labeling patterns of alanine and valine were of interest as they are derived from pyruvate and therefore can be used to distinguish between whether glucose is metabolized through the ed pathway or the emp pathway. nobacter, amycolatopsis balhimycina produces the glycopeptide balhimycin. the balhimycin aglycone is identical to the aglycon of vancomycin, which is a commercial glycopeptide. as a. balhimycina appears to be accessible to genetic modifications and the biosynthetic cluster responsible for balhimycin production is published, this genetically well-characterised academic strain can serve as a platform strain for production of vancomycin analogues derived through combinatorial biosynthesis. the understanding of the physiology of this microorganism is essential for the efficient accumulation of potentially commercial secondary metabolites. the bacterium is capable of growth in a fully defined minimal medium, with the production of balhimycin. the strain was grown at either nitrogen or phosphate limitation and balhimycin accumulation was followed at these conditions. flux analysis of balhimycin production by amycolatopsis balhimycina based on c labelling experiments was performed. the zygomycetes blakeslea trispora and phycomyces blakesleeanus accumulate beta-carotene, ubiquinone (coenzyme q), various different sterols, and other terpenoids, all of them produced via the mevalonate pathway. these fungi are used or could be used for the industrial production of these terpenoids, edible oil, chitosan, and various organic acids. by measuring the specific radioactivity of terpenoids made from radioactive mevalonate, leucine or acetate in the presence of excess glucose in wild types and mutant strains we have concluded that these fungi have separate subcellular compartments for the production of carotene, sterols and triacylglycerols. the terpenoid moiety of ubiquinone is synthesized in the same compartment as ergosterol. these compartments contain separate pools of all their common metabolites, beginning from acetyl-coa. mevalonate carbon atoms do not find their way back to general metabolism, i.e., these fungi lack the "shunt" pathway. the compartments are regulated independently. the very large variations in carotene content caused by many environmental and genetic changes are not accompanied by variations in the ubiquinone content. the ubiquinone content increases when the cultures grow on leucine or acetate as carbon sources and is not affected by illumination. phycomyces, but not blakeslea, increases the production of ubiquinone in presence of oligomycin. lincomycin, produced by streptomyces lincolnensis, is important, clinically used antibiotic. its gene cluster consists of putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmra, lmrb, lmrc). the organization of transcription units was determined. the analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed in early stages of cultivation. previous analysis of biosynthetic pathways of lincomycin and functionally different anthramycin antibiotics (anthramycin, sibiromycin, tomaymycin, mazetharmycin and porothramycin) indicates that the genetic information on the lincomycin and anthramycin biosynthesis should share common elements (genes), both biosynthetic and regulatory. hybridization experiments demonstrated presence of several analogues of lmb genes involved in the biosynthesis of anthramycin produced by streptomyces refuineus and porothramycin produced by streptomyces albus. effect of various calcium salts on the erythromycin production by saccharopolyspora erythraea m. rostamza , a. noohi , j. hamedi * : department of biology, faculty of science, science and research branch, islamic azad university, tehran, iran; microbial biotechnology lab., department of biology, faculty of science, university of tehran, tehran, iran. e-mail: jhamedi@khayam.ut.ac.ir (j. hamedi) calcium carbonate has the positive effect on the erythromycin, however, because of its low water solubility, caused to clogging the spargers of fermenters and fouling the microfilters. in this research, various soluble calcium salts was added to the fermentation medium and their effect the growth of saccharopolyspora erythraea and erythromycin production was studied in the complex medium consisted soy bean meal, dextrin and starch as major ingredients. the fermentation conditions were rpm, days at • c. the results obtained showed that there is no significant difference between erythromycin concentrations in the medium containing calcium lactate and calcium carbonate. however, erythromycin concentrations in the other calcium salts containing media were less than to calcium carbonate containing medium. optimum concentration of calcium lactate for erythromycin production was g/l. lincosamides and its derivatives are clinically important antibiotics. comparison of gene cluster coding for lincomycin biosynthesis and newly identified cluster of genes for celesticetin biosynthesis revealed new information on functions of several genes common for both biosynthetic pathways. the celesticetin gene cluster was identified by screening the cosmid library of streptomyces caelestis with heterologous probes based on lincomycin biosynthetic genes involved in a part of biosynthesis shared by both antibiotics. sequence analysis of the cluster revealed putative orfs, out of which are lincomycin biosynthetic genes analogues, four are specific for celesticetin biosynthesis and one codes for resistance. the gene cluster is bounded with transposase genes on both sides. in order to clarify function of three putative regulatory genes of lincomycin biosynthesis, the insertional inactivation with the pcr targeting system in streptomyces lincolnensis was done and resulted in differently reduced production of lincomycin. larsen cmb biocentrum-dtu, technical university of denmark, kgs. lyngby, denmark we present a new algorithm called "x-hitting" for automatic identification of novel bioactive compounds based on full spectroscopic characters of highly complex mixtures of natural product. one of the most dramatic advances in recent drug discovery has been the increase in screening capacity throughput and data handling. therefore, analysis has become the bottleneck in the drug discovery process. the algorithm presented here is investigated and demonstrated on identifying potentially new bioactive compounds. in addition method is shown to have a high performance for automatic identification of known structures. these tasks are referred to as new-hitting and cross-hitting, respectively. finally, the receiver operating characteristics (roc) is introduced to the research field as an important tool for evaluating the performance of the "compound predictor". through examples it is shown, that known cross-hits are identified with high proficiency, and that the new-hitting works on finding new targets represented by analogues and structurally new compounds. a gene encoding a ␥-butyrolactone autoregulator receptor that have a common activity as dna-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation in streptomyces was cloned from a natamycin producer, streptomyces natalensis, and its function was evaluated by in vivo. pcr using the primers designed from two highly conserved regions of streptomyces autoregulator receptors gave a -bp band, the sequence of which revealed high similarity to the expected region of a receptor gene. by genomic southern hybridization with -bp insert as a probe, a -bp intact receptor gene (sngr) was obtained from s. natalensis. in vivo to clarify the function of sngr, a sngrdisrupted strain was constructed, and a phenotype was compared with the wild-type strain. the sngr disruptant started natamycin production h earlier and showed a . -fold-higher production of natamycin than the wild-type strain. in addition, sporulation was earlier and -fold abundance. the phenotype indicates that the autoregulator receptor protein of s. natalensis acts as a primary negative regulator of the biosynthesis of natamycin and is related to regulation of sporulation. exploring the biocatalytic potential of the novel thermostable baeyer-villiger monooxygenase: phenylacetone monooxygenase daniel e. torres pazmiño , gonzalo de gonzalo , gianluca ottolina , giacomo carrea , dick b. janssen , marco w. fraaije , biochemical laboratory, groningen biomolecular sciences and biotechnology institute, university of groningen, nijen- baeyer-villiger monooxygenases (bvmos) represent useful biocatalytic tools as they can catalyze reactions which are difficult to achieve using chemical means. however, only a limited number of these atypical monooxygenases are available in recombinant form. using a recently described protein sequence motif, a putative bvmo was identified in the genome of the thermophilic actinomycete thermobifida fusca. the nadph-dependent and fad-containing monooxygenase is active with a wide range of aromatic and aliphatic ketones and sulfides. genetic and kinetic data suggest that phenylacetone is the physiological substrate of the enzyme. previously, it was reported that this bvmo exhibits only a moderate enantioselectivity with (r,s)-␣-methylphenylacetone. this poster we will show an overview of the biocatalytic potential of the enzyme as explored so far. interestingly the enzyme has been found to perform highly enantioselective oxidations with a range of ketones and sulfides. this again indicates that this novel thermostable oxidative biocatalyst can be a useful tool for the synthesis of chiral building blocks. the enzyme s-adenosylhomocysteine hydrolase (adohcyase) catalyzes hydrolysis of s-adenosylhomocysteine (adohcy), an inhibitor of transmethylation reactions, into adenosine (ado) and homocysteine (hcy). the catalysed reaction is reversible, and the equilibrium is strongly displaced in direction of the synthesis of adohcy when reaction occurs in vitro. nevertheless, its biotechnological application resides in the synthesis of antivirals. for it, we selected between different producing microorganisms of the enzyme, the gram positive bacterium corynebacterium glutamicum. after designing the specific oligonucleotides, the gene was expressed in escherichia coli using the expression system pet a+ with iptg. the electrophoretic analysis under denaturing conditions, shows a clear induction and over-expression of a protein with a mw of kda. on the other hand, the immobilization of this recombinant enzyme in a solid support allows to use it as a catalyst for the synthesis of adohcy. the enzyme was purified by imac thanks to the presence of n-terminal × his tag end, and immobilized in eupergit c for the optimization of the production of adohcy, a product of high value. sequencing, cloning, expression, purification and characterization of a novel cytochrome p monooxygenase from rhodococcus rubber luo liu, rolf d. schmid, vlada b. urlacher institute of technical biochemistry, stuttgart university, allmandring , stuttgart, germany. e-mail: itbvur@itb.unistuttgart.de (l. liu) the cytochrome p monooxygenases are heme-containing proteins, which catalyze a wide range of oxidative reactions (werck-reichhart and feyereisen, ) . a monooxygenation activity was observed for the strain rhodococcus ruber dsm . a p -like gene fragment was amplified by pcr using degenerated primers. for identification of regions that flank this p -like dna fragment, the method "directional genome walking using pcr" was applied (mishra et al., ) . the full size gene encoding a cytochrome p enzyme was amplified by pcr from genomic dna and cloned into the vector pet a(+) for heterologous expression in escherichia coli bl (de ) cells. the enzyme was purified using metal affinity chromatography. the primary protein structure suggests, that this enzyme is a natural self-sufficient fusion protein consisting of a ferredoxin, a reductase and a p monooxygenase. the reductase activity was determined using an exogenous electron acceptor cytochrome c. the reductase domain of this p monooxygenase showed a strong preference for nadph over nadh. the substrate spetrum was investigated. in the presence of nadph the p enzyme shows hydroxylation activity towards -ethoxycoumarin, naphthalene, indene, acenaphthene, toluene and fluorene. mishra, r.n., singla-pareek, s.l., nair, s., sopory, s.k., reddy, m.k., . directional genome walking using pcr. biotechniques , - . werck-reichhart, d., feyereisen, r., . cytochromes p : a success story. genome biol. ( ), . - . . selective production of monoglyceride consisted of conjugated linoleic acid by penicillium lipase yomi watanabe , , yoshie yamauchi-sato , toshihiro nagao , satoshi negishi , tadamasa terai , takashi kobayashi , rolf d. schmid , yuji shimada : osaka municipal technical research institute, osaka, japan; stuttgart university, stuttgart, germany; the nisshin oillio group, ltd, yokosuka, japan; osaka institute of technology, osaka, japan conjugated linoleic acid (cla) is a group of c fatty acid (fa) containing two conjugated double bonds. it is expected to prevent cancer, adipositas, atherosclerosis etc, and is commertially available in the primary form of free fa, containing almost equal amounts of cis, trans-and trans, cis-cla. it is therefore desired to be converted to a palatable form. for this purpose, we have previously proposed two enzymatic ways to convert cla to monoglyceride, an emulsifier, with penicillium camembertii lipase; ( ) sequencial esterification-glycerolysis, ( ) esterification at low tem-perature. these methods, however, are time-and energy-consuming. esterification of cla with glycerol under ambient pressure by the lipase produces equal amounts of mono-and diglycerides. in contrast, it was newly found that the reaction under reduced pressure supressed the formation of diglycerides and achieved to produce % monoglyceride at % esterification. improving the thermal stability of cellobiohydrolases i (cel a) from t. reesei by site directed evolution frits goedegebuur , lydia dankmeyer , peter gualfetti , brad kelemen , edmundo larenas , paulien neefe , pauline teunissen , colin mitchinson : genencor international bv, archimedesweg , cn leiden, the netherlands; genencor international inc., page mill road, palo alto, ca , usa genencor international has been working to produce improved enzyme products for economic conversion of ligno-cellulosic biomass to fermentable sugars. most of this work was performed under a subcontract with the u.s. department of energy for cellulose cost reduction for biomass conversion. cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. cellobiohydrolases play a key role and all effective cellulase mixtures contain a large excess of cellobiohydrolases over endoglucanases, suggesting that it is the exoglucanase activity that is limiting. the fundamental dependence of reaction rate on temperature predicts that large increases in performance, and decreased enzyme cost, would be achieved if the enzymatic conversion could be operated at elevated temperatures. industrial strains of trichoderma reesei produce cellulases at very high levels and low cost. however, t. reesei cbh (hypocrea jecorina cel a) does not have sufficient stability to survive and perform at high temperatures. this poster shows the thermal stability improvement in t. reesei cbh by site directed evolution. sites with increased thermal stability properties were combined and evolved in high temperature stable cbhi variants. the evaluation of lipases as biocatalysts for organic chemistry can be carried out, at laboratory scale, by using soluble enzymes for biotransformations in aqueous media. however, the industrial exploitation of such an enormous potential should require a suitable protocol for immobilization of lipases. the binding of lipases on suitable pre-existing supports should greatly improve the performance of industrial reactors allowing us a continuous use or re-use of such interesting biocatalysts. in addition, lipases, like most enzymes, are not perfect chemical catalysts. lipases may be unstable and they may not have the optimal activity nor the optimal enantio or regioselectivities. in this way, immobilization of lipases, together with its relevance for the performance of each different industrial reactor, could be also used as a tool to improve and optimize some of these parameters. that is, immobilization of lipases, far from an already solved problem, constitutes an exciting field of research in the promising area of industrial bio-organic chemistry. in this work we would like to present useful immobilization methods of several lipases. the lipase immobilised were used for enzymatic hydrolysis of peptidomimetics of structure a. type of immobilzation used can changed the enantioselectivity of biocatalyst prepared. the absolute configuration of products b and c obtained in enzymatic reactions were assigned by chemical correlation. two-component flavin-dependent monooxygenases form an interesting class of flavoenzymes. they consist of two separate proteins; a monooxygenase component, which catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing component, which reduces flavin (fad or fmn) using nad(p)h as an electron donor. a well-known example of this class of monooxygenases is styrene monooxygenase (otto et al., ) . due to the ability to form enantiopure epoxides, which are relevant building blocks for the pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for biocatalysis. while screening a metagenomic library for oxidative enzymes, an indigo-producing clone was found. sequencing the particular clone revealed an inserted fragment of environmental dna encoding a two-component monooxygenase ( many investigations over the recent years have been directed to the production of natural aroma compounds. through biotransformation and bioconversion, aroma compounds considered as "natural" can be produced starting from monoterpenes, generating high value products as rose oxide. rose oxide is found in small amounts in some essential oils such as bulgarian rose oil and geranium oil. (−)-rose oxide is an impacting flavor compound and has a small threshold: . ppb. application of agro-industrial residues in bioprocess on one hand provides alternative substrates, and on the other hand helps solving pollution problems, that might be caused by the disposal of this residue in nature. the liquid cassava waste, is originated by the pressing of cassava roots. it is considered as a "harmful" pollutant waste due to its high organic charge and presence of cyanide. on the other hand, can be considered rich in nutrients that can be used in other applications. it was found that sporulated surface cultures of penicillium sp. were able to convert citronellol into cis-and trans-rose oxides. other bioproducts were , -dimethyl- -octen- , diol and , -dimethyl- , -epoxy- -octanol. no chemical oxidation or auto-oxidation products were detected in liquid control broths. the experiments were conducted at • c and rpm. when the medium was cassava, the production of rose oxide, , -dimethyl- octen- , -diol and , -dimethyl- , -epoxy- -octanol were insignificant reaching trace amounts. but when the mycelium developed in cassava medium and than transferred to mineral medium (citronellol as c-source) the concentrations of rose oxide increased dramatically, reaching mg/l for the cis-isomer and mg/l for trans-isomer. a mechanistic mathematical model of enzymatic degradation of avicel and phosphoric acid swollen cellulose (pasc) has been proposed. the model is based on the degree of polymerization (dp) of starting substrate, and follows its decline with time, to the final end product -glucose. three enzyme classes, namely, endoglucanase (eg), cellobiohydrolase (cbh) and ␤-glucosidase (bg) are all individually incorporated in the model. the model is, additionally, taking into account cooperative action of the involved enzymes, as well as effects of enzyme inhibition by end-products, cellobiose and glucose. to be able to describe the complex process of enzymatic hydrolysis with a set of differential equations certain assumptions needed to be introduced. those assumptions represent the simplification of an up-to-date knowledge of both substrate and enzyme structure, but also enzyme mode of action. for example, one of the often asked questions is: "what is happening with shorter cellooligosacharides (dp and up) laying on the surface of cellulose chain? are they being adsorbed to the core cellulose chain or partly solubilized to a hydrolysis broth?" to give answers to these questions and confirm mathematical modeling real enzyme hydrolysis data are needed. in this work, four well characterized, highly purified mono-component enzymes from humicola insolens (two eg and two cbh) and one bg from aspergillus niger were used to hydrolyze avicel and pasc. by careful choice of catalyst, some enzyme specific characteristics like presence or absence of cellulose binding domain will also be incorporated into the model. hydrolysis experiments were initially performed by distinct mono-component enzymes, to confirm the basic characteristics of each of the enzyme classes. soluble hydrolysis products (dp - ) were analyzed by hplc and detection of non-soluble, higher-dp polysaccharides was performed by technique of polysaccharide analysis using carbohydrate gel electrophoresis. optimisation of halogenase enzyme activity k. muffler , m. retzlaff , k.-h. van pée , r. ulber : technische universität kaiserslautern, germany; technische universität münchen, germany; technische universität dresden, germany. e-mail: muffler@rhrk.uni-kl.de (k. muffler) halogenases provide the opportunity of a regioselective and stereospecific halogenation of organic subtrates in contrast to the class of haloperoxidases. these enzymes allow a gentle synthesis of halogenated organic molecules and are capable to halogenate in specific positions (hammer et al., ; keller et al., ) , whereas traditional organic synthesis often failures or mainly leads to byproducts (hasegawa et al., ) , e.g. halogenation of tryptophan in other positions than position . our current research is focussed on tryptophan- -halogenases, because the -br/cl-tryptophan could be applied as a pharmacologically attractive precursor of serotonin. in our work we describe the optimization of an enzyme assay respectively the enzyme activity. for this purpose we use a genetic algorithm. the responsible gene of the fadh dependent enzyme was cloned from streptomyces sp. origin into a pseudomonas fluorescens strain. however, the optimization procedure was done with the purified his-tagged tryptophan- -halogenase, which was easily obtained from the crude extract of the lysed cells by application of immobilized metal affinity chromatography. the application of algorithms allows an optimization of the multidimensional search problem leading to a global optimum in the search space in contrast to the traditional used one-factor-at-a-time method. latter often failures, because this method does not reflect possible influences the parameters can have on each other. effect of organic solvent type on the enantioselectivity of candida rugosa lipase in the hydrolysis of racemic naproxen methyl ester in biphasic reaction system serpil takaç, deniz mutlu department of chemical engineering, institute of biotechnology, ankara university, tandogan, ankara, turkey hydrolysis of racemic naproxen methyl ester to produce s-naproxen was carried out in the biphasic system using isooctane, cyclohexane, hexane and toluene with candida rugosa lipase after stirring in the phosphate buffer (ph . , . m) for h at + • c and centrifuging. the hydrolyses were carried out in shaking flasks for h at rpm and • c with the initial substrate concentration of . m. the concentrations of the enantiomers of racemic naproxen methyl ester in organic solvents and those of naproxen in buffer solution were determined with hplc. it was found that the enantiomeric excess for substrate (ee s ), enantiomeric ratio (e) and conversion (x) decreased in the following order: isooctane > cyclohexane > hexane > toluene. the enantiomeric excess for product (ee p ) was found to be the same for isooctane, cyclohexane and hexane where the lowest ee p was obtained in toluene. the highest ee s , ee p , e and x values achieved in isooctane at the residence time of h were , , , and %, respectively. this study was supported by ankara university biotechnology institute (project no: ). fusarium fujikuroi (gibberella fujikuroi mating group c) produces multiple secondary metabolites such as gibberellins and bikaverin. gibberellins are terpenoid hormones that induce growth and regulate various stages of development in plants. they have numerous applications in agriculture industry. bikaverins are polyketides that have toxicity against different organisms because they inhibit respiration. regulation of polyketide and gibberellin synthesis by nitrogen has been intensively studied in fusarium but little is known about their regulation by carbon source. our main interest is to understand the regulation of biosynthesis of these compounds in f. fujikuroi imi . to investigate this regulation the organism was grown in high nitrogen medium under submerged conditions and then transferred to nitrogen-free media having various concentrations of different carbon sources. the gibberellin production was not affected significantly. on the other hand bikaverin was synthesized enormously when sucrose was used as the only carbon source. high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentration. the bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. the effect of the c source on the expression of key genes, cps/ks (copalyl diphosphate synthase/kaurene synthase) for gibberellin and pks (polyketide synthase) for bikaverin biosynthesis is currently under investigation. ment of dormancy and loss of viability in seeds with the passage of time, it lacks any systematic propagation from seeds and is typically propagated through rhizomes. this restricts large scale cultivation of this plant. in vitro propagation of plants is an effective means for rapid multiplication of species, in which conventional methods have limitations. in the pesent study we have analysed the role of various growth promoters and the effects of dark and light incubation on germination of n. alba seeds. the results indicate that in vitro propagation of n. alba from seeds can be applied as an efficient method of multiplication. the study was funded by the state planning commisson (dpt) turkey and the university of ankara vide projects no. . this research was performed for developing of biological treatment process of odor gas such as mek, h s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over % was obtained by biofilm formation. at ppm of inlet odor gas concentration and s of retention time, the removal efficiency was and % in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over % at the operational conditions above s of retention time. post soviet countries are going through the transition stage and are extremely sensitive to new technology, economics or social changes and globalization processes. that is why decision-making and system of regulation of the use of gmo's are very sensitive to number of factors. three levels of factors, the most influential to decision-making: global, regional and local; are identified at the research paper. global level depends on external policy of leaders of gmo regulations. usa and eu have the biggest influence on transition countries, though their positions completely differ. as usa was leader in inventing gmos, it is lobbing newly created biotechnological industry. in eu and other european countries lobbing of biotechnological industry was not as strong as in usa. thus, their national law is stricter. world trade organization and international agreements are also part of global level. regional level. geographical position of country is also very important because every regulation system depends a lot on regulation that is implemented in neighboring countries. countries do not exist in vacuum; they are linked territorially, politically, economically and socially with neighboring states. regulation systems of the transition countries in the eastern europe can be divided into three types: those who have no system of regulation of gmo (belarus, romania, hungary and ukraine); who approved some variety that are treated as safe to the market (poland, moldavia and georgia) and who approved all of the gmos (bulgaria, croatia and russia [before ]). but even if a country declares not to use gmo it is rather difficult to control import of such products, because of the lack of the testing laboratories, corruption of the state employees, agreements on intellectual property, and institutional country problems. in spite of global and regional tendency of gmos related regulation the most important part is the local level, namely the national regulation system. depending on national level we are choosing priorities at higher levels. as an example of post soviet countries ukraine is taken, as ukraine is one of the largest countries and one of the biggest exporters of agricultural products in europe. research includes the analysis of attitude to gm product, their potential risks and benefits of three categories that influence decision-making the most: farmers, gm experts and non-governmental organizations. recombinant microorganism development for extracellular benzaldehyde lyase production hande kaya , pınar Ç alık , tunçer h. ozdamar : iblab, department of chemical engineering, metu, ankara, turkey; bre laboratory, department of chemical engng, ankara university, ankara, turkey. e-mail: e @metu.edu.tr (h. kaya) in this study, the extracellular production of the benzaldehyde lyase (bal, ec . . . ) that catalyses the synthesis the enantiopure -hydroxy ketones for drug syntheses, by bacillus sp., was aimed. for this purpose, the signal dna sequence of an extracellular bacillus enzyme, i.e., serine alkaline protease, was fused in front of the bal gene (accession number ax ) from pseudomonas fluorescens biovar i, using pcr-based gene splicing by overlap extension (soe) method. b. licheniformis (dsm ) chromosomal dna was used as sap gene (accession number x ) template for the synthesis of sap signal sequence. bal gene was amplified by using the plasmid carrying bal gene, puc ::bal. thereafter, the signal peptide of sap with its own promoter was fused in front of the bal gene by soe method. the hybrid gene first cloned into puc plasmid, thereafter sub-cloned into pbr , pmk and phv shuttle vectors. the escherichia coli-bacillus plasmids carrying the hybrid gene pre(subc)-bal was transferred into bacillus subtilis npr− apr−, and bacillus licheniformis. the influence of the host bacillus species on bal production on a defined medium with glucose was investigated in bioreactor systems. for each of the recombinant (r-) bacillus species, effects of initial glucose concentration on cell growth and bal production were investigated; and, physiological differences and similarities between the wild-type and r-bacillus species are discussed. thereafter, the benzaldehyde lyase production capacities of recombinant e. coli and b. subtilis are compared in terms of cell concentration and bal volumetric and specific activities. for the comparison bal gene was cloned into prseta vector which is under the control of strong t promoter and expressed in e. coli bl (de ) plyss strain. the variations in by-product distributions with each recombinant organism and yields are also discussed. phosphoketolases (ec . . . ) are thiamine diphosphate (thdp)-dependent enzymes, that play a crucial role in the pentose phosphate pathway (ppp) of heterofermentative and facultative homofermentative lactic acid bacteria, and of the d-fructose phosphate shunt of bifidobacteria. reports affirm that cellulomonas flavigena can use the ppp when is cultured under anaerobic conditions. a genomic library of c. flavigena constructed in -zap express vector was screened. four positive clones were isolated, in vivo excised and the resulting pbk-cmv phagemids, each containing a . kb insert, were characterized by restriction analysis and dna sequencing. the open reading frame (orf) of the dxylulose -phosphate/d-fructose -phosphate phosphoketolase gene, xpkl, was located from nucleotide to . the xpkl orf encoded a amino acids-residue polypeptide (xpkl) with a calculated molecular mass of , da, a value coincident with that estimated by comparative sds-page (about , da). a putative ribosome binding site (gggagc) is present - nucleotide upstream of the translational start of the xpkl polypeptide. the c. flavigena xpkl polypeptide sequence was % identical to dxylulose -phosphate/d-fructose -phosphate phosphoketolase from bifidobacterium sp., bifidobacterium gallinarum, gloeobacter violaceus and bifidobacterium adolescentis. this analysis also revealed highly conserved regions. lactococcus lactis is a main diacetyl-producing bacteria by citrate metabolism in dairy products. the transport of citrate in these bacteria is dependent on citrate permease that is encoded by citp gene. previous studies of the citqrp operon in escherichia coli mutants showed that citp message is considerably stabilized in rnase iii mutant. so, in the context of the citrate metabolism research, the characterization of the lactococcal rnase iii enzyme is very important for the dairy industry. rnase iii is an endoribonuclease which has an important role in rrna processing and control of gene expression. with the aim of studying lactococcal rnase iii we have cloned the rnc gene from l. lactis ssp lactis il in the broad host range pls rgfp vector. this plasmid includes the gfp gene, encoding the green fluorescent protein (gfp), cloned under the control of the pm promoter, that is inducible by maltose. maltose induction of the lactococcal rnc expression showed a -fold increase of rnc transcription from this plasmid. activity assays for lactococcal rnase iii were standardized using crude extracts and a substrate specific for b. subtillis rnase iii. the results showed that this substrate was specifically cleaved by lactococcal rnase iii and its activity induced by maltose. lac-rnc was cloned in pet vector and the corresponding six-histidine-tagged rnase iii protein was overproduced in e. coli bl (de ) strain by iptg induction. the protein was purified by affinity chromatography using hplc system and was shown to be active by in vitro activity assays using the lac-rnase iii specific substrate mentioned above. we have also cloned lactococcal rnc gene and studied its expression in an e. coli rnc deletion mutant ( rnc). complementation assays performed in e. coli demonstrate that the lactococcal rnase iii (lac-rnase iii) is able to process rrnas and to regulate the levels of polynucleotide phosphorylase (pnpase). these results demonstrate that the lactococcal enzyme is able to substitute the ec-rnase iii not only in the rrna processing, but also in the processing of mrnas. the amount of lactococcal rnc transcript in an e. coli rnc strain was . -fold higher than in the wild type strain, suggesting that the e. coli rnase iii triggers the degradation of the heterologous rnc mrnas. the results obtained have shown that lac-rnase iii is an interesting enzyme for biotechnological purposes. objectives: the pharmaceutical and food industry has an increasing demand for selectively glycolized active agents. in our application isomaltose can be synthesized by immobilized dextransucrase, which transfers a glycosyl residue from sucrose (substrate) to glucose (acceptor). as the reaction proceeds, isomaltose can act as an acceptor and is converted into undesired follow-up products called isomalto-oligosaccharides, imos. we investigate on two approaches to avoid imo formation, selective adsorption of isomaltose (ergezinger et al., ) and the co-entrapment of dextranase adsorbate, which breaks imos down to isomaltose. results and conclusions: the first part of our research concerns the adsorption of dextranase on bentonite, which complies with langmuir model. at complete saturation ( . g g − ) our immobilisate exhibits an activity of , u g − . a kinetic analysis does not reveal significant differences between the adsorbed and free form of enzyme (k m,bentonit : . ± . m versus k m,free : . ± . m). thus, bentonite displays a high binding capacity paired with favorable kinetic properties. beyond that we investigate the activity of dextransucrase in co-immobilisates, which is reduced during coimmobilization due to interactions with the adsorbate. among various co-immobilisates, the one containing dextranase bound to preblotted bentonite imparts the highest activity ( % as compared to control: immob. dextransucrase). the molar yield coefficient of coimmobilisates y isomaltose/sucrose surpasses coefficient of control by %. further on we will characterize mass transfer of dextranase substrate into alginate matrix as well as bentonite-dextransucrase interactions. and kefir. a second approach is to use yeast as a production organism to produce natural folates for fortification. here we investigate and discuss the folate content in skq n, a diploid strain of saccharomyces cerevisiae, when cultured in different media and at different stages of growth. the aim is to gain a basal knowledge of the folate production profile, forms of folate produced and degree of leakage to the surrounding medium, in relation to the culturing medium and physiological state of the cells. danisco innovation, danisco a/s, langebrogade , po box , dk copenhagen k, denmark we at danisco a/s (copenhagen, denmark) have revealed a new starch degrading pathway by the discovering several new enzymes and metabolites in fungi and algae. these new enzymes include glucan lyases, dehydratases and tautomerases, which proved to be useful in biocatalysis. these new metabolites proved to be useful as both antioxidants and antimicrobials for food and non-food applications. this pathway is named as anhydrofructose pathway of starch and glycogen degradation. this technology is referred to as the anhydrofructose technology. diet is evolving from nourishing populations via providing essential nutrients to improving health of individuals through nutrition. modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. as a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. nutrigenetics asks the question how individual genetic disposition, manifesting as single-nucleotide polymorphisms (snps), copy-number polymorphisms (cnps) and epigenetic phenomena, affects susceptibility to diet. nutrigenomics addresses the inverse relationship, i.e. how diet influences gene transcription, protein expression and metabolism. the mid-term objective of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) to define a "healthy" phenotype. the long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. the major challenges for -omics in nutrition and health still lie ahead of us, some of which apply to -omic disciplines in general while others are specific for -omic discovery in the food context: (i) the integration of gene-and protein expression profiles with metabolic fingerprints is still in its infancy as we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) -omics in nutrition must be particularly sensitive: it has to reveal rather many subtle than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. metabolomics, the comprehensive analysis of metabolites, is an excellent diagnostic tool for consumer classification. the great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. this feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating dietary responders from non-responders, and, last but not least, (d) discover bioactive, beneficial food components. independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. it is evident that not only comprehensive analyses with one discovery platform (lateral integration of information) are required but also vertical integration between different -omic levels are indispensable for a deeper understanding of disposition, health, environment and diet (desiere, ) . a major "vertical integration issue", to date unresolved, is given by different timescales of transcript production, protein expression and metabolite generation (nicholson et al., ) . the transcript machinery usually responds fast to an external stimulus (seconds to minutes), the proteins may be expressed within minutes to hours (and have a halflife from minutes to even months) and metabolites vary significantly during the day and depend on latest dietary input. this means that data, which seem to correlate qualitatively (e.g. reflecting the same pathway), may not necessarily be related time-wise. rather, they may represent different responses at different time points and, possibly, to different stimuli. comprehensive -omic analyses is an essential building block of "systems biology", which can be defined as follows (clish et al., ) : systems biology is the comprehensive analysis of the dynamic functioning of a biological system (cell, organ, organism or even ecosystem) at gene, protein and metabolite (or higher organizational) level, achieved by comparison of two defined biological states of this system, typically before and after perturbation. while a comprehensive list of components (genes, proteins, metabolites) of a given biological system is a pre-requisite for this kind of research, the main reasoning for the "system view" is that only information on the interactions between the components gives clues to function of the entire network. a systems biology approach has recently demonstrated the power of proteomics to dissect immunity and inflammation. toll-like receptor recognition and signalling was elucidated and showed, how bacterial "barcodes" are read and interpreted in order to trigger an adapted immune response (aderem and smith, ) . in order to address some of the challenging objectives of -omics-driven nutritional research, we have addressed (a) the effect of early antibiotic administration on the maturation of intestinal tissues, (b) protein discovery in human milk, (c) the effects of polyunsaturated fatty acids on gene expression and lipid profile in the liver, and (d) biomarkers for intestinal stress. (a) antibiotics and gut maturation: the effects of early administration of antibiotics on intestinal maturation were assessed at the gene expression level in a rat model. (b) human milk: rapid enrichment and iterative, consolidated identification of immunologically relevant milk proteins was achieved through the employment of restricted-access media and a tailored proteomic strategy (labéta et al., ; lebouder et al., ; panchaud et al., ) . (c) fatty acids and liver transcriptome/lipidome: epidemiological studies have correlated higher intakes of poly-unsaturated fatty acids (pufas) with lower incidence of chronic metabolic disease. the molecular mechanisms regulated by pufa consumption were examined assaying the liver transcriptome and lipid metabolome of mice fed a control and a pufa-enriched diet (mutch et al., ) . (d) gut stress markers: as a first step, we catalogued protein expression along the jejunum, ileum and colon of the rat intestine and found gut segment-specific proteins (marvin-guy et al., ) . the innovative combination of a neonatal separation model with proteomic analysis allowed us to study, whether early life psychological stress may impact the adult gut neuromuscular protein expression and the approach revealed specific protein biomarkers. omics for engineering lactic acid bacteria willem m. de vos wageningen center for food sciences, wageningen university, the netherlands. e-mail: willem.devos@wur.nl. url: http://www.wcfs.nl/ lactic acid bacteria (lab) are high at-rich gram-positive bacteria that have a well-established record in industrial food fermentations where they contribute to conservation, flavour and texture. in addition, several lab are used as food-grade hosts for the production of enzymes, peptides or metabolites. finally, lab are exploited in functional foods that contribute to the health and well-being of the consumer. a variety of metabolic engineering approaches have allowed for the improvement of many attributes of lab. these approaches have been facilitated by the possibility of uncoupling of growth and metabolite production in lab, the wealth of genetic tools that allow modulation of gene expression in a dynamic range, and the determination of several complete lab genomes (de vos et al., ) . we have developed lactobacillus plantarum as a paradigm for lab engineering by experimental and modelling approaches, the application of functional and comparative genomics, and the implementation of other post-genomics avenues (kleerebezem et al., ; smid et al., ; de vos et al., ) . examples of optimizing the production of vitamins and other cofactors, the impact of these engineering approaches on the global transcription and metabolite profiles, and determining the l. plantarum activity in the human host will be discussed. solanum tuberosum (potato) is the fourth major crop worldwide and used for food, feed and biotechnological applications. to fully realize the biosynthetic potential for production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. a total of , sage (serial analysis of gene expression) tags of nt representing , different tags were analyzed. the tags seen or more times were assigned a tentative function by comparison to homologous genes. contrary to the transcript profile of rice seedlings (gibbings et al., ) the storage organ of potato is not dominated by transcripts encoding storage proteins. transcripts for four types of protease inhibitors, a metallothionein and a lipoxygenase were more prominent than patatin isoforms. the lactic acid bacterium lactococcus lactis is used extensively in the production of fermented milk products. during cheese production the bacterium experiences many changes in its immediate environment, as a result of its own reactions. the most severe change is the accumulation of lactic acid, which changes the ph of the medium until growth is totally inhibited. we have focused upon a survey of these dairy related stress responses, as a means of constructing more robust strains. when l. lactis starter cultures are produced in rich media, they will experience an initial period with purine limitation after being added to the milk substrate, a stress condition that in several studies have been found to induce cross resistance towards a number of other stresses. we have analyzed both general purine nucleotide (atp and gtp) and specific gtp limitation in chemi-cally defined medium, using both proteomics and transcriptomics. the differential expression analyses were performed with a custom designed dna microarray of pcr amplified probes. the two stress conditions resulted in very different stress responses, both at the transcriptomic and proteomics level. from a new study on the temporal expression pattern of l. lactis during growth in milk, we present preliminary data showing differential expression of genes and proteins of the purine stress stimulon as well as other stress stimulons. cell physiology of the yeast saccharomyces cerevisiae glucose repression mutants ∆snf , ∆snf and ∆snf ∆snf was studied in batch and glucose limited chemostat cultivations. detailed physiological studies were performed on cells grown in batch using glucose, galactose, or glucose-galactose mixture as a carbon source. during growth on glucose-galactose mixtures it was shown that after glucose was consumed, galactose consumption remained repressed for about h in ∆snf or ∆snf mutants, and for more then h in ∆snf ∆snf mutant, whereas it only lasted h in wild-type cells. the global transcriptional response in the glucose repression mutants was studied using chemostat cultures. s. cerevisiae wild type and the mutants were grown in glucose limited aerobic chemostats at a dilution rate of . h − . biological triplicates were performed for each strain. to identify transcriptional responses of the glucose repression mutants, statistical tests, clustering method and a model-driven analysis method were used. the global transcription data analysis experiments showed that genes involved in hexose transport, carbohydrates metabolism, respiration, and signal transduction were differently expressed in ∆snf and ∆snf mutants comparing to wild type cells. combination of gene expression data and gene-scale metabolic model indicated changes in the metabolic sub-networks among studied glucose repression mutants. genomics technologies have recently been introduced into food and nutrition science for identifying targets of molecular actions of nutrients as well as non-nutrient components of foods. changes in the transcriptome, proteome and metabolome have been determined for assessing the molecular actions of zinc as an essential micronutrient and of flavonoids in processes such as colon carcinogenesis and atherosclerosis. zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. zinc deficiency effects in human cells and an animal model (rats) were analyzed by microarrays and showed that a low intracellular zinc concentration caused major alterations in the steady-state mrna levels of several hundred target genes-dependent on the tissues studied including liver, brain, muscle, intestine and kidney. proteome analysis from the same samples by d-page followed by peptide mass fingerprinting via maldi-tof-ms identified similarly a large set of proteins with altered expression level but allowed a common theme of action to be identified. although pleiotropic in first view, the obtained pattern of zinc-affected genes/proteins may represent a reference for defining the zinc regulon in mammalian cells. flavonoids occurring in large number in plant species are considered protective agents in a variety of processes including inflammation and cancer development. we have studied the effects of around selected flavonoids in a screening program and identified for compounds such as flavon, genistein or quercetin by genomics technologies their putative mode of action in colon cancer models and endothelial cells. as part of a collaborative effort employing human endothelial cells and blood mononuclear cells from a human intervention trial with soy isoflavones (genistein/daidzein) the effects of the flavonoids on the stress-response to oxidized ldl and homocystein was analyzed. a set of markers of anti-inflammatory and anti-apoptotic activity was identified for genistein and daidzein and cell biological studies confirmed that both compounds prevented programmed cells death in stressed endothelial cells. in the food processing industry, unwanted presence of extremely heat-resistant bacterial endospores creates major problems due to their capability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. screening of spoilage isolates using genomic typing techniques to visualise putative genome-based biomarkers allowed us to classify strains according to the degree of thermal resistance of their spores. in addition, we showed that sporulation in the presence of ingredients rich in calcium ions promotes thermal resistance of developing spores and correlates with the expression of specific (marker) genes (see oomes and brul, ) . finally, the molecular program that forms the basis of spore germination has been assessed using genome-wide expression analysis. noticeably genes involved in dna-repair were transiently expressed in germinating wild-type spores. also, surprisingly, it was found that spores contain significant levels of ribosomal and messenger rnas. degradation of these rna molecules upon spore thermal injury was found to be characteristic for their thermal resistance and predictive for their subsequent outgrowth behaviour. this finding is currently being patented. the information on spore presence, predictions of their thermal resistance and process survival chances, is used to structure a process management system to facilitate optimal food quality assurance and allow for real time analysis in case of the need for quality control. the information on spore presence, predictions of their thermal resistance and process survival chances is now being integrated. this is used to formulate the conditions for a process management system with state of the art food production quality assurance, which allows for real time analysis in case of the need for quality control. the interplay of the pectinase spectrum of aspergillus niger as revealed by dna microarray studies elena martens, jac benen, johan van den berg, peter schaap fungal genomics group, laboratory of microbiology, wageningen university, dreijenlaan , ha wageningen, the netherlands. e-mail: elena.martens@wur.nl (e. martens) the saprobic fungus aspergillus niger is an efficient producer of extracellular enzymes many of which show carbohydrate modifying activities. these enzymes have gras status and therefore are widely used in the food and feed industry. after determination of the genomic sequence of a. niger by dsm, it was estimated that only a fraction of the potential of secreted enzymes is currently characterised. database mining using the proprietary genome sequence has resulted in the identification of more then genes encoding enzymes involved in the depolymerisation of the back bone and the site chains of the complex polysaccharide pectin. additional enzymatic activities required to remove methyl and acetyl esters, present in pectin were also observed. by using dna microarrays we have sought to gain insight into the complex regulation of all the genes involved in pectin degradation. a. niger was cultivated on sugar beet pectin and on the monomeric constituents of pectin, viz. galacturonic acid, rhamnose and xylose. subsequently the corresponding transcriptomes were analysed. we will report on our findings concerning the regulation of the expression of the genes involved in the degradation of pectin and its main constituent-galacturonic acid and the consequences for the interplay of the encoded (novel) enzymes. since reactive oxygen species (ros) are formed in all living organisms a wide range of antioxidative enzyme systems are present to keep the system in balance. when an animal is slaughtered the cellular anti-oxidative capability is reduced, resulting in an accumulation of ros followed by an increased oxidation of dna, lipids and proteins. generally lipid oxidation is a well-known problem, causing increased rancidity during prolonged storage, of especially fatty fish. the implications of protein oxidation are, however, more unclear also in respect to quality decay of fish. protein oxidation causes a wide variety of amino acids modifications, where of the most studied is carbonylation of proline, argenine, lysine or threonine. these carbonyl groups can be labelled with , -dinitro-phenylhydrazine. combining two-dimensional gel electrophoresis with immunoblotting enables the detection of carbonyl groups for each single protein. the results presented here, reveal that both during frozen storage and tainting of rainbow trout protein oxidation/carbonylation increases, furthermore there is an increase in oxidation/carbonylation for distinct proteins. anne-marie neeteson european forum of farm animal breeders, benedendorpsweg , wl oosterbeek, the netherlands society is concerned about food, animal welfare, food safety, new technologies, scientists and industry. these elements are all present in genomics for farm animals. therefore, it is important to build awareness in scientists and industry, start a dialogue with stakeholders and society, and to be transparent in a pro-active way. this paper will address the issues at stake for scientists and industry, when it comes to genomics and animal health. it will combine the results of imperical, ethical and sociological efforts in three eu funded projects. (c) the proper use of genomics in relation to infectious diseases in production animals, and the role of the scientist in the development in new technologies in this field, are being addressed in european animal disease genomics network of excellence (ead-gene, http://www.eadgene.org/). some observations are that: ( ) genomics does not concern changing the gene. however, acceptability of any discovery dealing with living beings and edible products, is not obvious just like that! animals have a symbolic and emotional load. ( ) genes are still related to eugenics, in the mind of people. genes as such cause reluctance, but it is seen as positive if the use of medication can be reduced, and if animals will be better resistant to disease. ( ) consumers are in favour of consumer education, compulsory labelling and the imposition of minimum standards. the inclination to pay more for foods produced according to desired standards relates closely to income level. ( ) animal welfare is the major issue citizens mention as a concern. the focus of breeding organisations on productivity should be counterbalanced by serious attention to the animal's needs in order to avoid unnecessary negative impact on the welfare of the animals. ( ) when technical specialists and lay people communicate, they tend to use different languages: they use the same words, but with rather different interpretations. so transparency of breeding practices and clear definitions of terminology will be essential for effective communication among all stakeholders. ( ) food safety and human health are the major concern for most people, when it comes to making a choice. during the latest decades research within the field of animal genomics has in general been following the same strategies as those used within the field of human genomics, although with much less resources. the porcine genome has been characterized intensively through the development of linkage maps, comparative maps and physical maps. until a few years ago it had not been anticipated that it would be possible to embark on whole genome sequencing of animals genomes. however, because of technological developments and much lower costs for sequencing, several animal genomes have now been assembled/are on the way to being assembled. the initial step towards sequencing the porcine genome was taken by the sino-danish pig genome project. the efforts within this project have now generated approximately . million genomic shotgun sequences and . expressed sequence tags (ests). the shotgun sequences have been included in a three-species alignment to make an initial evolutionary analysis. the results show that pig is much closer to human than mouse is. the ests represent -end sequences from a total of non-normalized cdna libraries. based on assembly and annotation of the ests the structure of the porcine transcriptome has been analysed. the relevance of assembling the porcine genomic sequence is justified both from the perspective of sustainable animal breeding and from the fact that the porcine model is an important research platform because of the anatomical, physiological, biochemical and metabolically similarities with man. examples of functional genomic studies both aimed at sustainable animal breeding and aimed at exploiting the pig as a model for medical studies will be discussed. genomics refers to global, systematic and high throughput approaches that allow collecting large amount of data and thus offer new possibilities for analysis and understanding biological processes. we will present some new knowledge related to reproduction in farm animals resulting from three different strategies. ( ) functional analysis of gene and protein expression: the transcriptome and the proteome analysis allowed to identify new genes and proteins whose expression is associated with processes of ovarian follicular growth and atresia as well as oocyte maturation in bovine and porcine species, maturation of spermatozoa in the different compartments of epididymis. farm animals produce food as cost effectively as possible, however this may have negative side effects for their health and welfare. trade off processes between production on one hand and reproduction and health on the other hand play a crucial role. the principles of selective breeding for the best of naturally occurring variation has proven to be able to balance an increased level of production and quality of life for the animal. every year, the economic value of the genetic gain achieved by the breeders and carried over to the producers is . % of the economic value of eu farm animal production. consequently, a conservative estimate of the gain from animal breeding is, every year d . billion in europe. recent developments, such as the sequencing of the genomes of the human, chicken and cow, together with high throughput laboratory techniques, means that there are new opportunities to enhance quality of life. the goal of this paper is to give an overview of the options offered by genomics for enhanced quality of life with focus on identifying relevant gene variants and technologies for large scale tracking and tracing. selective breeding for the best of naturally occurring variation remains the same as in traditional systems, but by pinpointing the relevant gene variants along genomics it is possible to identify directly the animals best selected for high production without comprising health and welfare. the combination of full genome sequences, software tools, study of functional physiological processes cost-effective high-throughput snp genotyping and comparative mapping have the (proven) potential to identify relevant gene variants, e.g. pork color, boar taint, general disease resistance. functional mutations have direct option of application in breeding programs. unfortunately this is not the case for genetic markers due to cost of genotyping and inconsistent phenotypic effects. new technologies for snp genotyping are cost effective and enable large scale genotyping ( . of animals/day). a selection of the best technology and strategic use of these opportunities enable tracking and tracing. the application of this technology offers new opportunities for quality of life, both for animal and humans. background: studies have shown that prebiotic and probiotic consumption alters the gastrointestinal flora, modulates the immune system, inhibits genotoxicity and has a protective effect on colon carcinogenesis. however, the effect of synbiotic consumption on these parameters in subjects at risk of colon cancer has not until now been investigated. aim: to determine if a synbiotic (prebiotic and probiotics together) modulates cancer risk biomarkers in human subjects at risk of colon cancer. methods: a -week randomised, double blind, placebo controlled, ethically approved trial of a food supplement containing lactobacillus gg, bifidobacterium bb- and raftilose synergy (prebiotic) was performed in colon cancer subjects who had undergone 'curative resection'. faecal and blood samples were obtained before (t , week ) midway through (t , weeks) and following intervention (t , weeks). rectal biopsies were obtained at t and t . the effect of synbiotic consumption on the faecal flora was assessed using standard plate count techniques. genotoxic damage was measured in single cells derived from biopsies using the comet assay. fw was prepared by diluting faeces : in dmem, ultracentrifugation and sterile filtration. the genotoxic (comet assay) and cytotoxic potential (almar blue assay) of fw was determined. peripheral blood mononuclear cells were isolated from blood and cytokine production in vitro assayed by elisa. natural killer cell cytotoxic activity and the phagocytic and respiratory burst activity of monocytes and granulocytes in whole blood were determined by flow cytometry. results: in the synbiotic group faecal numbers of bifidobacteria significantly increased (p < . ) and lactobacilli increased although not significantly (p = . ) while coliforms decreased (p < . ). enterococci, clostridium perfringens and bacteroides were unaffected. in the placebo group bifidobacteria decreased (p < . ), the other bacterial groups were unaffected. in biopsies genotoxic damage was increased in the placebo group at t versus t (p = . ) but was unchanged in the synbiotic group. the genotoxic and cytotoxic potential of fw was unaltered. synbiotic consumption significantly increased (p < . ), ifn-␥ production by pbmcs but il- , il- , il- , and tnf-␣ production was unaffected. natural killer cell, phagocytic and respiratory burst activities were unaltered. conclusion: synbiotic consumption did not have a strong immunomodulatory effect on the systemic immune system in this study, nor did it influence the genotoxic and cytotoxic potential of fw. however, synbiotic consumption altered the composition of the gut flora to a more beneficial composition as well as protecting against genotoxic damage in vivo, suggesting a protective effect of synbiotics against colon carcinogenesis. emmaÅrsköld, malin svensson, halfdan grage, peter rådström, ed w.j. van niel applied microbiology, lund institute of technology, lund university, p.o. box , sweden lactobacillus reuteri is used today in a variety of dairy products as a probiotic bacterium. several lactic acid bacteria have the ability to produce different kinds of exopolysaccharides (eps), which have the potential to be used as an alternative biothickener. however, the yield of eps is too low to be profitable in the food industry. to optimise the environmental conditions for eps formation a l. reuteri strain was chosen for a factorial design study. the factors used in this experiment were temperature ( - • c), ph ( . - . ) and sucrose concentration ( - g/l); for each factor three different values were chosen. the strain was grown in batch mode using a semi-defined medium at constant ph and sucrose as the carbon source. the results obtained with fermentations revealed that the highest eps formation was found at • c, ph . and g/l of sucrose. sucrose did not further affect the eps formation above a concentration of g/l. temperature and ph were significant for the eps formation, but only temperature was significant for growth. a central composite design study was chosen for further optimization of the ph and sucrose concentration for maximum eps formation. also the gene expression of the sucrase enzyme responsible for eps formation was investigated using qrt-pcr. the data were used to develop a model for growth and eps formation. dendritic cells (dc) play a pivotal immune regulatory role in the th , th and treg cell balance. dc are present in the gut mucosa and may thus be target for modulation by gut microbes. here, we screened a large panel of human gut-derived lactobacillus and bifidobacterium spp. for dc polarizing capacity: bone marrow-derived murine dc were exposed to lethally irradiated bacteria and cytokine and dc surface markers were analyzed. substantial differences were found among strains in their capacity to induce proinflammatory cytokines, while the differences for anti-inflammatory cytokines were less pronounced. bifidobacteria were weak il- , il- and tnf-␣inducers, while both strong and weak cytokine-inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- , il- and tnf-␣ production induced by otherwise strong cytokine-inducing strains, while il- production remained unaffected. those lactobacilli with greatest capacity to induce il- were also most effective in up-regulating surface markers. surface marker up-regulation was however reduced in the presence of weak il- -inducing strains. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. cell surface-associated glycolytic enzymes from lactobacillus plantarum v mediate adhesion to human epithelial cells and extracellular matrix proteins s.m. madsen, j. glenting, a. vrang, p. ravn, h.k. riemann, h. israelsen, m.r. nørrelykke, a.m. hansen, m. antonsson, s. ahrné, h.c. beck bioneer a/s, hørsholm dk- , denmark among the main selection criteria of lactic acid bacteria for probiotic use, the ability to adhere to intestinal epithelial cells, mucus, or extracellular matrix proteins is considered important. using a proteom based approach we identified a group of novel surface proteins that are non-covalently bound to the cell wall of the probi-otic bacterium lactobacillus plantarum v. surface proteins were extracted and analysed by gel electrophoreses followed by mass spectrometry analysis. the surface proteins included glycolytic enzymes like, e.g glyceraldehyde -phosphate dehydrogenase, which usually is a typical intracellular enzyme. a collection of lactobacillus species was screened and the phenomenon of surface-associated glycolytic enzymes was found in many of the analyzed species. this is to our knowledge the first example of surface-associated glycolytic enzymes in probiotic bacteria. however, in pathogenic bacteria these enzymes are well known and their surface localization is involved in adhesion to human epithelial cells and invasion. we suspect that the presence of these enzymes on the surface of probiotic bacteria could prevent adhesion of pathogenic bacteria and possibly also involve other probiotic activities such as immune modulation. binding studies showed that the surface-associated glycolytic enzymes of lb. plantarum v were able to bind to caco- intestinal cells and extracellular matrix proteins like fibronectin. scientific and industrial interest on exopolysaccharides (eps) synthesized by microorganisms and on their chemico-physical properties has been quickly growing in the last years. many strains of lactobacilli produce eps allowing them to adhere to human mucosae and therefore to have probiotic effects such as the stimulation of immune response and even antitumoral activity of these molecules has been claimed. futhermore prebiotic actions of eps beneficially affect the human host health improving the properties of indigenous microflora. in this research we have studied two novel interesting lactobacilli strains: lactobacillus plantarum dsmz and a particular human isolated strain of lactobacillus crispatus that have probiotic potentialities and are good eps and l(+)-lactic acid producers. the aim of this work has been to characterize these strains and their metabolites (eps, organic acid and bacteriocins), to state them as probiotics and to study their adhering ability on human cells. we have studied the physiology of l. plantarum and l. crispatus in shaking flasks as well as their optimal fermentation conditions to obtain high cell density cultures suitable for use as starters in the food industry and eventually for probiotic preparations. fermentation experiments have been performed in a bioreactor equipped with microfiltration (mf) modules using a semidefined medium and various carbohydrates in different culture conditions (aeration, temperature). the kinetic of eps production has been followed and according to results both strains have shown a growth-related production ranging from to mg/l. in vitro studies concerning the ability of these strains to adhere to human mucosae are in progress as well as the structural characterization of the exopolisaccharides. previous studies have shown that compression coating improves the storage stability of freeze-dried lactobacillus acidophilus, although this stability is related to the degree of cell injury, which in turn is related to the compression pressure used. compression coating has also been found to improve the survival of freeze dried l. acidophilus during exposure to simulated gastric fluid (sgf). the aim of the present work is to create a compression coated l. acidophilus formulation, with targeted release at the terminal ileum and beginning of the colon in the human gastrointestinal tract. dissolution studies were performed using a phosphate buffer with a ph of and . , to simulate gastric fluid and intestinal fluid (sif), respectively. cell viability was monitored using multi-parameter flow cytometry (mpfc), together with traditional cfu/ml counts. mpfc was used to identify live, dead and stressed cell populations, using the fluorescent stains propidium iodide (pi), , -dihexylocarbocyanine iodide (dioc ( )) and to-pro- . results show that an enteric coating material, eudragit l - , is both suitable for compression coating, and enhancing the survival of cells when exposed to sif. pectin usp has also been shown to promote targeted release of the cells. the opium poppy papaver somniferum contains more than tetrahydrobenzylisoquinoline-derived alkaloids. it is the source of the narcotic analgesics codeine and morphine, which accumulate in specialized internal secretory cells called laticifers. in the aerial parts of the plant, the laticifer cells are anastomosed, forming an articulated network. laticifers are found associated with the vascular bundle in all plant parts. the morphinan alkaloids morphine, codeine and thebaine are found both in roots and in aerial plant parts and specifically accumulate in vesicles within laticifers. the benzo[c]phenanthridine alkaloids sanguinarine and -hydroxysanguinarine are found in root tissue. the syntheses of sanguinarine and of the tetrahydrobenzylisoquinoline latex alkaloid laudanine are completely understood at the enzyme level. nearly all enzymes of morphine biosynthesis have also been described. in more recent years, cdnas encoding enzymes of alkaloid biosynthesis in p. somniferum have been isolated and characterized. the cell-specific localization of several of the enzymes of morphine, sanguinarine and laudanine biosynthesis has also been described. with knowledge of many of the gene of alkaloid formation and their sites of expression, the metabolic engineering of p. somniferum for tailored alkaloid profiles is now being undertaken. an agrobacterium tumefaciens-based transformation and a regeneration protocol have recently been developed specifically for narcotic tasmanian cultivars. the various cdnas encoding genes of alkaloid biosynthesis in p. somniferum are being systematically reintroduced into the plant to achieve engineered plants with altered alkaloid profiles. the first results have now been obtained with sense and antisense genes stably expressed in a regenerated tasmanian cultivar. the ultimate goal of exploiting the genes of alkaloid biosynthesis is to produce transgenic medicinal plants of specific alkaloid content that would facilitate commercial production and improve our understanding of the factors that regulate biosynthesis as well as provide experimental systems with which to investigate the ecological role of alkaloids in planta. integrated transcript and metabolite profiling for gene discovery in plant natural product pathways richard a. dixon, lahoucine achnine, bettina deavours, mohammed farag, marina naoumkina, lloyd w. sumner plant biology division, samuel roberts noble foundation, ardmore, ok , usa the rich diversity of chemical structures found in the plant kingdom arises in large part from a limited number of basic chemical scaffolds (e.g. terpene, polyketide) that are modified by a limited number of chemical substitution types (hydroxylation, glycosylation, acylation, prenylation, o-methylation, etc.) . much of the diversity is brought about by the substrate-and/or regio-specificities of the substitution enzymes. in contrast to the large collections of gene sequence and transcript level data available on-line, little detailed information exists on the plant (secondary) metabolome. promiscuity of substrate specificity in vitro may complicate attempts to assign functions to genes of secondary metabolism accessible to researchers through various cdna library collections. using the isoflavonoid and triterpene pathways in medicago species as examples, we describe how integrated metabolite and transcript profiling approaches can aid functional genomics, help explain metabolic regulation, and provide tools for assessing the impacts of genetic modifications in plant secondary metabolism. focused and non-targeted approaches were used to assess the impact associated with introduction of new high flux pathways in arabidopsis thaliana by genetic engineering. transgenic a. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of cyp a , cyp e , and ugt b from sorghum bicolor accumulated % dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. in a similar manner, plants expressing only cyp a accumulated % dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. in contrast, insertion of cyp a plus cyp e resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific uv protec-tants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. the accumulation of new glucosides in the plants expressing cyp a and cyp e , was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. the pleiotrophic effects observed in plants expressing sorghum cyp a and cyp e were complemented by retransformation with s. bicolor ugt b . accordingly, insertion of high flux pathways directing synthesis and intracellular storage of high amounts of natural products is achievable in transgenic plants with marginal inadvertent effects. arabidopsis thaliana-distinct function in gene transcription dynamics? jeppe madura larsen, brian stougaard vad, søren mølgaard, kell andersen, mads n. davidsen, klaus d. grasser department of life sciences, aalborg university, aalborg, denmark in recent years it has been shown that introns to some extent can regulate expression in the eukaryotic cell. insertion of introns in the utr in arabidopsis genes has shown to increase gene expression at both rna and protein level. in order to investigate if utr introns have distinct characteristics, we analyse these in the well annotated arabidopsis thaliana genome published by the arabidopsis genome initiative. , loci annotated with full-length cdnas were analysed and loci ( . %) containing utr introns were isolated. we studied if the genes containing these introns showed different patterns in alternative splicing and gene function (gene ontology classification) compared to the remaining genes not containing this intron type. of the isolated loci ( . %) contained only one utr intron and these were used for further analysis, where it was investigated if the utr introns had a characteristic size distribution. genes containing transcripts with utr introns were more subjected to alternative splicing ( . % versus . %) and had a tendency to be more involved in cell regulatory functions compared to genes without this intron type. it was also found, that utr introns was characteristically size-distributed. we identified thee predominant sizes of approximate , and bp compared to only one for orf introns. this suggests widespread multiple splicing events in utr introns. the results presented here suggest that utr introns have distinct characteristics and function in gene transcription dynamics. cytochrome p monooxygenases appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites in microbial, animal and plant cells. in particular, cytochrome p scc catalyzes the conversion of cholesterol into pregnenolone-the precursor of all steroid hormones in mammalian steroidogenic tissues. cytochromes p are also involved in the biosynthesis of different plant steroid derivates that play important role in regulation of plant growth and development. therein, investigation of possible influence of cytochrome p scc expression on plant regulatory system is of a great interest. this report devoted to the investigation of transgenic tobacco plants, which have been generated by the transformation with recombinant plasmid pgbp f constitutively expressing cyp a cdna of the bovine cytochrome p scc . the transgenic state of the plants was confirmed by southern blot analysis. transgenic plants are phenotypically different from the control ones. in particular, they obtained a substantially higher growth rate and are larger than wild type plants. we have demonstrated that incubation of fragments of the transgenic plants leaves in [ c]-labeled cholesterol containing medium results in formation of the radioactively labeled product with chromatographic mobility corresponding to pregnenolone. the presence of this metabolite in the steroid fraction of lipid extracts obtained from the transgenic plants leaves was confirmed by gas chromatography mass spectrometry (gc-ms) method. the data obtained indicate that cytochrome p scc synthesized in transgenic plants displays its specific catalytic activity. biotechnological production of glucose isomerase enzyme with streptomyces olivochromogenes for production of fructose syrup from hydrol m. hashemiravan, a. sadat barikani department of food science and technology, azad university (pishva, varamin unit), institute of food science and agriculture, tehran, iran the use of glucose isomerase for isomerization and production of fructose syrup was performed by selected industrial strain of streptomyces olivochromogenes ptcc . growth of microorganism and production of enzyme in different culture media was studied, and effects of different parameters such as phosphate and aeration was evaluated. the growth of microorganism at • c, caused a production of high amount of enzyme. the production of enzyme was considered in two culture media (a and b). medium (a) was selected for the higher production amount of enzyme. the highest amount of enzyme production was seen in medium a, which was . giu/ml, after h. the use of baffles in culture flasks, increased the amount of enzyme production, four times more. the production of enzyme was increased, . times more, in phosphate deficient medium. the cells containing enzyme (intra cellular glucose isomerase) was separated by centrifuge, and extraction and release of enzyme was performed by ultra sonication, that is a physical-mechanical method. this method released about . % of total intracellular enzyme. the best length of time for sonication was found to be min. experiments showed that optimum ph and temperature of the enzyme were . - and • c, respectively. the highest activity of the enzyme was observed at ph . for up min. at the time of isomerization reaction the existence of magnesium ions showed to be necessary and omission of this ion cause a decrease of enzyme activity in isomerization process, but this effect was not necessary for the enzyme activity results showed that treatment of glucose syrup at temperatures of , and • c, by the enzyme, caused %, % and % of isomerization, respectively. efficiency increase of high acetic acid production with the use of acetobactereace iranian native strains mutation m. hashemiravan , a. alirezasadat barikani : department of food science and technology, azad university (pishva, varamin unit) institute of food science and agriculture, iran; young iranian researchrer's club, tehran, iran the first step in the present research is the isolation of acetobactereace native strains from fruits (such as grape or apple) and fresh vinegar. this separation has been done with the use of effective isolation methods and optimized mediums. ten strains were isolated effectively, the bio chemicals test were performed, each of them were detected, classified and nominated with a special code. two methods have been used for mutation: (a) mutation with the ultraviolet radiation; (b) mutation with nitrous acid. in the first method the microbial cells were treated with us radiation for different periods of , , and s, and the effect of the uv mutation was assessed. as a result, the period of s was determined as the optimum mutation periods. in the second method, the microbial cells were first washed with the acetate buffer . m with the ph of . , then ml nitrous acid . m was added and was mixed for - min. finally the sampling was done in the periods of , , , and min and was transferred to a plate containing the medium of ethanol-phenol red-agar. the two methods have been compared with each other. each method has its own advantages and disadvantages. the mutation pathway in method (b) is more stable and conducted, while mutation with the uv radiation method, change the position of thiamine-cytosine bases absolutely randomly. finally, the best mutant site was inoculumed with the medium containing alcohol %, acetozyme gz . g/l, acetozyme d g/l, acetic acid . % and l double distilled water. the acetic acid was produced in the rate of g/ ml. also the mutant strains were detected with scanning electron microscope (sem) and interesting photographs were taken from mutant cell. the performed experiments were planned with the use of taguchi statistical method (qualitek ). this research has been performed in the irost as the thesis of ph.d. in food biotechnology industry. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with . % (w/v) epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph . ) or chloride (ph . ) as leading ion and -amino-caproic acid (ph . ) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic danalysis. genotypical differences affecting the response of pisum sativum to differing boron/iron applications e.e. hakki, u. zeynep, m. hamurcu, a. tamkoc, m.b. babaoglu, s. gezgin department of field crops, faculty of agriculture, selcuk university, kampus, konya , turkey. e-mail: eehakki@selcuk.edu.tr (e.e. hakki) boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plantation areas of turkey. both defficiency and toxicity problems exist in a total of about % of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant aquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. increase in sulfite production by accelerating sulfate uptake in brewing yeast t. fujimura, y. kodama, y. nakao, n. nakamura, w. miki institute for advanced technology, suntory ltd., mishima-gun, osaka - , japan sulfite plays a role as an antioxidant, which stabilizes beer flavor. therefore, it is important to control the sulfite concentration during fermentation. sulfite is produced as an intermediate in the sulfate assimilation pathway in yeast. we have already reported that over-expression of a lager yeast-specific ssu gene, encoding a sulfite efflux pump, leads to increase of sulfite production (fujimura et al., ) . in the present work, we have clarified that there are two types of sul genes (scsul and non-scsul ) each encoding a high affinity sulfate permease in lager brewing yeast. eighty percent and % identity are found by comparing the dna sequences and the deduced amino acid sequences, respectively. a comparative functional analysis of the two genes has been performed aimed at achieving further increases in sulfite production by accelerating sulfate uptake. over-expression of scsul and non-scsul has been achieved by transformation of lager brewing yeast, saccharomyces pastorianus. experiments have been done with and without expression of non-scssu . the resultant transformants have been evaluated by fermentation tests in wort. over-expression of either scsul or non-scsul failed to show significant effect on sulfite formation. a combination of over-expression of non-scsul and non-scssu resulted in two-fold higher sulfite production compared with overexpression of only non-scssu and four-fold higher compared with the parental strain. these results suggest that the non-sc-gene types significantly contribute to sulfite production in lager brewing yeast. , t., et al., . functional phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyse the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. possible applications of phytases have been suggested in animal nutrition to increase mineral bioavaliability and to decrease phosphate pollution in area of intensive life stock management and in human health. zea mays is one of the cereals that contain high amount of phytate as the major phosphate storage compound. over bacteria were isolated and screened for phytases from the halosphere, rhizosphere and endophyte of malaysian maize plantation. the phytase activity of the isolates was screened by a modification of the ammonium molybdate method. the highest extracellular phytase activity was detected from bacteria that isolated from the endophyte of the maize root. in this paper, results for isolates chosen for media, temperature and ph optimization will be presented. production of plant proteinase from jack fruit seeds (artocarpus integrifolis) and its influence on rheological and sensory characteristics of low fat yogurt el-sayed el-tanboly dairy sciences department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, . (t ), . (t ) and . (t ) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period ( days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t showed more less firm after days of storage being . g/ g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t and t of and mpa s, respectively, compared with control of mpa s at days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t gained the highest scores ( points) followed by the control ( . points) after days of storage, while yogurt of t showed a low scoring being . from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of . units of plant proteinases/ml milk. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions g/l from white distilled lees and . g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. toasted wine was traditionally produced in galicia, northwest of spain. nowadays this technique is being recovered. grapes after harvesting are air dried in order to concentrate sugars, acids and flavor compounds. raisings are pressed to obtain a must with high sugars concentrations. two different grape wines were prepared concentrating the sugars up to and brix, respectively. in order to get a better knowledge of the problems involved, synthetic media simulating the grape musts were prepared. theses musts were used to optimize the initial sugar concentration, the amount of nutrients required, the optimum temperature to carry out the fermentation and the influence of the type and amount of yeast. under the best conditions some fermentations with grape must were carried out to produce wines with intense aroma and flavor notes and high residual sugar concentrations. in this studies, bacillus sp. e strain was isolated from koreanstyle fermented soybean paste and it was producing the biological response modifier (brm). the brm activated the b cell selectively. it was identified the bacillus licheniformis e . the brm was purified by ion-exchange chromatography and gel filtration. chemical properties of brm: molecular weight of brm was estimated to be about , , da. sugar content of brm was . % (w/w) and glucosamine ( . mol%) was the high level. protein content of brm was . % (w/w) and serine ( . mol%) was the high level. infra-red absorption spectrum was showed the characterization of glycoprotein. biological properties of brm: the brm which isolated from fermented soybean paste was similar to that of bacillus licheniformis e by immuno-fluorescence assay. we confirmed that the brm was capsular substance of b. licheniformis e . potato nitrogen concentrate (pnc) is a highly viscous liquid with high complex nitrogen content produced from the protein-fraction in potato starch extraction. the concentrated extract is rich in minerals and ␣-amino nitrogen. although ␣-amylase nowadays is mainly produced exploiting bacillus production systems there is still considerable demand for fungal ␣-amylase from aspergillus oryzae origin. the aim of the experiments to be reported here was to investigate, if pnc can replace commonly used complex nitrogen sources in the production of fungal ␣-amylase. the following data have been measured in pnc pretreated by diluting to / and clarifying by centrifugation. total-n: . g n/l; ␣-amino-n: . % (w/v) (as glycine); soluble protein (bradford): . mg/l (as bsa); total carbohydrates: . g/l; reducing sugars: . g/l; dry weight: . % (w/w). in the following experiments nitrogen sources were replaced on the basis of their ␣-amino nitrogen content. the carbon source for all experiments was maize starch. the formation of ␣-amylase by a. oryzae atcc in shake flasks -using pnc (centrifuged or not), yeast extract, malt extract, casein hydrolysate or meat extract -was compared to "standard" cultivation with corn steep liquor. the experiments showed only small differences in ␣-amylase titers using complex nitrogen sources. no remarkable differences were observed in the resulting biomass. in general no differences in enzyme productivity and biomass formation could be seen after h of incubation. especially the bench top bioreactor experiments indicated an optimal fermentation time of about h. cultivations of a. oryzae atcc were carried out in bench top bioreactors. comparing cultivations in a medium with pnc as the sole complex nitrogen source to one containing csl as such no significant differences both in the formation and amount of ␣-amylase and the fungal growth were observed. thus pnc might be able to replace complex nitrogen sources such as csl or even the more expensive yeast extract and casein hydrolysate in fungal amylase production systems. . ) is involved in the metabolism of inositol and catalyzes the conversion of d-glucuronic acid to l-gulonic acid with nadph as a cosubstrate posterior to the oxidation of inositol to glucuronic acid by the enzyme inositol oxygenase. although the yeast sporobolomyces oryzicola (nakase and suzuki, ) is not able to grow on inositol as the sole carbon source, intracellular glucuronate reductase can be found in cells grown in a medium containing d-glucuronic acid. the enzyme could be a useful tool in the design of a specific quantitative assay for glucuronic acid, e.g. in so called energy drinks. the organism was grown in media containing either glucose and glucuronic acid or only glucuronic acid and difco yeast nitrogen base. whereas growth on both media was similar in shake flask culture, hardly any growth in either medium was observed in bench top bioreactors. the influence of dissolved oxygen tension was investigated and the relevant data will be shown. the formation of intracellular glucuronate reductase activity by sp. oryzicola is inducible by media containing glucuronic acid. no activity is found in cells grown in a medium containing only glucose as the carbon source. besides the activity against d-glucuronic acid, activities against -ketogluconate and -at very low levelsagainst galacturonic acid and the lactone of glucuronic acid were detected. the enzyme activity is stable up to • c. the ph has relatively low influence on the activity against glucuronate, whereas the reduction rate of -ketogluconic acid is optimal at ph . - . with significantly lower values at ph . and . , respectively. data on the kinetics of the conversion of both glucuronate and -ketogluconate will be shown. nakase, t., suzuki, m., . j. gen. appl. microbiol. , - . the multiple nutritional and functional impacts of food fermentation on human health have been widely accepted (reddy and pierson, ; hugenholtz et al., ) . however, the related role of the involved microorganisms to the nutritional effect from the fermented food is still not well defined and the mechanisms involved are still largely unknown. the present study was to investigate iron bioavailability in carrot juice fermented by two selected lab strains, l. pentosus fsc and ln. mesenteroides fsc . after digestion by gi enzymes, the juice was supplied to fully differentiated caco- cells to study iron uptake and transepithelial transport by caco- cells from the digested juice. our data revealed strain specified changes in iron bioavailability in carrot juice fermented by these two strains. after in vitro digestion with pepsin and pancreatic-bile enzymes, the best yield of soluble iron was from ln. mesenteroides fsc fermented juice. surprisingly, the l. pentosus fsc fermented juice yielded about five times higher uptake iron as compared to fresh juice, while ln. mesenteroides fsc fermented juice was not significantly different from the fresh juice. interestingly, the transepithelial transferred iron across the cell line was however better from ln. mesenteroides fsc fermented juice than from l. pentosus fsc fermented juice. to summarise, our study showed that level of soluble iron after in vitro digestion does not necessary indicate iron absorption, especially in the case of lab fermented food. data on improved iron uptake from l. pentosus fsc fermented juice indicated exiting of promoter(s) for iron absorption in such juice that is not related to the production of organic acids and lowering ph effect. peng zhang, herve vanderschuren, martin stupak, wilhelm gruissem institute of plant sciences, zurich, the tropical root crop cassava (manihot esculenta crantz) is a major source of food for approximately billion people worldwide. in sub-saharan africa, more than million people rely on cassava as their major source of dietary energy. in many parts of africa and latin america, cassava leaves are a vegetable source for daily uptake. cassava is grown mostly by poor farmers under marginal environmental conditions and in areas where few other crops can sustain competitive yields. the crop is therefore fundamental for subsistence farming and food security, but it is also very susceptible to stresses common in the areas and conditions where it grows. in many parts of africa, reliable cassava production is strongly impacted by infections with the african cassava mosaic geminiviruses (cmgs), a rapidly spreading disease that causes large yield losses. in the coastal areas of east africa, cassava production now is threatened by another devastating disease, cassava brown streak disease (cbsd). cassava plants are also frequently attacked by many pests, such as cassava hornworm and stemborers. several reports also indicate that greater leaf longevity, especially under drought conditions, could be important for increasing yields and/or the stability of production in cassava, as well as improve the access to an important nutrient source. conventional breeding efforts have attempted to address the constraint to cassava production, but with limited success. the new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava. these new technologies have the potential to make cassava much more productive, a better source of nutrients, and profitable to grow, hence, greatly contributing on the sustainable development of tropical agriculture. recently we have developed biotech cassava with value-add traits, including resistance to cassava mosaic virus, prolonged leaf life and insect resistance. new strategies are also explored to increase protein content of cassava storage roots. we are currently undertaking pilot studies with two teams of leading scientists and experts for projects to test acmv-resistant transgenic cassava lines in africa and lines with extended leaf retention at ciat, colombia under field conditions. this development of substantially equivalent improved transgenic cassava lines is part of a larger study to analyze the need, effectiveness and biosafety of biotech cassava for agricultural production. the goal of the pilot studies will be the development and coordination of a broader project that produces important and novel scientific results, valuable information on the need and impact of biotechnology at the subsistence farming level, and a sound scientific basis for the development of guidelines for biosafety assessments and release of transgenic organisms into the environment and agricultural production in africa and latin american countries. this study was conducted to reveal the effects of different pretreatments on obtaining haploid plants by using the anther culture in pepper capsicum annum l. cultivars demre sivrisi and sirena. buds were collected at uninucleate microspore stage. anthers collected from buds were cultured in ms medium containing different hormones and hormone concentrations. experiment results revealed that when sirena anthers were pre-treated cold at + • c for h and kept in darkness at • c for a period of week gave good results. in the case of demre sivrisi anthers were pre-treated cold at + • c for h and kept in darkness at • c for a period of week gave good results. on the other hand no cold pretreatment to anthers resulted with low embryo formation. similar results were also observed on the anthers kept at • c for week as callus was produced in some petri dishes but no regeneration was observed. as a conclusion, since no cold pretreatment to anthers resulted with low embryo formation it is possible to say that cold pretreatment should be applied to anthers in pepper another culture studies. the yeast d. hansenii ufv- was tested in this work in batch experiments in synthetic media at constant initial substrate concentration ( g l − ) under variable oxygenation conditions. to get additional information on its fermentative metabolism, a stoichiometric network was proposed on the basis of the general knowledge available in the literature on xylose metabolism in pentose-fermenting yeasts and the specificities of xr and xdh activities in d. hansenii and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. it can be stressed that under strongly oxygen-limited conditions xylitol production was negligible, whereas under semi-aerobic conditions maximum xylitol production (p max = . g l − ) and yield (y p/s = . g g − ) were obtained. a progressive decrease in these parameters was observed under fully aerobic conditions, suggesting that xylitol-producing yeasts require limited oxygen conditions, which is species-dependent. the proposed model, which utilizes the experimental specific rates of substrate consumption and product formations, allows estimating the main bioenergetic parameters. besides, it proved to be an effective tool to investigate different metabolic situations and showed how they can influence the flux distribution of the carbon source and the bioenergetics of this biosystem. the effect of disinfectants on fungi anne svendsen, pernille skouboe bioneer a/s, hørsholm dk- , denmark prevention of mould spoilage of foods can only be carried out successfully, if the species, which are actually spoiling the food product, are known. a very limited number of fungal species has been associated with the spoilage of each food category. proper disinfection of production facilities is very important to avoid mould spoilage. resistance of moulds to disinfectant treatments are known and different species have shown different response to the same disinfectant. to obtain proper disinfection it is important to know the resistance of the spoilage fungi against different disinfectants. in this study the effect of disinfectants on the spoilage fungi of cheese, rye bread, liver paté and fruit juice was investigated. in collaboration with five food companies the dominating species responsible for spoilage of each food product were isolated and used for testing. commercial disinfectants and disinfectants "under development" were tested. tests were performed in suspension and on surfaces, the methods used were modified after en and en . considerable variability in fungicidal effect among the species was observed. some disinfectants were ineffective at low temperature. some disinfectants showed different effect in suspension and on surfaces, resulting in an effective kill in suspension and almost no effect on surface. the identification of effective disinfectants in the food industry includes: ( ) testing against the specific spoiling species of the food product, ( ) testing on surface, not only in suspension, ( ) test parameters adapted to the food manufacturing plant. intensified research efforts in recent years confirm the major importance of the microbial flora in the gastro-intestinal tract for human health. ingestion of prebiotic oligosaccharides increases the number of the desirable bacteria like bifidobacteria and lactobacilli in the colon. we are looking at beta-galactosidases from lacto-bacillus spp. for the production of galacto-oligosaccharides (gos) because we speculate that the enzymes of probiotics will form gos with high prebiotic potential. in this present study, purified betagalactosidases of selected lactobacillus strains were used for the production of gos from lactose. different enzyme reactor set-ups, both discontinuous and continuous, were tested and compared. temperatures up to • c and ph values between and . were required for satisfactory enzyme stability during the process. enzyme source, substrate concentration and the level of substrate conversion were found to be critical process parameters for gos yields and composition. yields of up to % (w/w) of total sugars were achieved when the initial lactose concentration was g/l. capillary electrophoresis (ce) and hplc with pulsed amperometric detection were the analytical tools for investigating the influence of reactor type, enzyme source and conversion level on gos composition. the prebiotics market is increasing rapidly and is expected to more than double until to about million d world-wide. therefore, the development of enzymatic processes on an industrial scale is a high priority goal of our research. starter addition does not always succeed in improving standardisation and quality of the complex sensory properties of traditional fermented foods. in many cases the added strains do not grow as well as the environmental strains present in the production plant. here, a method of geometric simplification (by dichotomy) of a complex ecosystem found on a raw milk livarot ( strains) was tested on cheese curd. by a limited number of cultures, successively, out of , out of and out of strains were selected on the basis of two criteria (i) respect of the taxonomic proportion, (ii) generation by the daughter ecosystems of an odour close to the one of the mother ecosystem. finally a sub-ecosystem of strains gave an odour similar to the one of the more complex mixture. the use of molecular methods (pcr-sscp) permitted to follow the main species growing. mother and daughter ecosystems were characterized by sensory analysis and gc-ms. probably because of an important redundancy of the strain functions, the method was very efficient. this method may permit to improve a lot the set up of mixture of strains and species used in fermented food industry. effect of the dilution rate on the exopolysaccharide production by bifidobacterium longum atcc c. shene, m. rubilar, s. bravo universidad de la frontera, chemical engineering, av. francisco salazar , casilla -d temuco, chile exopolysaccharides (eps) producing lactic acid bacteria are used in dairy industry (cheese and yogurt) due to the rheological properties that these compounds confer to the products. preliminary results also suggest the use of eps as health-promoting (anti-tumor and immunostimulatory actions) ingredients. bifidobacteria are grampositive bacteria natural inhabitants of the gut of warm-blooded animals and man. a number of investigations have shown that bifidobacteria promote host health mainly because of the reduction proliferation of some pathogenic bacteria through acid synthesis. in this work results obtained in the experiments carried out to test the capability of b. longum atcc to synthesize eps are presented. continuous culture fermentations were carried out at dilution rates between . and . h − . composition of the culture media was that of the mrs broth. biomass concentration presents higher values ( . - . g l − ) at dilution rates between . and . h − . biomass growing at these rates is difficult to pellet and adheres to the fermentor walls behavior that was not observed at other growth conditions. eps from cultures grown at these rates were preparated and fractionated. authors wish to thank the chilean conicyt for the economical assistance given through the project fondecyt . high pressure-low temperature (hplt) inactivation processes were performed on bacillus subtilis vegetative cells at various conditions. at atmospheric pressure, lowering the temperature to as low as − • c was found to have minor anti-microbial effects. upon application of high pressure various phase transitions occurred in the microbial suspensions under study. after pressure treatment at - mpa, cells were plated under optimal conditions to assess cell viability. treatments at - mpa and − • c were the most effective in inactivation. in these cases, ice i-iii solid-solid phase transition was observed. in addition, we hypothesised that intracellular thawing (solid-liquid phase transition) had already occurred while the extracellular surrounding was undergoing solid-solid phase transition. this double effect is suggested to be key in mediating the observed large drop in viability. we speculate that more cells survived after treatment at − • c compared to the same treatment at − • c because both the extra-and intracellular surrounding remained fully frozen. at − • c a solid-solid phase transition was observed when pressure was higher than mpa. a metastable state of ice i was observed at mpa treatment. results from the current study will be presented (see also shen et al., ifset in press) . the data call for a mechanistic evaluation of the effects of hplt as an anti-microbial treatment. such data are currently being gathered and will be used in defining optimal hplt process conditions for the food industry. the influence of saccharomyces cerevisiae, kloeckera apiculata and candida pulcherrima mixed cultures on the selected alcohols formation during model fermentation pawel satora, tadeusz tuszynski department of fermentation technology and technical microbiology, food technology faculty, agricultural university, cracow, poland. e-mail: psatora@ar.krakow.pl (p. satora) for the study five yeast species were chosen, isolated from successive stages of plum fruits spontaneous fermentation: from the beginning (candida pulcherrima, kloeckera apiculata, saccharomyces cerevisiae w ), middle (s. cerevisiae w ) and final fermentation (s. cerevisiae k ). to characterize the potential influence of yeast mixed cultures on the selected alcohols formation, wick-erham synthetic medium ( % glucose) was fermented by mixed cultures of two and three yeast species. after distillation, ethanol, propanol, isobutanol, isoamyl alcohols, hexanol and -phenylethanol were determined using gas chromatography. findings were compared with the results obtained after monoculture fermentations. the use of mixed cultures resulted in increasing of glucose utilization rate, ethanol and fusel alcohols formation (except propanol) and decreasing of methanol synthesis. the samples fermented using two yeast species characterized higher (about %) amount of volatile compounds in relation to monocultures. it takes note of especially high level of ethanol (av. . g/dm ), methanol ( . mg/dm ) and isoamyl alcohols ( . mg/dm ). the positive feature of triple cultures using was limitation of methanol and fusel alcohols synthesis that was accompanied by relatively high ethyl alcohol production (av. . g/dm ). the consumption of sugar syrup becomes increasingly significant in industrial processes due to economic advantages and the easy of use. the production of sucrose syrup using enzymatic hydrolysis represents the safest alternative, once the reaction does not produce any toxic or undesirable substance. this work consists on the production of sugar syrup by immobilized inulinase from kluyveromyces marxianus, with two alternatives process: (a) syrup enriched with fructooligosaccharides or solely with glucose and fructose. the process is comprised by the following stages: production and purification of the enzyme in optimized conditions, immobilization of the enzyme in solid support and the conversion of sucrose in a fixed bed bioreactor with the immobilized enzyme. the final composition of the product can be a mixture of glucose, fructose, sucrose and fructooligosaccharides or a mixture of fructose and glucose, according to the operational conditions. the bioreactor can be operated continually for approximately months with the same biocatalyst. the product from this process is ideal for applications in the food products such as sweet, candies, chocolates, yogurts, etc. besides, the prebiotics properties of the fructooligosaccharides, is a beneficial stimulant of the intestinal flora, which gives to the product a functional property. studies on plant microbial interactions using azotobacter sp. as bio-inoculants towards soil fertility baljeet singh saharan faculty of biotechnology, jcdm college of engineering, sirsa , india. e-mail: baljeet.saharan@gmx.de, baljeet br@yahoo.co.uk (b.s. saharan) high nitrogen fixing, phytohormone producing isolates of azotobacter, azospirillum, acetobacter and pseudomonas were used as inoculants on wheat and cotton with varying doses of nitrogen under field conditions. bio-inoculants were selected on the basis of yield, dry weight and survival rate of bacteria under field conditions. seeds of wheat variety wh were treated with different biofertilizers using nitrogen level of , and kg ha − and one level of p, i.e. kg ha − in field along with control. under field conditions, maximum yield was obtained with azotobacter chroococcum e at ( ± . kg ha − ) as well as kg ha − ( ± . kg ha − ) followed by a. chroococcum ht ( ± . kg ha − ) and avk ( ± . kg ha − ). whereas, with kg ha − highest yield was observed with mac ( ± . kg ha − ) followed by e ( ± . kg ha − ) and avk ( ± . kg ha − ). maximum height at kg ha − was observed with mac inoculation ( . ± . cm) followed by avk ( . ± . cm) and ht ( . ± . cm). various chosen strains were tested with desi (hd ) and american cotton (h ) under similar pot and field conditions as for wheat in the following season. plant height and yield were determined at the time of harvesting whereas survival rate was monitored at various intervals of time. survival rate of inoculated bacteria was determined after , , and days. highest survival rate was observed in mac (( . ± . ) × ), which decreased after and days, respectively. ( . ± . ) × , ( . ± . ) × with mac and ( . ± . ) × and ( . ± . ) × with ht , respectively. maximum boll weight was with avk ( . ± . g boll wt. plant − ) followed by pseudomonas ( . ± . g), ac ( . ± . g) and ala ( . ± . g) boll no. plant − was maximum with ala and avk ( ± . plant − ) followed by pseudomonas ( ± . plant − ). maximum height and dry matter was obtained with pseudomonas ( . ± . cm) and avk ( . ± . cm) with variety hd under field conditions. net saving of % nitrogen was observed using a. chroococcum (e and avk ) bioinoculants for wheat and cotton, respectively. to characterize the antioxidative properties of tempeh-fermented food prepared from vicia faba (l. kontu) with the use of rhizopus oligosporus, the sulfhydryl groups content and surface aromatic hydrophobicity of albumins were investigated. the results obtained for tempeh albumins were compared with raw vicia faba and bovine serum albumin (bsa). these results indicate that tempeh fermentation increased antioxidative activity of albumins. the measurements of antioxidative activity were carried out with the use of , -diphenyl- -picrylhydrazyl (dpph) and , -azinobis-( ethylbenzothiazoline- -sulfonic acid (abts). the albumins of faba bean-tempeh have possessed much higher activity for scavenging free radicals as measured with the dpph and abts ( . % and . %) than raw seeds ( . % and . %) and bsa ( . % and . %), respectively. it has been also found that tempeh fermentation process increased . times sulfhydryl groups content ( . m/mg of albumins) as compared to raw seeds ( . m/mg of albumins). the tempeh albumins have possessed lower surface aromatic hydrophobicity than raw seeds ( . fi and . fi, respectively). orange peel characterization and generation of fermentable sugars solutions for the biotechnological production of food additives b. rivas , j.m. domínguez , p. torre , j.c. parajó : department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, ourense, spain; department of chemical and process engineering, genoa university, via opera pia , genoa, italy. e-mail: brivas@uvigo.es (b. rivas) the citrus processing industry generates in mediterranean area around millions tonnes of orange peel as byproduct from the extraction of citrus juices in industrial plants. in order to avoid ecological problems and provide an extra profit, this residue was studied in order to generate a suitable substrate for the fermentation process oriented to the production of food additives. orange peels were characterized and the data collected allowed quantifying a % of this waste. soluble sugars ( . %), cellulose ( . %) and pectin ( . %) were identified as more important fractions. this material was submitted to two hydrolysis techniques, prehydrolysis (with diluted sulfuric acid) and autohydrolysis (with water) under different experimental conditions. autohydrolysis was selected as the most appropriate technique for the production of suitable fermentation media. finally, the liquors obtained at • c and liquid:solid ratio of g/g, containing . g/l of sugars, without additional nutrients, were employed to citric acid production by aspergilus niger cect (atcc , nrrl ) . the influence of the addition of calcium carbonate and methanol were studied. under the best conditions an effective conversion of sugars into citric acid was attained, showing the viability of the production of fermentable solutions from this industrial waste. today there is an increasing interest in using high gravity fermentation in brewing. high-gravity fermentation involves production of beer wort of up to • p or even higher and results in beer that has more consistent product quality. the main aim of this study is an increased understanding of how brewer's yeast respond to the various stress factors imposed during high gravity beer fermentation and the consequences these stress factors have on the gene regulation and its consequences on the metabolite levels (both intra-and extracellular). higher attenuation of the wort will be achieved by two different techniques: by the addition of highly fermentable adjuncts such as sucrose or glucose syrups and by mashing with addition of microbial enzymes such as pullulanases and glucoamylases. in the first part of the study model fermentation conditions are established, where the sugar uptake and product formation can be studied in details. characterization of the carbohydrate profile is analyzed by hplc. as flavour changes may occur at higher gravities, it is important to study changes in formation of secondary metabolites, especially esters. transcriptome and metabolome analysis will be used to establish how the stressful conditions prevailing under high gravity fermentations may influence the secondary metabolism in saccharomyces cerevisiae. furthermore, analytical aroma characterization of final beer will be studied by spme and gc-ms. detailed analysis of the effect of different stress factors on the cellular response using dna arrays and metabolite profiling will be carried out. dna arrays will be employed to evaluate if specific metabolic pathways are up-regulated or down-regulated as a consequences of the stress factors. naringin, a bitter compound that occurs in citrus fruit juices, may be converted to a nonbitter form by enzyme hydrolysis. the enzymatic complex naringinase was produced in aspergillus niger cect cultures with naringin as inducer (pérez-mateos et al., ) . crude extracts from a. niger and purified naringinase from penicillium decumbens were immobilized into a polymeric matrix of polyvinyl alcohol (pva) hydrogel cryostructured in liquid nitrogen. the operating stability of the pva-naringinase beads was tested using synthetic citric juice (gray and olson, ) . immobilized enzymes reduced % the naringin content at • c and ph . . furthermore, immobilized preparations from aspergillus and penicillium could be re-used through six cycles ( h) remaining % and % catalytic efficiency, respectively. financial support from "ministerio de ciencia y tecnología" and feder (no. agl - /ali). gray and olson, . j agric. food chem. , - . pérez-mateos, et al., ( ), . otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box , campinas, cep - são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was to identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during , and days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the medium was composed by, cwb:cr were mixed with distilled water and transferred into ml capacity erlenmeyers flasks and auto-claved at • c for min. the medium was then inoculated with spores ( . × ) and the flaks were incubated at • c. tannase was assayed according to the methodology of mondal et al. ( ) . according to the statist analyses, the optimum conditions to produce tannase was the range of temperature ( - • c); tannic acid ( . - %); residues percent (coffe: wheat bran) ( : ) and days fermentation time. the enzyme production increased . times more enzyme production than that was obtained before this optimization. yeast and lactic acid bacteria are two major microbial groups of the most fermented products. a large variety of fermented foods and beverage are made by the activities of both yeast and lactic acid bacteria, simultaneously or successively. during the spontaneous mixed fermentation of lactic acid bacteria and yeast population, it is extremely difficult to control microbial species due to the complexity of the microorganism involved. therefore, we have compared the antimicrobial activity of chitosan against two lactic strains, lactobacillus plantarum and lb. brevis, and yeast strains, saccharomyces cerevisiae to investigate the possible use of non toxic biopolymer chitosan for selective control in mixed culture. the lactobacilli were more sensitive to the inhibitory activity of chitosan than s. cerevisiae. the results suggest the possible use of low molecularweight-chitosan for the control of food fermentation in which both groups of organisms frequently occur together. the effect of vegetable oils on astaxanthin production of phaffia rhodozyma and xanthophyllomyces dendrorhous csaba vágvölgyi, gyöngyi lukács, miklós takó, Árpád csernetics, tamás papp department of microbiology, faculty of sciences, university of szeged, p.o. box , astaxanthin ( , -dihydroxy-␤,␤-carotene- , -dione) is one of the most important carotenoid product. it is used primarily as food colorant and animal feed additive. their effective antioxidant properties linked to a preventive action on various types of cancer and an enhancement of the immune response could lead to expanded commercial applications. among the natural microbial source available, the closely related red pigmented yeasts phaffia rhodozyma and xanthophyllomyces dendrorhous are of great biotechnological interest. these yeasts have desirable properties as biological sources of pigment, including rapid metabolism and producing high cell densities in fermentor, but the commercial production of astaxanthin is limited by the relatively low content in wild-type strains. the purpose of this study was to determine whether the different vegetable oils had an effect on the carotenoid production in p. rhodozyma. effects of media supplemented with corn germ oil, wheat germ oil, sesame-seed oil, palm oil, pumpkin-seed oil, coconut grease, olive oil (extra virgin), olive oil (sanza), sunflower-seed oil and cottonseed oil were tested. studies were performed on both a phaffia and a xanthophyllomyces strain. yeast was grown in yeast-pepton-glucose liquid medium complemented with the appropriate vegetable oil in different concentrations ( . - , v/v, %) . after four days the total carotenoid production was determined spectrophotometrically, and it was referred to dry cell mass. palm oil increased significantly the carotenoid production of the phaffia strain, while a similar effect on the xanthophyllomyces strain could be observed with coconut grease. composition of carotenoid compounds in the strains was determined by thin layer chromatography. lutein is considered a nutraceutic compound that has developed an increasing interest since it is one of the two carotenoids that are located in the macula of the human eye. its consumption is associated with the prevention of age related macular disease (amd). industrially, lutein may be produced using a saponification step of a mixture of lutein diesters that are previously extracted with hexane from natural sources. our proposal is to improve the process by catalyzing the same reaction using microbial lipases during the extraction step with hexane. additionally, the use of supercritical fluids represents an extension of enzymology in non-conventional media with process and environmental advantages. this work was developed using extracts from marigold flower (tagetes erecta) in hexane and supercritical carbon dioxide (sc-co ) where the lutein esters were hydrolyzed by two commercial lipases: lipase b from candida antarctica (novozym ) and lipase from mucor miehei (lipozyme rm m). in particular, we focused our interest in the role of water in the system. interestingly our results show an inverse dependence of the initial reaction rate with respect to the initial water activity (awi) for both lipases, a phenomena that seems to be related to the partition of substrates and products in the solid support)and the hexane phase as a function of water. when sc-co was used as solvent an increase in the consumption rate of lutein diesters occurred, reaching conversions of % in h. for hexane, the same conversion was reached after h. this result suggests a significant effect of the media on the reaction that can be related to shifts in the partition of compounds that bring the substrates in closer contact with the enzyme. this work also demonstrates that lutein hydrolysis seems to be another potential application of commercial immobilized lipases in the food/nutraceutical market. the enzyme of interest in this work is ␤-galactosidase from lactobacillus sp. (ec . . . ). ␤-galactosidases catalyze the hydrolysis and transgalactosylation of ␤-d-galactopyranosides (such as lactose). an attractive biocatalytic application is found in the transgalactosylaction potential of these enzymes which is based on the catalytic mechanism of ␤-galactosidases. the products of transgalactosylation, galacto-oligosaccharides, are non-digestible carbohydrates which meet the criteria of 'prebiotics' and therefore have attracted increasing attention. to produce these 'prebiotic' galactooligosaccharides, an inexpensive and efficient process is desired. immobilization of the enzyme ␤-galactosidase on an insoluble support is an attractive tool to make the process of lactose conversion more economical because the enzyme can be recovered and reused during continuous operation. in this present study, we aimed at immobilizing ␤-galactosidase from lactobacillus sp. by covalent linkages on two solid supports which are commonly used for protein immobilization: chitosan and eupergit c. the protein-binding capacity, the immobilization yield, ph and temperature dependency of activity and stability, and the kinetic parameters of immobilized enzymes were studied. higher activity retention of the immobilized enzymes over a broader ph range and at higher temperatures compared to those of the free enzyme was observed. the immobilized enzymes were evaluated in terms of transgalactosylation activity and stability for a to introduce of foreign genes for the important crop plants such as rice, we need a reproducible efficient procedure for regeneration of the calli through somatic embryogenesis. for this intention, we established the best callus induction medium for tarom mahalli and deilamani cultivars and created the method that the regeneration frequency was reached to %. calli were induced from scutellar tissues of mature seeds on ms medium supplemented with three level of , -d ( , . and mg l − ) and n medium supplemented with five level of , -d ( . , , . , and . mg l − ). for deilamani cultivar the best medium was n with . mg l − , -d and for tarom mahalli the same medium with mg l − were the best. in subculture media, sucrose was used instead of maltose. for regeneration analysis of plantlets, we used two-factorial experiment in base of crd; one factor was regeneration media with six levels (ms medium supplemented with five amount of kinetin and: naa (mg l − ) [( : ), ( : ), ( : . ), ( : ), ( : ), respectively] and . mg l − , -d and mg l − bap). other factor was dehydration process with three levels (without dehydration, dehydration with two layers of filter paper for min [prior to transfer to the regeneration medium], and third factor was factor of with substitution of sucrose with maltose [after weeks; sucrose: maltose]). we conclude that maltose due to changing in osmolarity proceeding can elevate the regeneration frequency to %. therefore, type of carbon source is critical in callus induction and regeneration. márová ivana, hrdličková jana, kubešová jitka, kočí radka, vidláková tereza faculty of chemistry, brno university of technology, purkyňova , brno, carotenoids are the most widespread natural pigments with important biological activities and applications mainly in food and feed industry. at present many ways including genetic engineering are developed to reach higher production of naturally formed carotenoids using microbial producers. in this work cloning and expression of crt gene cluster from pectobacterium carotovorum in recipient bacterial strain e. coli dh ␣ as well as in yeast strain s. cerevisiae was tested. plasmid vector phsg with inserted crt genes was used for transformation of chemically competent e. coli dh ␣ cells, while in s. cerevisiae shuttle vector paur was used. transformants were selected based on resistance to antibiotics, formation of orange-coloured transformant colonies, analysis of recombinant plasmid size and lc/ms analysis of carotenoids produced by recombinant cells. the yield of individual carotenoids (lutein, beta-carotene, lycopene) obtained from various bacterial transformants was several fold higher than in natural producer (lutein: . - . g/g of d.w., beta-carotene: . - . mg/g of d.w.). the highest yield obtained in transformed strain was . g/g of lutein and . g/g of beta-carotene. the yield of biomass and carotenoids in. transgenic s. cerevisiae was comparable to some industrial red yeast strains ( . mg of total carotenoids + mg ergosterol/l; g/l of biomass). so, transgenic yeasts could be suitable for large scale production of carotenoids and/or enriched biomass, while transgenic bacterial producers are perspective above al for high production of rare carotenoids as lutein or lycopene using transformation by specific genes of crt gene cluster. this work was supported by the project msm of the czech ministry of education, youth and sports. two forms of grape seeds, whole and powdered forms, were heated at four different temperatures- , , and • c. after heating, grape seeds were extracted with % ethanol ( . g grape seed/ ml of % ethanol), and total phenol contents (tpc), radical scavenging activity (rsa) and reducing power of the extracts were determined. thermal treatment of grape seed increased the antioxidant activity of extracts. the maximum tpc and rsa of whole grape seed extract (wgse) were achieved when the seeds were heat-treated at • c for min, while that of powdered grape seed extract (pgse) were at • c for min, and were greater than that of the non-treated control. according to the gc-ms analysis, several low-molecular-weight phenolic compounds were newly formed in the wgse heated at • c for min. these results indicated that antioxidant activity of gse was affected by heating conditions (temperature and time) and physical conditions of grape seeds at the time of heat treatments. analysis of the unexpected phenotypic consequences associated with plant transformation jonathan latham, allison wilson, ricarda steinbrecher econexus, , canon frome court, ledbury hr td, uk. e-mail: jrlatham@gn.apc.org (j. latham) transgenic plants often exhibit unexpected phenotypes. such phenotypes could arise from pleiotropic effects associated with the transgene, or they could arise from other sources. a recent econexus report underlined the potential for the process of plant transformation to result in genetic damage to the transformed plant (genome scrambling-myth or reality? transformation-induced mutations in transgenic crop plants: http://www.econexus.info/). the report showed that mutations arising at the site of transgene insertion are often substantial, frequently resulting in loss or rearrangement of chromosomal dna and insertion of multiple superfluous dna fragments. unintended mutations were also documented at other locations in the plant genome. such transformation-induced mutations could provide an explanation for unexpected phenotypes in transgenic plants. we decided to survey regulatory documents and the scientific literature for instances of unexpected phenotypic consequences arising in transgenic plants. this poster documents the preliminary results of our survey. it is intended to assess the range and frequency of unexpected consequences and to examine whether there is sufficient data available to determine their origin. it is our belief that investigating the origin of these unexpected phenotypes should be a principal aim of biosafety research. biotechnological production of metabolites such as carotenoids could be of high interest because of their antioxidative and antimutagenic activities in human body. production of these metabolites by microbial cells is dependent on cultivation conditions. so, presence of exogenous stress in cultivation environment could stimulate biosynthetic pathways of desired metabolites. two non-conventional yeast strains, rhodotorula glutinis and sporidiobolus salmonicolor, were chosen for study of carotenoid production useful in feed industry. hydrogen peroxide, sodium chloride and/or their combinations were used as exogenous stimulators of carotenoid pathway. presence of exogenous stress led to important overproduction of pigments as well as of supplementary studied substances (ergosterol, glycerol). higher adaptability of yeast cells was observed not only in cultivations with one type of stress. combination of stress factors in cultivation media induced significant increase of pigment formation. moreover, under controlled conditions in laboratory fermentor s. salmonicolor produced about eight-fold amount of ␤-carotene ( g/g) in medium with % nacl and mm h o than in control sample. similar result was observed in r. glutinis cultivated in presence of % nacl in inoculation medium only ( g/g of ␤-carotene). the use of stressed biomass of red yeasts in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition. this work was supported by the project msm of czech ministry of education. the culture ph significantly affects mycelial growth and morphology, exopolysaccharide (eps) formation, and their molecular properties during submerged cultures of a medicinal mushroom ganoderma lucidum. when the culture ph shifts from to , mycelial growth ( . g/l) and eps production ( . g/l) were favorable compared with other ph-control strategies. the mycelial morphology was also significantly varied upon culture ph: a feather-like pellets were found when the ph was controlled shifting from to at day , which was regarded as undesirable morphological form for eps production. compositional analyses revealed that the ratios and chemical compositions of the eps formed in bottom or top fractions of ethanolic precipitates were significantly different upon culture ph. the molecular characteristics of the eps were further investigated using a size exclusion chromatography/multi-angle laser light scattering (sec/malls) system. plant ␤-n-acetyl-hexosaminidase (hex) (ec . . . ) is reported to have diverse physiological roles like fruit ripening, degradation of reserved glycoproteins in germinating seed and chitin-elicited lignification. in this paper we report the purification and characterization of hex from korean ginseng roots. after extraction with citrate-phosphate buffer, hex was purified to homogeneity using ion exchanger chromatography, hydrophobic interaction chromatography and gel filtration. its molecular weight was determined using gel filtration and mass spectrometer. enzymatic parameters were studied with -methyl-umbelliferyl-n-acetylglucosaminide as substrate. the effect of heat stress and weak organic acids on escherichia coli and a comparison of its recovery by the plate count method and flow cytometry monica s. talsania due to the importance of microbiology for human health, methods have been developed to enumerate viable bacteria. dilution plating is seen to be the 'gold standard' for proof of a cells viability. however, the success of this method relies on post sampling growth, which is limited by our ability to grow cells in the laboratory. additionally, stressed or sub-lethally damaged cells, remain undetected. single cell measurements can provide rapid detailed physiological information, and the assessment of population heterogeneity. this work compared the recovery of stressed e. coli as measured by the number of cfu/ml and by multi-parameter flow cytometric analysis. weak organic acids and high temperature-short time processing (htst) were used to stress the cells both methods commonly used during food preservation. it was shown here that flow cytometry is a powerful tool for the enumeration and detailed analysis of any non-culturable microbial population, which is important because cytotoxic compounds and heat stresses used in food preservation often have a growth inhibiting effect but not necessarily a lethal one. paul g. kovalenko molecular biology & genetics nasu, zabolotnogo str. , kyiv, ukraine the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of weeks. mean transformation frequency ranged from % (for up to % (for , ). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba tl-dna and the s gus gene showed an average of more than %. these obtained root cultures were additionally elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g. uralensis were obtained by infection of a. rhizogenes have produced gl at an yield of . % dry weight on the period of culture as a days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels ( . g/l) of the total flavonoids production have been identificated on the strains which transformed by lba . this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. ayse gul nasircillar akdeniz university, biology, akdeniz univ. faculty of art-science, biological department, antalya, turkey mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and mg/l , -dichlorophenoxyacetic acid ( , -d) or mg/l naphthalenacetic acid (naa). the developed calli and regenerated plants were maintained on , -d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + , -d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. the glycolytic enzyme triosephosphate isomerase (tpi), which catalyses the interconversion of the triosephosphates dihydroxyacetone phosphate (dhap) and glyceraldehyde- -phosphate (gap), was studied for its control on glycolysis and mixed acid production in lactococcus lactis il . we constructed a number of l. lactis strains in which the tpi activity was modulated from % to % of the wild-type level. the enzyme was found to be present in high excess with % tpi activity supporting % of the wildtype glycolytic flux, and with % of the wildtype tpi activity the glycolytic flux was essentially unchanged. measurements of the upstream metabolites glucose- -phosphate (g p), fructose- , bisphosphate (fbp) and dhap were essentially unchanged for tpi activities from % to %, and only in the strain with % tpi activity we observed a significant increase in the intracellular dhap concentration. homolactic product formation was preserved throughout the interval of tpi activity studied, though a small increase in the amount of acetate and formate production was observed in the strain expressing tpi at the lowest level ( % tpi activity). the finding of an increased mixed acid pattern under intracellular conditions with a high dhap concentration is in contrast to earlier data from literature, which indicated that the triosephosphates play an important role in regulation of pyruvate metabolism in l. lactis with a negative effect on the mixed acid flux. we have recently shown that alcohols induce the adhesion of l. monocytogenes at low temperatures, presumably accompanied by enhanced exopolysaccharide (eps) production. however, little is known about the mechanisms involved in the formation of biofilm and eps by l. monocytogenes. in the present project, we show that deletion of selected regulatory and up-regulated genes did not abolish attachment, though the degree of alcohol-induction in some cases was affected. we are applying bioinformatics to search for homologues in l. monocytogenes of known eps genes from various gram positive bacteria. this has revealed candidate genes involved in the synthesis of eps, such as genes encoding glycosyltransferases. moreover, we are at present performing dna microarray analysis for the egde strain grown at • c in the presence of . % isopropanol. this data should, combined with the bioinformatic results, give us a good indication of the genes involved in alcohol-induced surface attachment. repetitive-pcr (rep-pcr) was applied in research on non-starter lactic acid bacteria (nslab) in cheese. we first showed that strains previously differentiated by pulsed field gel electrophoresis (pfge) also could be differentiated by rep-pcr. this was partially due to slight changes in the pcr conditions that allowed reproducible amplification of - kb bands. more than bands were obtained for most strains. a clear differentiation was also obtained between lactobacillus paracasei, lactobacillus plantarum, lactobacillus curvatus and lactobacillus danicus (a new species found in danish and estonian cheeses and traditional starter cultures). we found that this technique is highly reproducible, e.g. identical profiles in three different pcr-machines, two different dna isolation procedures, and different trained personnel. we applied the developed rep-pcr technique to confirm that survivors after heat treatment, were the actual strains introduced and not due to post-pasteurization contamination. we also showed that when we added a cocktail combination of five strains as protecting cultures to cheese, two to three members of this cocktail was dominating the cheese nslab microflora. in control cheeses without the cocktail in most cases other strains dominated, but in a few cases we were able to show cross-contamination between cheese vats. these data indicate that the rep-pcr will be useful to follow development of adjunct cultures as well as provide a reproducible subspecies (e.g. strain) differentiation. rep-pcr is a much quicker and less labour requiring procedure than pfge, and is apparently a much more reproducible technique than what has been seen for rapd. dynamic modeling of lactococcus lactis metabolism and its dynamic behavior for lactate secretion and regulatory characteristics jinwon lee, ui sub jung, hye won lee department of chemical and biomolecular engineering, sogang university, seoul, south korea, - . e-mail: jinwonlee@sogang.ac.kr (j. lee) dynamic metabolic model for lactococcus lactis has been developed in order to analyze a time-dependent behavior of lactate secretion mechanism and probe its regulatory roles. the model was used to compare and analyze the lactate metabolism through in silico simulation and in vitro experimental measurements most of all pyruvate branch point seems to play a major role in producing lactate, and the results of metabolic control coefficient analysis recommend to increase lactate dehydrogenase activity and to decrease nadh oxidase activity. for obtaining more realistic data, we have added some measured flux data including some intermediate metabolites. by combining the simulation results and experimental measurements, we could establish more reliable and robust systematic lactate secretion model. in addition, an efficient parameter estimation method was used to test the exactness of the reported kinetic parameters. what to choose -the fast or the detailed -strategy to get informative profiles of secondary metabolite produced by fungi in culture. chemo-diversity and lead discovery calls for high throughput techniques, but do we need columns will direct infusion esi-ms (dims) do the job. the latter may give matrix effects and lacks resolution resulting in loss of information, while lc-ms analysis takes time and challenge the data processing. results from nano-esi dims and lc-ms analyses of the same extracts important penicillium species are compared. these results illustrate advantages and problems using these techniques for rapid profiling of fungal secondary metabolites, reviling that matrix effects in dims do not seriously hampers detection of important metabolites while the specificity and certainty, for e.g. de-replication is much higher in lc-ms. phenotypic classification of fungi is essential in food biotechnology ulf thrane center for microbial biotechnology, biocentrum-dtu, søltofts plads , technical university of denmark, dk- kgs. lyngby, denmark. e-mail: ut@biocentrum.dtu.dk fungi are of great importance in food and food production. the intended use of fungi as cell factories for production of food ingredients is an upcoming issue in food biotechnology; however, this brings up a possible contamination with mycotoxins as a major issue. a reliable identification of the producer strains is crucial as a correct identification at species level following an updated taxonomy is the key to information on functional characters, e.g. useful metabo-lites and potential mycotoxins, growth conditions, resistance, etc. unfortunately, many mycological reports do not specify the taxonomy used or do not pay sufficient attention to taxonomical systems based on classification by functional characters-in contrast they are using a nucleotide sequence based phylogeny, which conveys little -if anything -about function of the organism. this situation is a major challenge for biotechnologists and mycologists in the years to come and will be highlighted by illustrative examples. the commercial interest in functional foods containing sufficient amounts of living probiotics is paralleled by the increasing scientific attention to the beneficial effect in the digestive tract. a daily intake of viable cells is proposed to ensure probiotic effect on consumer's health. one of the approaches which seems to be feasible to enhance probiotic viability and stability is to improve the fermentation conditions. during batch fermentation the viability of lactobacillus gasseri decreases after reaching a maximal value apparently indicating cell death. in this work, the apparent loss of viability can be avoided during fed-batch fermentation. a three-fold increase in viability is obtained when nutrient concentration was controlled compared with the viability reached in batch cultures. as a consequence, higher biomass concentration and lower specific lactic acid production were obtained. a mathematical model was developed to simulate and describe the effect of nutrient limitation on growth, viability, glucose consumption and lactic acid production. contribution to the metabolic adaptation to food restriction in rabbits (preliminary results) s. van harten , s. borges , p. cravo , l.a. cardoso : instituto de investigação científica tropical, cvz, lisboa, portugal; instituto de higiene e medicina tropical, lisboa, portugal. e-mail: svharten@gmail.com (s. van harten) in order to understand metabolic differences between two breeds of rabbits (halop ab and oryctolagus cuniculus algirus) during food restriction, the activities and expression of key enzymes and hormones of the rabbit were studied. animals from each breed were divided in two groups (ad libitum and restricted), revealing the results a similar difference in glycemic levels between fed and underfed rabbits, with a restriction of % of ad libitum feeding in the wild animals (decrease of % lw) and % of that ingestion in the halop breed (decrease of % lw). the activities of glutamine synthetase and glutaminase show a higher reduction of these enzymes in the wild animals superior to that of the halop breed, compromising, in this way, the ammonium detoxification and the entry of residual carbonated groups of the protein catabolism into the krebs cycle. in the latter animals, a rapid mobilization capacity of triacylglycerols (tga) appears to exist, with a rapid catabolism of fatty acids leading to their oxidation. the wild breeds' results reveal a rise of circulating tga, reflecting difficulties in the lipolysis and mobilization of nefa for oxidation. in these underfed animals, phosphoenolpyruvate and pyruvate suffered a large increase and oxaloacetate a decrease. the halop breed revealed results that indicate a diminution of glycolisis, being glucoses' energy substituted by carbonated chains of lipolysis and protein catabolism. hormone results showed a higher decrease in insulin, t and igf- in the underfed halop animals. in order to confirm the biochemical results, relative quantification of enzyme expression was studied by real time-pcr. since the introduction of genetically modified (gm) crops in , the area under their cultivation has globally increased from . million hectares in to . in . the number of countries adopting gm crops also rose from one country, the usa, in to in . despite numerous successes public opinion still questions the ecological, moral, ethical considerations and issues concerning altering the natural state of the organisms. in this study, a survey of food shoppers' knowledge, attitudes and perceptions of gm foods was carried out in food outlets in nairobi. the food outlets were determined by simple random sampling. using systematic sampling, shoppers were interviewed at targeted imported food products. focus group discussions were also conducted with farmers at city markets. the survey reflected views of a systematic sample of shoppers in seven food outlets between november and december of . it revealed knowledge at %, with positive attitudes and good perceptions towards gm foods (χ = . , d.f. , p < . ). seventy nine percent of shoppers were willing to buy and consume gm foods (χ = . , d.f. , p < . ). cross-tabulation of shopper's position on various issues raised in the survey showed a strong correlation between the respondents' respective knowledge, attitudes, and perceptions (r = . ). nineteen percent of food sampled tested positive for gms. poisson statistics were used to calculate the number of sample sequences. the statistical tools were obtained from spss version . . the results of this study will be of great interest in determining the use and adoption of gm crops in kenya. it will also guide the development of national foreign food policy on gm foods. the technology should be embraced as soon as it is acceptable to alleviate, drought, famine and hunger estimated to be affecting . million kenyans today, mostly children. consumers and gm foods: the case of turkeyÖzlenÖzgen , mustafa yildiz : department of family and consumer sciences, school of home economics, university of ankara, ankara , turkey; department of field crops, faculty of agriculture, university of ankara, ankara, turkey the future development of food biotechnology depends on consumer acceptance. scientists are aware that consumer attitudes will have a crucial impact on the process of the food biotechnology. because, food is one of the central features in human life. consumers' attitudes and trusts in the institutions will determine how gene technology will be used in food sector, in the future. recently, research concerned with consumer aspects of gm foods accelerated. but in turkey, the literature that deals with this subject is very limited and sparse. therefore, this research was carried out on the turkish consumers with the purpose of analyzing the consumers' awareness, assessments about benefits-risks, market place and labelling, and trusts in institutions, towards gm foods. this study was based on interviews with consumers who have recently purchased from major malls, during shopping hours. of the four major malls, voluntary male and female consumers were included in the research if they had main or secondary responsibility for household shopping. the questionnaire form was applied to subjects through face-to-face individual interview. the data were analyzed by using statistical methods according to explanatory variables, including age, gender and educational level. findings indicated that consumers' awareness and views about gm foods were connected to selected demographic characteristics. the results of this study can be important for consumer educators, marketing managers and policy makers. benefit-risk perceptions and moral beliefs of turkish consumers towards transgenic productsÖzlenÖzgen , haluk emiroglu , mustafa yildiz , ayşe sezen taş : department of family and consumer sciences, school of home economics, university of ankara, ankara, turkey; faculty of law, university of bilkent, ankara, turkey; department of field crops, faculty of agriculture, university of ankara, ankara, turkey the use of biotechnology in production has generated considerable debate involving the benefits-risks and moral beliefs associated with its use. consumer acceptance of genetically modified product is a critical factor will affect the future of this technology. this study was planned and conducted to determine the relationship between product/process related benefit perceptions, product/process related risk percentions and moral beliefs of consumers towards transgenic products. a total of university educated consumers, consisting of males and females, employed at the ministries selected by random sampling method in ankara, were included into study. interview techniques were used in the gathering the research materials. the interview instrument had been prepared considering previous research and literature. answers given to sentences typed likert were scored, used "varimax analysis technique" for validity. in order to test the reliability of questionnaire were calculated "cronbach alpha" as inner consistency coefficient. the t-test were performed for determining the differences dependent on gender and age variables between product related benefit perceptions, process related benefit perceptions, product related risk perceptions, process related risk perceptions and moral beliefs of consumers. the examination of relationships between product/process related benefit perceptions, product/process related risk perceptions and moral beliefs of consumers was made by correlation analysis technique. it is thought that the results of this study are important both for scientists and social scientists. the application of the dna recombinant technology for food production is generating a great debate in our society with the participation of scientists trying to explain the way of obtaining these new foods and which are their implications; environmentalist groups and anti-biotechnology associations that systematically are against to the application of this technology; legislatives bodies and the public in general, represented by consumers' organizations that expresses their right to be informed. considering that university students will be the future professionals and consumers their opinion on this topic will be decisive in its success or failure, this research is aimed to performed a global and comparative study of the agrobiotechnology perception by students from different areas of knowledge and studies. this study was carried out during the academic years - , being analyzed a total of valid surveys. the designed questionnaire included questions relatives to: evaluation of the own knowledge and interest on the topic; evaluation of the information sources mainly used by university students to obtain nutritional information; the opinion about gm food labelling; risks/benefits perception; purchasing intention and support of biotechnology. results obtained showed that % of the university students interviewed have a clear positive perception of biotechnology, mainly the students of the health sciences area. these students understand the scientific terminology and they use the university as the main source of information. they support the development of the biotechnology and they consider that in a future it will report them benefits. other group ( %) has a clear negative perception, they are mainly students from law and art history. they do not understand the scientific terminology, they consider that biotechnology will cause them risks, and as a consequence they don't have intention of buying these foods. the technology of the dna recombinant can be also used to introduce in the plants genome the gene that codes a protein of interest for their use as antigen. the application of agrobiotechnology has allowed the development of a new generation of vaccines that try to reduce or to eliminate the inconveniences of the classic ones. for the new vaccines design, the detailed knowledge of the biology of the pathogen is considered. with this knowledge the genes implied in virulence can be inactivated or modified selectively. the term "edible vaccines" it is usually applies to the use of edible parts of the plants (tubers, fruits, leaves, etc.) genetically modified with the purpose to produce specific components (antigens) of a pathogen (virus, bacteria, etc.) against which is wanted to protect a person or animal. however, oral is not the best vaccination route since the quantity of antigen for an efficient immunization it is usually high, being also needed, the co administration of an adjuvant that stimulates the immune answer. on the other hand, it is also important to highlight that the levels of antigen accumulation in transgenic plants is usually lower than the necessary ones. another problem is the irregular accumulation of the antigen in the different parts of the plants, thus difficult the appropriate control of the doses. tannin is polyphenolic component having some antioxidant properties and exists in many plants and fruits. in pomegranate juice this component causes turbidity and haze. during fruit juice clarification by conventional gelatin method, all poly phenolic substances which are responsible for antioxidant activity are removed and as a result the quality of the product is reduced. in the present study tannase enzyme (tannin acyl hydrolase; ec . . . ) was used to decompose tannin to gallic acid and glucose and as the result the amount of turbidity of the juice is decreased, however, the antioxidant properties remain unchanged since tannin is not decomposed and not separated in the juice as it occurs in the gelatin method. the amount of gallic acid in pomegranate juice samples before and after addition of tannase was measured using hplc tests and the optimum temperature, the enzyme and juice contact time, ph, and solvent concentration for clarification of pomegranate juice were obtained as • c, ph = . , h and mm citrate buffer, respectively. the potential benefits of enzymatic clarification of pomegranate juice, that is preservation of antioxidant activity and hence increasing the quality of the fruit product, in comparison to that of conventional clarification method by gelatin introduce a new technique in turbidity and haze removed in tannin containing fruit juices. the objectives of this study are to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against escherichia coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. in lactococcus lactis the enzymes phosphofructokinase (pfk), pyruvate kinase (pk) and lactate dehydrogenase (ldh) are uniquely encoded in the las operon. we have applied metabolic control analysis to study the role of this organisation. earlier work showed that ldh at wildtype level has zero control on glycolysis and growth rate but high negative control on formate production (c j formate ldh = − . ). we find that pfk and pk have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. pk has high positive control on formate (c j formate pk = . − . ) and acetate production (c jacetate pk = . − . ), whereas pfk has no control on these fluxes. decreased expression of the entire las operon resulted in a strong decrease in growth rate and the glycolytic flux. increased las expression resulted in a slight decrease in the glycolytic flux. at the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. the sum of control coefficients for the three enzymes individually was comparable to the control coefficient found for the entire operon at the wildtype level; the strong positive control by pk almost cancels out the negative control by ldh on formate production. the analysis suggests that co-regulation of pfk and pk provides a very efficient way to regulate glycolysis, and co-regulating pk and ldh allows the cells to maintain homolactic fermentation during regulation of glycolysis around wildtype level. bovine chymosin is used extensively in cheese production because of its specificity and low proteolytic activity. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. we isolated and characterized the prochymosin cdna from the abomasum of milk-fed kid goats. this cdna predicts a polypeptide of amino acid residues, with a signal peptide and a proenzyme region of and amino acids, respectively. the caprine preprochymosin has % and % identity with the corresponding lamb and calf sequences. the cdna fragment encoding prochymosin was fused in frame to the killer toxin signal sequence in a constitutive vector, and to the ␣-factor signal sequence-flag in an inducible expression vector. kluyveromyces lactis pm - c, k. lactis sel , characterized by a "supersecreting" phenotype, and saccharomyces cerevisiae bj were transformed with the recombinant plasmids. activated culture supernatants of yeast transformants showed milk-clotting activity. the flag-prochymosin fusion was purified from bj culture supernatants by affinity chromatography. after activation at acid ph, proteolytic activity assayed toward casein fractions showed that the recombinant caprine chymosin specifically hydrolyzed -casein. the recombinant caprine enzyme could be an alternative milk coagulant in cheese making. lipid accumulation in schizochytrium g / s was studied under batch and continuous culture. different glucose and glutamate source concentrations were supplemented in a defined medium. during batch cultivation, lipid accumulation occurred towards the end of the growth phase but ceased when cell proliferation stopped. under continuous culture, as dilution rate decreased from . to . h − , both cell dry weight and total fatty acid content (tfa) of the cell increased. with a constant dilution rate of . h − , nitrogen limitation induced lipid synthesis ( % tfa) as described for other lipid-accumulating organisms. however, with carbon-limited conditions, some lipid accumulation was still possible, the tfa being %. finally, the batch and continuous culture methods are compared for docosahexaenoic acid ( : , n − ) production. the objectives of this study is to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against e. coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. nitrite-oxidizing bacteria catalyze an essential step of nitrogen elimination in biological wastewater treatment. recently, novel and yet uncultured nitrite-oxidizing nitrospira-like bacteria were found to be abundant in municipal and industrial wastewater treatment systems where they outcompete nitrobacter, which has long been considered as the organism responsible for nitrite oxidation in bioreactors. despite the importance of nitrospira-like bacteria for wastewater treatment and for nitrogen fluxes in natural ecosystems, little is known about their ecophysiology and interactions with other organisms. cultivation-independent molecular techniques were applied to investigate the diversity, distribution, and physiological and genetic features of nitrospira-like bacteria in nitrifying activated sludge and biofilm. a surprisingly high diversity of these organisms was found to exist in these engineered and in natural habitats. moreover, significant physiological differences could be identified among various phylogenetic sublineages in the genus nitrospira. quantitative co-localization analyses performed by novel image analysis software revealed that these metabolic features are reflected by the spatial organization of nitrifiers living in biofilm and activated sludge flocs. based on an environmental genomics approach the genome of a nitrospira-like bacterium found in activated sludge is being analyzed. results obtained so far point at unexpected physiological capabilities of this organism, and allow us to propose that nitrospira-like bacteria may also play roles in the bioremediation of (per)chlorate and chlorite. the activated sludge process is the most common way to remove organic matter, nitrogen and phosphorus from wastewater by microbiologically means. knowledge about the microorganisms involved is fundamental for optimisation of existing plants and development of new plants and process designs. many of the bacteria believed to be involved in nitrification, denitrification, biological phosphorusremoval, and removal of organic matter in full scale plants are now identified by use of molecular methods. recent developments in experimental approaches have allowed the study of the ecophysiology of these uncultured and potentially important bacteria, thus providing a better understanding of their function in full-scale activated sludge ecosystems. relatively few dominant species in each functional group (e.g. denitrifiers and polyphosphate accumulating organisms) seems to be present. some species appear to be very specialized regarding nutrient requirements while others are more versatile. a new method for mercury remediation from industrial wastewater based on the enzymatic reduction of mercury by live mercury resistant bacteria immobilized on the pumice particles has been developed in gbf, germany, and implemented in the industrial scale (unknown). the experience gained during operation of this instalation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the sorption of mercury from wastewater, by immobilization of the bacteria directly on the activated carbon. for this it was necessary to define several significant parameters of the activated carbon used and the sorption process itself. the paper presents results of the equilibrium and kinetics investigations of the process of mercury sorption from aqueous solutions onto seven different types of activated carbon. the effective diffusion coefficients in the particles were obtained from the transient-state experiments and the sorption isotherms, saturation capacity of the sorbents and its dependence on the temperature and ph were identified. then the hydrodynamic and sorption characteristics of the activated carbon bed in a laboratory-scale fixed-bed bioreactor were investigated in different process conditions (mercury concentration, volumetric flow rate, temperature, ph). the results (effective capacity of the bed, dispersion and diffusion coefficients, mass transfer coefficient) enable implementation of this bioreactor for modified, integrated process of mercury bioremediation from industrial wastewaters. research supported by the grant kbn t c . bacterial cr(vi) reductases convert the very mobile toxic cr(vi) to the less toxic and less mobile cr(iii). the ability to reduce cr(vi) was studied on cell extracts of ochrobactrum tritici strain bvl and microbacterium sp. strain a. both microorganisms were isolated from the same sample of chromium-contaminated sludge, taken from a wastewater treatment plant. while in the first case activity was found to be associated with the intracellular soluble extract, in the second case it was a process occurring extracellularly. cr(vi) reduction by the intracellular soluble extracts of strain bvl required the presence of nadh or nadph as electron-donor, while the extracellular fraction of strain a only used nadph. several studies were made on strain bvl intracellular soluble extracts. a k m of . m cr(vi) and a v max of . ± . nmol cr(vi) min − mg − protein were estimated from the lineaweaver-burk plot and michaelis-menten non-linear regression. the temperature and the ph optima for cr(vi) reduction were . • c and . , respectively. hyperthermus butylicus is an anaerobic hyperthermophilic crenarchaeon, isolated from the solfataric sea floor off sáo migel island, azores (zillig et al., ) . h. butylicus grows at up to • c (optimally between and • c) at ph . it can utilize peptides, polysaccharides, and other substrates, as carbon sources to produce acetate, butyrate, and n-butanol. the capability to produce enzymes (e.g. hydrolases, dna and rna polymerases, etc.) that can tolerate and function at temperatures • c higher than most other thermophilic archaea, renders h. butylicus of particular interest to the biotechnology industry. the complete genome sequence of h. butylicus was determined and it contains , , bp on a single circular chromosome. protein encoding genes were identified which use a high level of uug and gug start codons. many of these were assigned functions on the basis of sequence comparisons. our analyses revealed some unusual metabolic properties in h. butylicus. several sugar transporters were identified, although the set of genes required for glycolysis is incomplete. moreover, genes encoding enzymes converting glucose to trioses are absent and no genes encoding enzymes of the pentose phosphate cycle or the kdpg pathway were detected. the h. butylicus genome encodes many proteases and peptidases although the lon proteases, encoded in all other archaeal genomes, are absent. although it was reported that h. butylicus does not utilize free amino acids in the media, genes for amino acid transporters were identified, and several proteins involved in di-or oligo-peptide transport are encoded. genes encoding signal peptidases are absent. we will summarize gene products of special biotechnological interest. reference zillig et al., . j. bact. , - . hot genomics: insights in the thermophilic lifestyle of thermus thermophilus from its complete genome holger brüggemann , , anke henne , gerhard gottschalk : göttingen genomics laboratory, institute of microbiology and genetics, university of göttingen, germany; institut pasteur, unité de génomique des microorganismes pathogènes, paris, france thermus thermophilus is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment. recently completed genome sequences of two strains, hb and hb , provide a solid foundation for investigating many aspects of thermophilic lifestyle; these range from molecular stability determinants to key elements of organismic physiology. in addition, the species has considerable biotechnological potential; many thermostable proteins isolated from members of the genus thermus are indispensable in research and in industrial applications. the closely related genera thermus and deinoccoocus belong to a distinct branch of bacteria called the deinococcus-thermus group. genome comparison of t. thermophilus and d. radiodurans, a mesophilic organism, which exhibits high resistant to radiation, oxidative stress, and desiccation, is of particular interest for the identification and exploration of thermophilic determinants. a large number of orthologs with a high degree of sequence identity are shared between the two species. this opens the opportunity for comparative studies of conformational and chemical thermostability of proteins, as well as for the identification of specific traits for each organism, explaining their unique physiological properties and their intriguing differences in stress tolerance. although strains hb and hb share a highly conserved chromosome, striking differences can be found between their megaplasmids, which encode a huge proportion of genes not found in the genome of d. radiodurans. possible contributions made by the megaplasmids to a thermophilic lifestyle will be discussed. microorganisms that can live in high temperatures, extreme ph and high salt concentration are called extromophiles. extromophilic microorganisms have extended our knowledge and understanding of fundamental questions such as the origin of life. the ability to grow in extreme conditions and to produce stable proteins makes extremopliles very attractive for the researchers and also for the industry. extremozymes from extremophiles have a great economic potential in many industrial processes, including agricultural, chemical and pharmaceutical applications. concurrent development of protein engineering will increase the application of enzymes from extremophiles in industry. turkey has vast and various ecologi-cal areas, and so it has a broad microbial diversity. based on the extremophilles which defined in the scope of this project, halophilic microorganisms produced industrially important proteins were isolated from Ç amaltı saltern area in izmir, turkey. in this work, growth of isolates at different temperature, salinity and ph values were investigated to determine the effects of various growth conditions. eight isolates grow at ph between . and . and two isolates at . - . . they grow at temperature between and • c and salt concentration between % and %. the results of some phenotypic characters showed that they are gram (−) and oxidase,ürease, dnase and nitrate reduction are (−), and catalase (+). they used d(+) glucose, maltose, lactose, sucrose, l(+) arabinose, d(+) mannose, glycerol and four of isolates used d(+) xylose as a carbon source. the isolates resistant to erythromycine, ampicilin sulbactam, cefoxitin, penicillin, bacitracin, novabiocin, amikacin and sensitive to ceftazidine, ciprofloxacin, amoxycillin/clavulanic acid, imipenem, chloromphenicol, ceftazidime/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoperazone amoxicillin. this project was supported by tubitak through project tbag - t . the technology of producing renewable energy sources such as ethanol, methane and hydrogen from biomass holds the potential of creating in-house energy resources while lowering the emission of greenhouse gasses as demanded by the kyoto protocol. recently, goals were defined for the european union determining that . % of the transportation fuel has to come from biofuels in year . a large-scale implementation of biofuels into the transportation sector will demand that lignocellulosic biomass, which is found in a surplus throughout the world is used as the raw material for the production process. the presentation will include a comprehensive description of the special bio-refinery concept developed in denmark for production of biofuels and other valuable products from straw. the concept includes several innovative steps such as a pre-treatment method using wet oxidation, on-site production of enzymes and a continuous fermentation process using a genetic modified thermophilic bacterium. by co-producing several biofuels in the plant optimal use of the biomass has been assured and the price of for instance of bioethanol is getting close to conventional oil-based fuels. optimizing each step in the bio-refinery, while having the full integration in mind, will be the way to make an economical viable biofuel production. in the presentation we will present our road map for achieving this goal in the nearest future. replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. one such fuel, which has been found well suited, is ethanol. it may be produced from various lignocellulosic materials, such as forest and agricultural residues, which are fairly inexpensive. to compete with gasoline the production cost must be substantially lowered. ethanol production from lignocellulose comprises the following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling. the enzymatic hydrolysis and fermentation can either be run separately (shf) or combined into a simultaneous saccharification and fermentation (ssf). the latter has been shown to result in higher ethanol yields than shf. some of the most important factors to reduce the cost are: efficient utilisation of the raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. in the last years we have performed several studies on the hydrolysis and fermentation of various forest and agricultural residues in a mini-pilot to improve the overall yield of ethanol and to reduce the energy demand and production cost. steam pretreatment, with small addition of acid catalyst, has resulted in sugar yields close to % of the theoretical for various types of raw materials, e.g. spruce, salix and corn stover. the ssf has been developed and optimized to give high yield of ethanol. for spruce an ethanol yield of about % of theoretical based on the composition of the raw material has so far been obtained using a two-stage steam-pretreatment of so impregnated raw material followed by ssf. improvements of the ssf step, in the form of high dry matter content, recirculation of process streams and adapted yeast have resulted in ethanol concentrations around g/l leading to substantial reduction in energy demand and production cost. these improvements have been assessed by techno-economic evaluation to determine the effect on the ethanol production cost. the process has been further optimised by process integration to further reduce the energy demand. the ethanol production cost was estimated to be around . - . euro/l ethanol assuming a yearly capacity of tonnes raw material (dry matter). production of bioethanol from spent grain, a by-product of beer production sho shindo, tadanori tachibana, akita research institute of food and brewing, akita-city, akita - , japan. e-mail: shindo@arif.pref.akita.jp (s. shindo) the breweries generate one million tons of spent grain every year, and about % of the spent grain is recycled in japan. therefore, it is environmentally and economically significant to consider the production of ethyl alcohol as biomass energy using the spent grain from the breweries industry. ethyl alcohol production from spent grain with immobilized yeast cells was investigated. spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. when kg of wet spent grain was treated under the kg/cm pressure for min using a l steam explosion reactor, g of total sugar was obtained from the liquefied spent grain. furthermore, . % (w/v) of glucose, . % (w/v) of xylose, and . % (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. ethyl alcohol production was carried out by immobilized sacchromyces cereviseae and immobilized yamadazyma stipitis simultaneously from liquefied spent grain. both yeast cells were immobilized on the glass beads carrier. xylose and arabinose were consumed after glucose was consumed completely during ethyl alcohol production. . % (v/v) ethyl alcohol was produced from liquefied spent grain that was adjusted % of initial sugar concentration after days. the vegetable oils constitute a resource of renewable potential for the production of fuels, becoming a viable alternative when compared to the diesel from petroleum. among the vegetable oils, the extracted oil of the castor plant seeds is a promising alternative source because it is constituted mainly of the ricinoleic acid ( -hydroxy- octadecenoic) that represents % of the total constitution of the oil approximately. the biodiesel obtained from castor oil can be defined chemically as being a mixture of methyl esters or ethyl esters of carboxylic acids synthesized by transesterification reaction of the existent triglyceride and an alcohol of little chain through the use of alkaline or enzymatic catalysts. in this work, we described the results found in castor oil with different degrees of purity. initially, it was made a rheological characterization followed by structural characterization (rmn c, rmn h and infrared) and thermal characterization (dtg, dta and dsc) of the crude and refined castor oil. it has been also measured the hydroxyl tenor, acidity index, saponification index and iodine index in different oils. later, these results were used to evaluate possible differences in the quality of the biodiesel (ethyl esters) produced in the enzymatic alcoholysis of the castor oil catalyzed by lipases (novozym , liposyme rm im and lipozyme tl im). the degree of substitution of castor oil derivative was performed by titration with . n hcl and confirmed by tlc analysis and the results showed conversion rates about %. has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip ) and inositol tetrakisphosphate (ip ) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia converted ip into ip (myoinositol , , , , -pentakisphosphates) and another isomer, which is yet to be elucidated. in a denitrifying pilot plant reactor, a new obligately anaerobic ammonium oxidation (anammox) process with great potential for nitrogen removal for high strength wastewater was discovered. after transfer of the complex microbial community to a laboratory sbr system, a highly enriched population, dominated by a single anaerobic chemolithoautotrophic bacterium related to the planctomycetes was obtained. the bacterium was purified via percoll centrifugation and characterized as 'candidatus brocadia anammoxidans'. survey of different wastewater treatment plants using anammox specific s rrna gene primers and anammox specific oligonucleotide probes revealed the presence of at least four other anammox bacteria, tentatively named 'candidatus kuenenia stuttgartiensis', 'candidatus brocadia fulgida', 'candidatus scalindua wagneri' and 'candidatus scalindua brodae'. a close relative of the two scalindua species, 'candidatus scalindua sorokinii' was found to be responsible for about % of the nitrogen conversion in the anoxic zone of the black sea and in the benguela upwelling system along the namibian coast, making anammox an important player in the global nitrogen cycle. electron microscopic studies of all five anammox bacteria showed that several prokaryotic membrane-bounded compartments are present inside the cytoplasm, which are surrounded by unique ladderane lipids. hydroxylamine oxidoreductase, a key anammox enzyme, was present exclusively inside one of these compartments, named the 'anammoxosome'. unique peptides fragments of the purified hao were used to locate the hao gene in genome assembly of 'candidatus kuenenia stuttgartiensis'. the implementation of the anammox process in the treatment of wastewater with high ammonium concentrations was started at the treatment plant in rotterdam, the netherlands, where it is combined with the partial nitrification process sharon. the estimated price for nitrogen removal with partial nitrification and anammox is about . euro/kg n. gas lift reactors could sustain the highest anammox capacity at . kg n removed/m reactor per day. an alternative configuration of anammox is the oxygen-limited canon process in which aerobic ammonium-oxidizing bacteria protect anammox bacteria from oxygen and produce the necessary nitrite. maximum nitrogen removal with canon in gas lift reactors was . kg n/m reactor per day. using several different conditions and parameters, the competition and co-existence of aerobic and anaerobic ammonium-oxidizing bacteria were modeled. in addition to ammonia, urea was also converted after a -week adaptation in the canon system. recently it was shown that anammox bacteria can use organic acids as additional energy source. murray moo-young, wa anderson department of chemical engineering, university of waterloo, waterloo, ont., canada n l g bioreactors are central to the bioremediation of contaminated environments of water, air or soil. in all three areas of application, bioreactor design is critical to the development of new or improved processes. this overview focuses on the physical limitations of bioreactors caused by biological requirements. the information is based on our own research findings. the need for more applicationsoriented bioremediation research becomes apparent. for technoeconomic reasons, the airlift type has often been the bioreactor of choice for most bioremediations. however, lack of adequate understanding of the quantitative effects of operating conditions on its performance has been an ongoing concern. these effects have been characterized for engineering implementation. to enhance productivity, innovative pretreatment techniques of the polluted sources have also been developed using photocatalytic and chemical oxidation methods. case studies on petrochemical-contaminated water and soil reveal significant enhancement potentials. other studies on microbial biofilters for air bioremediation indicate that the active mass of the biological consortia is not sufficiently understood for rational design. analysis and retrofit design of wastewater treatment facilities using process simulation tools demetri petrides, alexandros koulouris, intelligen, inc., scotch plains, nj , usa. email: dpetrides@intelligen.com (d. petrides) process simulators have been used in the petroleum and chemical industries for over four decades to facilitate the design of new processes and optimize the performance of existing ones. similar benefits can be derived from the use of such tools in the environmental arena, particularly in the field of physical and biological treatment of municipal and industrial wastewater. specifically, process simulators can be used to evaluate and improve options for: ( ) more efficient removal of nutrients (e.g., organic nitrogen and phosphorous) that cause eutrofication, ( ) estimation and control of volatile organic compound (voc) emissions from open tanks, and ( ) more efficient removal and control of hazardous compounds. the potential benefits will be illustrated with cases studies involving both municipal and industrial wastewater facilities. the microbial reduction of metals has showed recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. under certain conditions, however, microbial metal reduction can also mobilise toxic metals with potentially calamitous effects on human health. some effluents present heavy metals as soluble compounds, several microorganisms have the capacity to precipitate these metals as insoluble compounds, and this fact allows the collection and separation of these metallic precipitates from contaminated medium. sulfate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from a solution. the purpose of this work was study the capacity of desulfovibrio sp. cultures to reduce mixtures of the heavy metals in presence or not of petroleum. for it the experimental design k (k = ) was carried out. the five studied factors were cr, cu, mn, zn and petroleum. the study was carry out with desulfovibrio sp. batch studies were performed in ml sealed bottles with different concentrations (cr(iii)- ppm, cu(ii)- ppm, mn(ii)- ppm, zn(ii)- ppm) of metal sulfate and g l − of petroleum. during batch incubation the dissolved concentration of metal studied in supernatant were decreased to undetectable levels for zn ( - %), however with cu ( - %), mn ( - %) and cr ( - %). the development of continuous process with sulfatereducing bacteria seems to be a suitable alternative to reduce metals in solution from contaminated media such as industrial or mine effluents. after these preliminary results, some experiments in course are focused to study that purpose. reduction of odour emissions from livestock buildings using a bioscrubbing system morten Øgendahl, nawaf abu-khalaf, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, dk- odense m, denmark. e-mail: tvede@bmb.sdu.dk (m. Øgendahl) a bioscrubbing system for reducing odour emissions from livestock buildings is presented. the bioscrubbing system consists of two separate units; an absorption column and a water purification module. the absorption column is mounted in the ventilations stacks in the livestock buildings absorbing odorants in the effluent air flow. the odorants are absorbed in a spray of droplets formed by a grid of high pressure nozzles in the inlet of the absorption column. the spray of droplets is extracted from the air flow and pumped to a centrally located water purification module, an inverse three phase fluidised bed bioreactor, where the bio-degradation of the absorbed odorants occurs. the bioreactor features a split sparging system for maximum mixing and aeration. the cleaned water is recirculated to the absorption column. an electronic tongue will quantify key odorants in the bioreactor. the absorption column is designed to be retrofitted into existing livestock building ventilation systems. the water purification module is constructed in standard size units simplifying scaling to match the requirements of individual applications. the total bioreactor volume is increased by increasing the number of standard bioreactors. this work describes a "light off" toxicity bioassay sensor based on whole cell genetically modified bioluminescent bacteria. the biosensor was constructed by mating between the environmentally isolated phenol-degrading acinetobacter sp. strain df and the plasmid putk that is an inc p␤ plasmid with the bioluminescence genes luxcdabe inserted into a genetic region involved in plasmid replication and transfer. subsequently, the bioreporter designated df /putk and used to investigate phenolics toxicity. among examined phenolics, pentachlorophenol, catechol and nitrophenol recorded the fastest effect on the bioluminescence of bioreporter df /putk over incubation period of min. the effect of various concentrations of phenol and its derivatives either in an individual, duple or triple mixture forms on the bioluminescence response of the constructed bioreporter df /putk were also examined. significant reduction of the bioluminescence was observed whenever a mixture contained pentachlorophenol, catechol and nitrophenol, respectively. to develop a system appropriate to commercialize, the constructed bioreporter df /putk was subjected for immobilization in microtiter plates using several entrapment gels. after a selection of materials was tried, lb/agar was chosen as the most suitable candidate material. characterization of key odour compounds in an air wet scrubber is presented. the key odour compounds represent five chemical groups, i.e. sulphide, alcohol, volatile fatty acids (vfas), phenol and indole. direct aqueous injection (dai) and solid phase extraction (spe) methods were used before injection of key odorants into the gas chromatography-flame ionisation detection (gc-fid). the dai and spe methods were efficient in the identification of odour compounds in the wet scrubber. the spe method had a high recovery and can be more effective in the identification of compounds at low initial concentration. however, dai showed a better linearity and a lower limit of detection (lod) than the spe method. the dai method was the method of choice for characterization, as it is cheaper, easier to handle and highly applicable. at least two odorants, phenol and -butanol, were quantified successfully using the dai method. their lod was less than their odour detection limit in the wet scrubber. dai method can be used as a reference measurement method for any further analytical application, e.g. electronic tongue. recent developments in biotechnology enabled the widespread use of microbial enzymes in textile, detergent, food and dairy industries and also in various environmental applications. microorganisms which live at extremes of temperature, ph and salinity, produce extremozymes that offer many exciting opportunities for their use in clean production. in this study, microorganisms were isolated from camaltı saltern area inİzmir, turkey. effect of medium salinity on the growth of these microorganisms was determined. seven out of isolates required salt for growth. the salinity ranges at which growth was detected were: - % for two isolates, - % for one isolate, - % for two isolates and - % for one isolate. the isolates were also screened for their capability of producing industrially important enzymes such as amylase, protease, lipase, xylanase and cellulose which are widely used not only in textile, detergent, food and dairy industries but also in various environmental applications. all of the isolates were found to be producers of both amylase and xylanase enzymes at varying salinity array within - % salt concentration range at ph . . extracellular protease activity was detected in the medium of all isolates grown at , , , and % salinity at both ph . (optimum growth ph) and ph . . out of isolates, , and were found to produce cellulase enzyme when the salt concentrations were , and %, respectively. at % salt concentration, only one isolate was found to be cellulase enzyme producer. none of the isolates were found to produce lipase enzyme at - % salt concentration range. this project was supported by tubitak through project tbag - t . chemical engineering department, middle east technical university, ankara , turkey. e-mail: ubakir@metu.edu.tr (u. bakir) glass and ceramic tiles are very widely used industrial materials. in most cases, periodical cleaning is required to maintain their optical properties such as transparency and visual aspects. because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. the application of these tiles in care facilities to reduce the spread of infections, in public and residental places to improve hygienic conditions are of general interest. in this study the aim is developing methods to apply thin film coatings on glass tiles to make them anti-bacterial by utilizing photocatalysis and investigating their anti-bacterial properties. semiconductors because of their reasonable band gap energies find great attraction through this purpose. the photocatalytic property of semiconductors are used in this process. oxidising radicals are formed on the coated surfaces and these radicals attacks the organic pollutants and bacteria on contact with the surface. titanium dioxide (tio ) coated surfaces are considered to be very effective against organic and inorganic materials, as well as against bacteria. in the experimental procedure coating solution is prepared by sol-gel technique. after pretreatment of surfaces, the coating solution is applied on the surfaces by dip-coating method. after appropriate thermal treatments, to achieve thin, dense and strong coatings, indicator microorganism is directly applied on the coated surfaces and illuminated under solar simulater light source. finally, the number of surviving microorganisms are determined. in this study, the effects of titanium dioxide (tio ), tin oxide (sno ) solutions and metal doping to these coating solutions on anti-bacterial function were investigated. as a result of this study, the number of escherichia coli that is used as indicator microorganism, on tio and sno coated glasses with respect to the control glass reduced by - % and - %, respectively. doping with metals increased the activity of the coatings, hence the number of surviving microorganisms decreased. activity of a methanogenic ecosystem during the primary contact with a solid support s. michaud, n. bernet, p. buffière, j.p. delgenès inra-lbe, avenue des etangs, f- narbonne, france in this paper, the biological activity during the first initial contact between a methanogenic sludge and a solid support was investigated in batch experiments, at different solid concentrations, using two different granular solid materials and with glucose as the main organic substrate. in all cases, the introduction of a solid material in a methanogenic suspended biomass induced a response of the anaerobic microorganisms, after a lag phase during which biological activity was not detected. this lag phase could be the consequence of a physical stress induced by the first contact between microbial cells and the solid surface. this lag phase was not observed when the biomass used originated from a biofilm reactor, i.e. using a biomass previously exposed to a solid material. a change in the metabolism of organic matter from catabolism and methane production toward production of other compounds could be observed, characterised by a sharp decrease of the methane yield in the anaerobic system. analyses of the gas and liquid phases did not show the production of any new gaseous or soluble compound as the biological end product of this activity. this suggests the production of non-soluble compounds by an anabolic pathway, which could indicate the initiation of biofilm formation. this metabolic activity was shown to be directly correlated to the ratio between the solid surface introduced and the microorganism concentration in the anaerobic culture (m g vs − ). from kinetic observations, it could be observed that acetogenic methanogenesis recovered more rapidly than syntrophic propionate and butyrate degradation. evaluating microbial diversity of hydrocarbon degrading bacteria cleantis braithwaite, howard rosser, tawfiq al-ibrahim, hussain, al-bandi research and development center, saudi aramco, dhahran, saudi arabia the analysis of microbial diversity with molecular methods is central to isolating and identifying new and potential biocatalysts resources for research and industry. the ability to degrade hydrocarbon components of petroleum is widespread among bacteria, and is an effective method for remediation of a variety of ecosystems. due to the high carbon content of oil and the low levels of other nutrients essential for microbial growth, treatment of oil with phosphorus and nitrogen is generally required to enhance the growth of hydrocarbon-degrading bacteria and to stimulate oil sludge degradation. in this research study, three types of oily sludges from a gas plant, refinery, and terminal facilities were treated with nutrients. to assess the microbial diversity, both biolog culture method and culture independent polymerase chain reaction (pcr,) denaturing gradient gel electrophoresis (dgge) methods were used. nutrient addition significantly improved oil sludge degradation. we identified and characterized several hydrocarbon degrading bacterial strains that have the ability to convert petroleum. these bacteria included representatives both gram positive and gram-negative genera. there were slight difference in the quantity and type of hydrocarbon degrading bacteria found in the three sites. this is the first molecular analysis of hydrocarbon degrading microbial population in saudi arabian operations. mussel adhesive proteins, including the -plus variants of foot protein type (fp- ), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. here we report the novel production of a recombinant mytilus galloprovincialis foot protein type variant a (mgfp- a) fused with a hexahistidine affinity ligand in escherichia coli, and its ∼ % purification with affinity chromatography. recombinant mgfp- a showed a superior purification yield and better apparent solubility compared to those of the previously reported recombinant m. galloprovincialis foot protein type (mgfp- ). the adsorption abilities and adhesion forces of purified recombinant mgfp- a were compared with those of cell-tak (a commercial mussel extract adhesive) and mgfp- using qcm analysis and modified afm, respectively. these assays showed that the adhesive ability of recombinant mgfp- a was comparable to that of cell-tak but lower than that of recombinant mgfp- . collectively, these results indicate that recombinant mgfp- a may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments. cresol, a monomethylated phenol, is an aromatic compound. the environmental protection agency (epa) has determined the carcinogenic potential of cresol. various options are being examined for the degradation of cresol because of their unavoidable large scale production and toxicity. many aromatic hydrocarbons can be used as electron donors aerobically by species of pseudomonas, thus leading to the ring cleavage of these compounds. in the present study, pseudomonas strains were isolated from activated sludge collected from sewage treatment plant. it was repeatedly transferred onto nutrient agar plate to check the purity of the culture. the organism was grown aerobically in an inorganic medium with p-cresol as the solitary carbon source. pseudomonas was confirmed by the expression of green pigment, gram staining and biochemical tests including koh, catalase, nitrate reduction and carbohydrate fermentation reaction. inoculation status was used to determine the rate of degradation of p-cresol. the effect of temperature on p-cresol degradation was studied. moreover, the effect of different concentrations of the aromatic compounds on pseudomonas as well as substrate variability was also documented. phenolic intermediates were estimated colorimetrically using -aminoantipyrene, folin-lowry method, uv spectrophotometry and hplc. the results indicated that pseudomonas could degrade up to mg/l of p-cresol within h. pseudomonas sp. exhibited good metabolic versatility and degraded other aromatic compounds including m-cresol and p-hydroxybenzoic acid. we conclude that this strain of pseudomonas has excellent potential for bioaugmenting the degradation of p-cresol-containing waste water treatment units. a considerable amount of waste cooking oil is produced by the restaurant industry worldwide. this poses a significant environmental and economic problem, since high oil and grease concentrations in the sewage system could lead to pipes occlusion and decreased efficiency in water treatment operation plants. therefore, sending these wastes to recycling companies or hazardous waste processors is usually required. yarrowia lipolytica, a well-known lipase producer, requires the presence of lipidic compounds (i.e. vegetable oils) to boost enzyme biosynthesis. in this work, the suitability of waste cooking oil as lipase inducer in submerged cultures of this yeast has been assessed. if successful, this procedure could allow both the degradation of an abundant waste and its valorisation as a raw material for the production of a high added value product. the microorganism was grown in a liquid medium to which various amounts of waste cooking oil were added. biodegradation degrees up to % (measured as decrease in cod) were obtained after days of treatment. also, initial glucose concentration in the basal medium seemed to influence the efficiency of the process. on the other hand, addition of waste oil led to a significant increase in lipase production (more than two-fold), compared to oil-free cultures. moreover, chain-length specificity of the produced enzymes was significantly different: high activity towards medium chain length esters was found, which hinted to the occurrence of both lipases and esterases. biodesulfurization: a documental review j. ferrer, simon bolivar university, environmental engineering lab. caracas, venezuela a documental review about larger interest aspects in biodesulfurization technique is showed. especifically, the investigation is related to general framework and the justification of this technique, degradatives pathways elucidated up to now, involved microorganism, important elements in development of bacterial desulfurization and progress areas, and future tendency. in situ bioremediation of a p-nitrophenol contaminated site and assessment of its community structure debarati paul, gunjan pandey, sumeet labana, rakesh k. jain institute of microbial technology, sector a, chandigarh , india. e-mail: rkj@imtech.res.in (r.k. jain) biodegradation of p-nitrophenol (pnp), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. bioremediation studies were carried out in pnp-spiked soil in small plots under natural field conditions using arthrobacter protophormiae rkj . role of carrier material was examined by immobilizing the bacteria on corncob powder prior to adding them to soil. these studies demonstrated successful removal of pnp by immobilized cells that were able to deplete pnp completely in days, whereas free cells were able to deplete % pnp in the same time period. monitoring the fate of released bacteria revealed fairly stable population of the cells when they were immobilized on corncob powder throughout the period of study. on the other hand, there was a decrease of . log units in colony forming units of free cells at the end of the study ( days). bacterial community structure and diversity was also studied for the pesticidecontaminated site wherein the effect of addition of an exogenous strain on the existing soil community structure and on soil functionality was determined using molecular techniques. as revealed by restriction fragment length polymorphism (rflp) studies different phylotypes could be identified on the basis of similar banding patterns. sequencing of representative clones of each phylopyte showed that the community structure of the pesticide-contaminated soil mainly constituted of proteobacteria and actinomycetes. terminal fragment length polymorphism (t-rflp) analysis showed only subtle changes in community structure during the process of bioremediation. bacteriocins encompass an array of structurally different molecules produced by a number of phylogenetically distinct bacterial groups and trigger the killing of the same or closely related species. the recombined escherichia coli strain harboring a bacterocin coding region of xanthomonas campestris pv glycines ra was disrupted to obtain cell homogenate. peptidic xanthomonas bacteriocins (pxb) were separated by lowering ph and adding salt. the resulting pxb's were partially purified using ion exchangers, gel filtration. two final active fractions, a and b, were obtained with a yield of . % and - -fold purification. the activity of pxb was stable at the ph ranging from . to . . andreja kresal, vanja kokol, vera golob textile department, university of maribor, , slovenia wastewater from textile dyeing industries is characterized by high chemical and biological oxygen demands (cod and bod) and intense color due to the extensive use of synthetic dyes. as dyes of complex aromatic structures are resistant to removal by the typical microbial population and may be toxic to the microorganisms present in the treatment plants, discharge of the wastewater to the treatment plants may lead to its failure. beside, direct discharge of these effluents into municipal wastewater plants and/or environment may cause the formation of toxic carcinogenic and/or unhealthy breakdown products. different chemical and physical methods (adsorption, coagulation-flocculation, oxidation, filtration and electrochemical treatments) for color removal have been proposed, but due capital costs and slow operating speed as well as huge amounts of sludge creation there is still a great need to develop an economic and effective method. the use of lignin degrading white-rot fungi and their enzymes (laccase, lignin peroxidase, manganese peroxidase) has attracted increasing scientific attention due their ability to oxidative degrade a wide range of recalcitrant organic compounds. in the contribution, the decolorization efficacy of different commercial textile reactive dyes (anthraquinone, azo, triphenylmethane) will be investigated after the treatment by laccase from trametes versicolor. in order to examine the reuse of enzymatically decolorized liquors, the ecological suitability and the toxicity of the degradation products after different time of enzyme exposure will be studied. this work was carried out within the scope of research project e! cawab. influence of heavy metals on growth and extracellular enzyme production of a trichoderma harzianum strain with biocontrol potential l. hatvani , l. kredics , a. szekeres , z. antal , l. manczinger , a. nagy , c. vágvölgyi : department of microbiology, university of szeged, p.o. box , h- szeged, hungary; hungarian academy of sciences, university of szeged, microbiological research group, hungary; pilze-nagy ltd. kecskemét, p.o. box , hungary. e-mail: kredics@bio.u-szeged.hu (l. kredics) trichoderma species are common soil inhabiting asexual filamentous fungi with teleomorphs belonging to the hypocreales order of the ascomycota division. besides the industrial and clinical importance of the genus, certain strains have been found to cause great losses in mushroom cultivation while other strains are well known to possess high antagonistic activity against several plant pathogenic fungi and therefore used as biocontrol agents. important mechanisms of antagonism include competition and mycoparasitism, which -among others -can be related to the fast growth of trichoderma strains and the production of several extracellular enzymes. the influence of certain, soil-occurring heavy metals on mycelial growth and the secretion of extracellular enzymes involved in competition and mycoparasitism was examined in this study regarding an effective, potential biocontrol isolate of trichoderma harzianum. the metal ions zinc, manganese, copper, iron, lead and mercury were applied at the concentrations of , , , , , and m, and dry mycelial weight as well as the activities of extracellular ß-glycosidase, cellobiohydrolase, trypsinand chymotrypsin-like protease and n-acetyl-glucosaminidase enzymes were determined. it was found that mercury totally blocked mycelial growth, while other metal ions exerted a much lower influence on growth. the presence of heavy metals did not have a significant effect on the activity of the examined extracellular enzymes with the exception of trypsin-like protease, which showed a four-to six-fold rise in activity in the presence of certain sublethal concentrations of copper. based on these results, our further aim is to develop copper-resistant derivatives by mutagenesis from trichoderma strains with biocontrol potential. since proteases play an important role in mycoparasitism, these strains could be applied within the frames of integrated pest management in combination with copper-containing fungicides, resulting in an enhanced level of crop protection even with reduced amounts of fungicides. this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. the significance of biocontrol agents (bcas) is that some of them possess good antagonistic abilities against plant pathogenic fungi. a significant number of the most prominent fungi for the purposes of agricultural application belong to the genus trichoderma. in previous studies, in vitro assays on agar plates were reported as the generally used method for the evaluation of antagonistic abilities, as the results of these assays are well transferable to the practical application. the aim of the present study was to develop an accurate, image analysis-based method for the evaluation of the biocontrol characters of bcas. randomly selected trichoderma isolates were tested against fusarium culmorum. in the currently developed method, the areas of the fungal colonies were calculated on petri dishes by measuring the occupied surface of the medium on digital images. the inhibition effect was recorded as the value of biocontrol index (bci), which was calculated from the ratio of the area of the trichoderma colony and the total area occupied by the colonies of trichoderma and the plant pathogen. the proposed method was tested for numerous parameters, and the results revealed that bci proves to be capable for the accurate measurement and scale of the biocontrol abilities of fungal isolates. this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. the effect of advanced oxidation processes and recirculation on biodegradation of leachates from aerobic landfills liliana krzystek, anna zieleniewska-jastrzębska, stanisław ledakowicz department of bioprocess engineering, technical university of lodz, - lodz, poland modern landfills are built and operated in a way which allows us to treat them as a special type of bioreactor. simulation of municipal waste biodegradation in lysimeters provides knowledge on basic processes that take place in an aerated landfill. the aim of aeration is to stabilise mainly biodegradable and nitrogen containing components and to reduce methanogenic potential. stabilised leachates from old landfills contain big quantities of refractive carbon compounds that cannot be removed by biological methods. in such case most advantageous is to apply advanced oxidation processes (aops). the objective of this study is an experimental simulation of a landfill aerobic stabilisation and the impact of aops and recirculation of leachate on the reduction of organic load. the performance of the processes was monitored by the reduction in time of basic indices of organic load (bod , cod, toc, vfa, tkn, n-nh + ) and changes in biogas composition. the simulation of aerobic landfill processes was carried out in lysimeters with a fixed bed of household solid waste stabilised during months in anaerobic conditions. leachates taken from the lysimeters were recirculated and subjected to advanced oxidation processes, i.e. ozonation and uv radiation with the addition of h o . experimental studies showed that the aerobic waste stabilisation was a very quick process. during a month the bed was stabilised, reaching a significant reduction of organic load indices. aeration of the lysimeters caused a quick reduction of mainly degradable organic substance (in terms of bod ) and n-nh + and vfa. the reduction of methanogenic potential of the landfill was even faster. the composition of gas at the outlet from the lysimeter changed and after one day already its content was similar to atmospheric air. a more frequent recirculation of leachates enhanced greatly the aerobic biodegradation. it was found that application of advanced oxidation processes (especially ozonation) contributed to a growing reduction of the organic load in the leachates from aerated lysimeters. the application of leachate ozonation resulted in a very high degree of reduction of organic compounds (up to %). the objective of the experimental study was to assess the effect of temperature on the extent of aerobic batch biodegradation of potato stillage with a mixed culture of bacteria of the genus bacillus. the experiments were performed at , , , , , , , , and • c, at ph , in five l l working volume stirred tank reactor (str) (biostat ® b, b. braun biotech international). the duration of the process was h. initial cod of the stillage amounted to . g o /l, the main carbon sources being reducing substances ( . g/l), organic acids (determined as their sum) ( . g/l) and glycerol ( g/l). at • c, no cod reduction or biomass increment was found to occur. at the other investigated temperatures, the reduction in cod measured after suspended solids (ss) separation varied from . % ( • c) to . % ( • c). without ss separation, cod reduction ranged between . % ( • c) and . % ( • c). this indicates that, in terms of the extent of cod reduction, the optimal process temperature was • c and that there was a local optimum at about • c. according to the temperature applied, the content of reducing substances decreased by . - %, that the organic acids by . - . %, and that of glycerol by . - %. the experiments also produced the following two findings: ( ) the rise in temperature brought about a decrease of biomass concentration in the str (measured as ss and bacterial number), and ( ) temperature was a factor affecting the demand for ammonia nitrogen (n-nh ), which was the highest at and • c. the high n-nh demand observed both over the higher and lover ranges of the investigated temperature should be attributed to the release of n-nh and to the large amounts of the biomass produced, respectively. the results obtained imply that the extent of potato stillage biodegradation with a mixed bacterial culture was high over a wide range of the investigated temperature. polychlorinated compounds such as tetrachloroethylene (pce) have become serious environmental pollutants. considerable attention has been paid to these organochlorine compounds. this paper describes the molecular analysis of dechlorinating gene in halorespirating bacterium and efficient bioremediation process. an anaerobic bacterium, that dechlorinates pce to tce, was isolated and identified as a species of the genus desulfitobacterium. a novel pce reductive dehalogenase (prda) gene from the desulfitobacterium sp. strain kbc- was identified. these prd genes, including membrane anchor protein, were classified as a novel type of pce reductive dehalogenase (approximately % homology with the general pce dehalogenase). according to the substrate utility of this strain kbc- and phylogenetic analysis of prda, the type of this microorganism may be expected to play the role of a primary degrader of pce in the environment. high efficient bioremediation process so called the restricted aeration system which means microaerobic/aerobic reciprocal bioremediation process was developed. strong modifications take place, as ammonia production with a subsequent rise of the ph value and a rapid heat evolution leading to temperatures of up to • c. little is known about the microbial community in the toscano cigar fermentation and its development as fermentation proceeds. the aim of this study is to investigate the microbial community composition, its dynamic and its influence on the toscano cigar production process. our results show that the fermentation could be divided into three different phases: initially yeasts are the predominant microorganisms while bacterial growth is partially inhibited; the middle phase is characterized by exponential growth of bacteria while yeasts disappear. in the final phase the microorganism population is mostly represented by sporigen microbial species. the occurrence of yeasts in the first phase could be attributed to their ability to grow at low temperature and low ph levels. the bacterial population flourishes after the yeast cells have reached a stationary phase and probably grows on residual nutrients and autolysing yeast cells. yeasts and bacteria involved in the fermentation process were isolated and characterized. the microbial community was investigated by a combination of phenotypic and molecular approaches. the phenotypic characterization was based on both colony and cell morphology. the isolates were then identified by rrna genes sequence analysis. finally, in order to clarify the role of the identified microorganisms in the production process, a preliminary biochemical characterization was carried out. biosensors have undergone rapid development over the last few years; in particular, in environmental field many biosensors using microorganisms and purified enzymes as biological component, were recently studied. benzene is present everywhere with high levels in the cities and sometimes, in petroleum processing plants. it is classified as carcinogenic compound of first class able to cause leukaemia. because the evaluation of benzene requires complex instruments and quite long analysis times, it is required to study alternative systems for benzene detection simple, fast and highly sensitive, such as biosensors. from pseudomonas putida mst, strain previously isolated in our laboratory and able to degrade benzene, we isolated genes encoding for benzene , -dioxygenase and cis- , -dihydrodiolbenzene dehydrogenase to use in the development of two different hydrocarbon biosensors based on microorganisms and on purified enzymes. the genes isolated were cloned in pvlt and we developed three microbial systems carrying: ( ) benzene dioxygenase, ( ) dihydrodiol dehydrogenase and ( ) benzene dioxygenase-dehydrogenase modified by pcr to obtain enzymes with histidine tag. the cloning was planned to construct recombinant strains able to overproduce the enzymes; the enzymatic activities will be evaluated both using whole cells and purified enzymes. study of operation condition of biofilter using fibril-form matrix for odor gas removal don-hee park, chonnam national university, this research was performed for developing of biological treatment process of odor gas such as mek, h s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over % was obtained by biofilm formation. at ppm of inlet odor gas concentration and s of retention time, the removal efficiency was % and % in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over % at the operational conditions above s of retention time. ozonated water is produced using an ozone generator in a container filled with cold water. it is useful for sanitizing the surfaces of various products for which heat or chemical treatment is inappropriate, such as fresh food products. in this study, we investigated the antimicrobial effects of ozonated water and electrolyzed ozonated water against escherichia coli, s. aureus, bacillus subtilis and yeast, saccharomyces cerevisiae for practical use in sanitizing various products. the results demonstrated that the electrolyzed ozone water was effective for the reduction of microbial population at relatively low concentration of ozone. also, the electrolyzed and the ozonated water showed synergistic antimicrobial effects. many xenobiotics can react spontaneously with thiol moieties of glutathione (gsh), forming gsh-conjugates, or via glutathione s-transferases (gst). these enzymes participate in detoxification of potentially harmful compounds from endo or xenobiotic origin. using saccharomyces cerevisiae as experimental model, we observed that cells mutated in the gtt or gtt genes showed twice as much cadmium absorption than the control strain. we proposed that the formation of the cadmium-glutathione complex is dependent on those transferases, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. the addition of glutathione monoethyl ester (gme), a drug that mimics glutathione (gsh), to gtt ∆ cells restored the levels of metal absorption to those of the control strain. however, with respect to gtt ∆ cells, addition of gme did not alter the capacity of removing cadmium from the medium. taken together, these results suggest that gtt p and gtt p play different roles in the mechanism of cadmium detoxification. by analyzing the toxic effects of this metal, we verified that gtt ∆ and gsh ∆ cells showed, respectively higher and lower tolerance to cadmium stress than control cells, suggesting that although gsh plays a relevant role in cell protection, formation of the gsh-cd + conjugate is deleterious to the mechanism of defense. furthermore, analyzing the harmful effects of other xenobiotic, menadione ( -methyl- , -napthoquinone), we have also observed that gtt p and gtt p isoforms play distinct functions in the process of cell protection as well as in drug remove, since both strains showed lethal phenotypes after direct exposure to mm menadione. however, after adaptive treatments (mild-heat or exposure to a lower menadione concentration), cells acquired tolerance to menadione stress, although the gtt ∆ mutant had still shown a higher sensitivity against drug toxicity. by analyzing the malondialdehyde (mda) produced in response to menadione, we observed that gtt ∆ cells exhibited increased levels of lipid peroxidation, indicating that, during menadione exposure, gsh-conjugates are formed by the same transferase isoform, gtt p, involved in cadmium stress. financial support: stint (sweden), cnpq and faperj (brazil). polycyclic aromatic hydrocarbons (pahs) are ubiquitous and persistent throughout the environment. they are generally distributed from both natural and industrial sources. many pahs can have a detrimental effect on the flora and fauna of affected habitats through uptake and accumulation in food chains, and in some instances, they induce serious health problems and/or genetic defects in humans. many research efforts have been expended to find a suitable method for remediation of soil and water environments contaminated with pahs. amongst them, the use of ligninolytic fungi is particularly suitable for the development of such processes, since they produce extracellular lignin-degrading enzymes (mnp, lip, laccase, . . .) which degrade a wide range of organic pollutants. coriolopsis rigida has been reported to produce extracellular laccase as the sole ligninolytic enzyme. this makes this fungus particularly suitable for the study of xenobiotics degradation by laccase. the purpose of this research was to obtain high laccase activities by c. rigida in solid state cultures and to determine their ability to degrade anthracene (typical pah). both in vivo and in vitro assays were performed. the former led to - % degradation in days depending on the culture conditions, whereas the latter showed a degradation percentage above % in days when low mediator concentration (hbt) was added to the reaction mixture. focus will be given on pressure-driven membrane bioreactors, gastransfer membrane bioreactors and the novel ion exchange membrane bioreactor (iemb). the latter concept has been developed and currently studied by our group. this process, based on integration of donnan dialysis with bioconversion of one or more target pollutants to harmless products, has been modeled and experimentally verified for the removal of various charged inorganic pollutants such as nitrate, perchlorate and bromate by mixed microbial cultures under anoxic conditions. tests of up to months showed a very good operational stability. the essential role of the microbial membraneattached biofilm, which develops naturally in this type of systems, will be also demonstrated and discussed. poly(lactic acid) (pla), which is one of biodegradable plastics, is depolymerized by hydrolysis and releases soluble monomer or oligomer of lactic acid. many bacteria can use the monomer and oligomer as an energy source or a carbon source. in this study, we applied pla to an electron donor for denitrification process of the previously developed bioreactor, which could remove ammonia from wastewater by simultaneously carrying out two biological processes, aerobic nitrification and anaerobic denitrification. a bench-scale bioreactor was constructed with a gel-plate containing pure-cultured cells of nitrosomonas europaea and paracoccus denitrificans and a pla-plate. the pla-plate was prepared by mixing three kinds of plas with different molecular weight and tricalcium phosphate to keep the constant release of the electron donor for a long term. batch treatment experiment with the bioreactor was repeated with an artificial wastewater containing ammonia for days. the bioreactor could remove nitrogen from the artificial wastewater at nitrogenremoval rate of approximately g n/day per square meter of gel-plate surface during the experiment period without an additional electron donor. the performance was equivalent to that obtained with our bioreactor using ethanol as electron donor for denitrification. the bioreactor using pla dose not need an additional pump for serving an electron donor (e.g., ethanol) and a hollow space for serving. therefore, the concept using solid electron donors like a pla would be effective our bioreactor to compact and simplify, and would be possible to develop a portable or disposable bioreactor. leucosporidium antarcticum as a source of enzymes for biotechnology arkadiusz wojtasik , , marianna turkiewicz , jaroslaw dziadek , pawel parniewski : centre for medical biology pas, lodowa street, - lodz, poland; faculty of biotechnology and food sciences technical university of lodz, stefanowskiego / street, - lodz, poland. e-mail: awojtasik@cbm.pan.pl (a. wojtasik) leucosporidium antarcticum is a psychrophilic yeast able to growth at low temperature. these microorganisms live in antarctic marine waters and are endemic to that cold environment. furthermore, l. antarcticum is also isolated from the digestive tract of antarctic krill euphausia superba. enzymes isolated from coldadapted microorganisms such as l. antarcticum having a specific activity at low temperatures ranging from to • c are considered for utilization at biotechnological applications such as bioremediation, production of polyunsaturated fatty acids of dietary significance and might be a source of industrially useful enzymatic proteins. the main goal of this study was to construct a cdna library of l. antarcticum. the partial cdna library was obtained and some of the clones were analysed. the sequencing analyses allowed us to find an approximately base pair nucleotide sequence which displayed a very high homology to disulfide bond chaperone belonging to the hsp family from psychrobacter sp. high similarity of that heat shock protein was found on an amino acid sequence level and was reaching nearly %. the main object of our further research is to clone hsp family protein gene and to obtain its expression in a mezophilic host strain. also, further clones will be analysed to find other interesting genes encoding the psychrophilic proteins. this work was partially funded by the kbn grant i / / . out of plant species found in the flora of turkey, about are endemic. beautiful flowering (geophytes) bulbous plants form an important part of this rich biodiversity. besides use as ornamental plants, these have great potential in perfume and pharmaceutical industry. genera of fritillaria, ornithogalum, muscari, bellevalia, tulipa, galanthus, sternbergia, crocus, arum and biarum have important and critically endangered species with high export potential that enters into this group. most of these are endangered and their collection from wild and export has been banned to conserve them. large scale production and conservation of these species could also be achieved by in vitro techniques. therefore bulb scale and immature embryo explants of sternbergia candida, s. fischeriana, muscari muscarimi, fritillaria imperialis and f. persica were cultured on different nutrient media supplemented with various concentrations of plant growth regulators using different culture applications. large numbers of bulblets were produced (over bulblets/explants) from single immature embryos on nutrient media in most species tested after months of culture initiation. regenerated bulblets were kept at • c for weeks and then transplanted to soil successfully. to our knowledge the present study is the first report for in vitro bulblet production from immature embryos of geophytes. the procedure described here provides a prolific bulblet production system that may form the basis of bioreactor culture and conservation of endemic and endangered geophytes. the commercial use of organofluorine compounds in industrial, pharmaceutical and pest-control applications has dramatically increased over the past few years, resulting in the introduction of numerous new organic compounds into the environment. organofluorine compounds are chemically very stable and are assumed to be resistant to biological degradation. given the chemical inertness of fluorinated organics, their bioactivity, and their potential for accumulation in the environment, it is important to understand their environmental fate and the mechanisms by which they might be degraded. examples of the biodegradation of fluorinated compounds in literature are scarce, being fluorobenzoic acids the most commonly reported. information on the cleavage of carbon-fluoride bonds in synthetic compounds is limited to fluoroacetate dehydrogenase. in this project we try to obtain more insight in the defluorination mechanisms by investigating the diversity of degradation routes for these compounds in several soil bacteria by making use of modern genetic tools. a gram-positive strain capable of aerobic biodegradation of -fluorophenol ( -fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. batch cultures were set up and substrate consumption, accumulation of intermediates and product formation were monitored. the consortium was able to use -fp up to concentrations of . mg l − and was able to utilize a range of other organic compounds. stoichiometric release of fluoride ions was measured in batch cultures suggesting that there is no formation of dead-end products during -fp metabolism. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene , nazif kolankaya : kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth ( % soytone, % d-glucose and . % cellulose pulp). maximal extracellular ligninase production was detected after days ( nkat). the optimum biobleaching conditions are • c and ph . , with days. in this condition p. versicolor decreased the kapa number from . to . and increased brightness from to . in -day treatment. boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plan-tation areas of turkey. both defficiency and toxicity problems exist in a total of about % of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant acquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. butachlor is one of the selective systemic herbicide toxins that are act by inhibition of protein synthesis. this toxin is used exclusively in the rice, barely, cotton and wheat farmlands. butachlor is belonging to chloroacetanilide herbicide group, which are consisting of butachlor, alachlor, acetochlor, metolachlor and poropachlor. in the view of bioenvironmental, butachlor is degraded in the soil by microbial activity. its stability is about - weeks. it is converted to the water-soluble derivatives in soil or water, with a slow evolution of carbon dioxide. because of butachlor is one of the herbicide toxin, it is inhibitor factor against growth of bacteria and microorganisms. microorganisms can be continuing their activities in the limited concentration of butachlor. therefore treatment of industrial wastewater consist of concentrated butachlor by the biological treatment is impossible and it is necessary chemical or physico-chemical treatment are used. in this research, biological treatment methods are used. in the biological treatment, an activated sludge system with volume of . l is used. in this method, butachlor with concentration of mg/l are treated. removal percent of butachlor for concentration of . and mg/l are calculated to . % and . %, respectively. removal percent of cod is also calculated to %. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized purification and downstream process of xylitol obtained biotechnologically from hemicellulosic hydrolyzate of corncobs b. rivas , p. torre , j.m. domínguez , j.c. parajó , a. converti : department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, ourense, spain; department of chemical and process engineering, genoa university, via opera pia , genoa, italy. e-mail: brivas@uvigo.es (b. rivas) biotechnological production of xylitol from lignocellulosic materials has been widely studied in the recent years with promising results that confirming the possible industrial application of this technology. xylitol purification from fermented broth is the limiting stage of this process. previous works suggest crystallization procedures in order to recovery xylitol from fermented synthetic solutions. the complexity of fermented hydrolyzate not allows direct crystallization. in this work, corncobs hydrolyzate obtained with autohydrolysis-posthydrolysis techniques, detoxificated with activated charcoal and concentrated was fermented to xylitol by d. hansenii. the fermented broth composition was . % of xylitol ( g/l), . % of other sugars and . % of other compounds that interferes in the crystallization process (as dry matter of the liquor). the fermented media was submitted to an absorption process with activated charcoal and concentrated until a xylitol concentration of g/l. the liquor was then submitted to a second step of precipitation with ethanol, the best results achieved in this study were obtained with an ethanol/liquor ratio of . in these conditions this treatment allows to remove a . % of the impurities. the resulting solution was evaporated and crystallized containing % of ethanol and a xylitol concentration of g/l. crystallization was performed at t = • c with slightly agitation. after h were separated xylitol crystals with a recovery yield of % and a purity degree of %. numerous publications have documented that only a minor number of the indigenous prokaryotic organisms found in complex environments such as the human intestine, biogas reactors, and soil are known, and probably only a fraction of this diversity can be accessed using traditional culturing techniques. some of the reasons for this are the lack of knowledge of specific growth conditions, specific nutrients, and obligate coculture requirements. also growth on a solid surface directly exposed to the atmosphere puts a very strong selective pressure on single cells supposed to develop into visible colonies. therefore, the knowledge of these microorganisms is scarce and generally limited to the s rrna genes that have been extracted from different environments and cloned for phylogenetic analyses. an obvious approach to circumvent these problems was the development of techniques based upon micromanipulation for isolation of single cells from complex mixtures. continuous development of modern microscopes in combination with the precision of a servo-powered micromanipulator and the development of the modern microscopic micro injectors used in ivf techniques has further aided the manipulation of single cells. this technique, however, does not solve the problems of the non-culturable cells, and other approaches are needed to gain more information about these organisms. an approach to the non-culturable cells could be genomic analysis of isolated single cells without preceding cultivation. this pcr-based technique is widely used for genetic analysis of human cells, but due to the small amounts of dna present in prokaryotic cells it has so far not been possible to produce identifiable amounts of dna from single cell amplification using conventional polymerases. a promising alternative used for amplification of small amounts of dna is the f dna polymerase operating under isothermic conditions. applying random hexamer primers, this polymerase carries out a multiple displacement amplification (mda) of high molecular weight dna template. in this study we demonstrate the successful application of mda for selective amplification of genomic dna from a single prokaryotic cell. the yield was > mg of amplified genomic dna corresponding to about a billion-fold amplification from a single cell. the technique was used to approach a large group of non-thermophilic archaea found in agricultural soil. our results show that combining mda with fluorescent in situ hybridization and cell isolation by capillary micromanipulation enables an unprecedented ability to investigate new species without cultivation. also this combination of techniques opens for studies of genetic heterogeneity within populations and processes such as horizontal gene transfer. precipitation of zn + , cu + and pb + at bench scale using biogenic hydrogen sulphide produced from the utilization of volatile fatty acids by sulphate reducing bacteria maria teresa alvarez , , carla crespo , bo mattiasson : department of biotechnology, center for chemistry and chemical engineering, lund university, p.o. box , s- lund, sweden; instituto de investigaciones fármaco bioquímicas, universidad mayor de san andrés, la paz, bolivia biological production of hydrogen sulphide (h s) from sulphate using sulphate reducing bacteria (srb) is popular within environmental biotechnology. srb require absence of oxygen, presence of nutrients required for growth and oxidizable organic substrates (to supply hydrogen atoms for reduction of sulphate). many organic wastes have been used as electron donors for the sulphate-reducers in the treatment of acid mine drainage (amd) including straw, hay, sawdust, peat, spent mushroom compost and whey, however, other wastes such as municipal organic waste can be used. the aim of this work was to study the possibility of using srb for the treatment of amd at bench-scale. this process involved three stages: the volatile fatty acid (vfa) production by hydrolytic bacteria from the degradation of vegetables and fruits, the production of h s through the utilization of the produced vfas by sulphate reducing bacteria and the precipitation of metals by using the biologically produced h s. the substrates used for vfa production consisted of tomato, papaya, apple and banana. the h s produced from the degradation of vfas was utilised for the precipitation of an artificial effluent simulating the heavy metal concentrations of a mine located at bolivian andean region, containing approximately mg/l of zn + , mg/l of cu + and mg/l of pb + . the maximum concentration of hydrogen sulphide obtained was approximately mm. removal efficiencies of %, % and % for zinc, cooper, and lead, respectively, were achieved in the present work. at • c during days. bacterial population was determined by counting in a neubauer chamber with optical microscope. sulfate concentration was measured by turbidity method and metal concentrations in the filtered supernatant were measured by icp-aes. the first part of study consists of determine the maximum concentration of each metal at which d. vulgaris and desulfovibrio sp. grow in similar way than control culture (without metal). both cultures tolerate: cr(iii) ppm, ni(ii) . ppm, zn(ii) ppm. the maximum precipitation percentages were approximately: % ( ppm cr(iii)), % ( . ppm ni(ii)) and % (for d. vulgaris- ppm zn(ii) and desulfovibrio sp.- ppm de zn(ii)). time to reach the highest precipitation was minor for mixed culture (desulfovibrio sp.) y all the cases. the next part was focused to study the precipitation percentage when metals are present in combination in the same metal levels (cr(iii)-ni(ii), cr(iii)-zn(ii), ni(ii)-zn(ii) and cr(iii)-ni(ii)-zn(ii)). the combination of metals does not affect significantly the bacterial growth and precipitation percentage of metals. this fact supposes an importance advantage so metals are commonly found together in the environment. future experiments are focused in development of this process in continuous operation mode. biosolubilisation and depolymerisation of coal has potential to produce a clean energy source or high value organic products from low rank coals such as lignite or sub-bituminous coal. these complex soluble phenolic compounds are of value as starting materials for biotransformation to value-added compounds such as antioxidants and flavourants. the bioprocess is carried out at ambient temperature and pressure and is perceived to be environmental benign. in the evaluation of coal solubilisation an important quantity for the assessment of process feasibility is the yield, i.e. the determination of the mass of product obtained per unit mass of coal solubilised. to date, results for coal biosolubilisation reported in the literature are qualitative or at best semi-quantitative, indicating trends with operating variables. the process kinetics has not been determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. in this paper, the use of an indirect method for the estimation of the growth and metabolism of fungal biomass by measuring co evolution and o consumption using an off-gas analyser is reported in the study of fungal coal solubilisation. coal determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. biosolubilisation was carried out in a stirred tank slurry bioreactor with working volume of . l. complete suspension of the coal particles of - m mean diameter was achieved at an agitation rate of rpm. growth yield coefficients based on coal and oxygen as well as maintenance coefficients were calculated from growth of the fungus under the same conditions using a non-coal carbon source such as glucose. these data were used to determine the stoichiometric coefficients for biomass growth, enabling the biomass production rate to be quantified in terms of co production rate and o consumption rate. a dna-chip platform for parallel detection of microorganisms related to biofilm in industrial systems and drinking water systems pernille skouboe, dorte lauritsen, kim holmstrøm bioneer a/s, kogle allé , dk- hørsholm, denmark. e-mail: psk@bioneer.dk (p. skouboe) an oligonucleotide microarray for simultaneous detection and identification of pathogenic bacteria related to technical water systems as well as drinking water has been developed. the approach is based on the use of a tandem hybridization technique with two ribosomal s rdna-pcr products, bp and bp long, generated from two consensus pcr reactions using conserved ribosomal primers end-labeled with cy and cy , respectively. the tandem hybridization technique implies an internally quality control for discrimination between target and non-target signals. the current prototype of the dna-chip platform includes oligonucleotide probes representing different genera (and subgroups of species), e.g. legionella, mycobacterium, aeromonas, campylobacter, vibrio and enterococcus. the platform has been used for detection and identification of species from pure cultures, and initial experiments with water samples from industrial systems have been performed. the potential as well as the limitations of using a dna-chip based detection format in its present form will be documented. particularly, its potential application as a rapid method for initial screening of environmental or food samples will be addresses. the aim is to reduce and optimize the number of samples required for traditional microbiological identification tests. in the course of a project for the development of a novel kind of a mycotoxin inactivating feed additive, the aim of this study was to isolate and characterize microorganisms with the specific ability to enzymatically break down and detoxify fumonisins, a group of structurally related fungal toxins, with fumonisin b (fb ) being the most abundant and -with respect to toxicology -also the most important representative of this group. these toxins are produced as secondary metabolites by some fusarium species such as fusarium verticillioides and f. proliferatum and are naturally occurring contaminants of cereal grains worldwide. they are found especially in maize and maize based products, and are known to be hazardous to human as well as to animal health. a natural feed additive, based on microorganisms and/or enzymes, should ensure the detoxification of fumonisins during feed uptake and digestion via microbial or enzymatic break down of these compounds, by that protecting the animal from the harmful effects of these mycotoxins. besides an extensive screening of microbial strains derived from strain collections, various different natural habitats were investigated for the presence of fb degrading microbial activity, such as intestinal contents of pigs, soil samples, and naturally fumonisin contaminated maize. while testing of nearly organisms from strain collections did not show positive results, fumonisin transforming activity could be detected in one soil sample and a number of maize samples. trials in order to isolate the respective fumonisin degrading microorganisms resulted in a number of strains, whose fb degrading activity could be proven. the most promising bacterial and yeast strains were further characterized with regard to a general taxonomic description, and to different aspects of their toxin degradation behaviour. approaching a more relevant in vivo situation, fb degradation trials in food-and feed-stuffs were conducted. further on, the applicability of the respective organisms as stabilized lyophilisates was investigated. arsenic is one of the most important global environmental pollutants and the toxicological effects are related to its chemical form and oxidation state. arsenite [as(iii)] is reported to be on average times more toxic than arsenate[as(iv)]. this work shows the ability of one strain of the species ochrobactrum tritici to grow in presence of several metals including arsenite, arsenate, selenite, selenate, tellurite and antimonite. its arsenite mic was determined as mm, whereas for arsenate, this bacterium could resist to concentrations upper than mm. we report the identification of two loci involved in high-level arsenic resistance. sequencing of the first locus identified four complete genes in the following order: arsr, arsd, arsa, arsb. the second locus containing genes for arsenic resistance was also characterized. each sequence has been compared with nucleotide and protein databank (blast programs) and significant homology with known orfs coding for arsenic resistance has been found. it is also possible that the phenomenon of high-level arsenic resistance in o. tritici could evolve other genes or loci. the ability of the ␣-proteobacterium o. tritici to tolerate high levels of arsenic in addition to other oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. this work is based on the mathematical modeling of kinetics of a thermophilic bacteria cultivation system. the cultivation proceeded by way of batch and continuous on the synthetic medium with the main carbon source-lactose. this medium simulated an industrial waste approximately. mixed thermophilic aerobic bacteria popula-tion, applied to the wastewater treatment (sludge v&k bystrice pod hostynem), was used to the inoculation. the cultivation system consisted of the laboratory fermentor biostat b (b. braun biotech) with working volume l and the control unit connected with a computer. it is possible the temperature, ph, aeration, stirring and foaming regulation. physical and chemical cultivation conditions were optimized. the chemical oxygen demand (cod), generally expressive the impurity level, was selected as the main parameter for the cultivations run classification. but into the mathematical model also the kinetics of biomass growth, lactose consumption, production of choice metabolites (acetate, lactate, succinate) and dissolved oxygen concentration was included. the modeling was located to two head distinguishable growth phases of the microorganisms. an optimization and identification of mathematical model parameters was practised in the software language psi/c. the difference between simulated curves and experimental data is not statistically significant on the relevancy level . (f-test). it took place the cod degradation at . % with the average yield coefficient y chsk/x . g cod/g biomass in the batch process with the air aeration only. there is a better way to the cod elimination (> . %)-the aeration of air enriched by the pure oxygen. in experiments with the continuous system was obtained the . % cod decrease after the steady state stabilization. this work was supported by project msm . fluorescence in situ hybridization (fish) of whole cells using oligonucleotide probes was applied to study the influence of low temperature and temperature reduction on the bacterial community of biofilm reactors for the removal of chlorophenols (cps). two packed bed reactors were set up for degradation of a mixture of -cp, -cp, , -dicp, and , , -tricp as sole source of carbon and energy at • c (ra) and • c (rb) and were inoculated with bacterial consortia adapted to these respective initial temperatures. the performance of the reactors was studied under different conditions of pollutant loading, aeration rate, and hydraulic retention times over months. total chlorophenol removal capacities of and mg l − day − were achieved in the bioreactors ra and rb, respectively, under a total pollutant load of mg l − day − . the population of ␤-proteobacteria was the major bacterial community of the biofilm ( - %) followed by the ␥-proteobacteria ( - . %). two bacteria with the ability to mineralize mg chlorophenols l − were isolated from the bioreactors and characterized as ralstonia basilensis and alcaligenes sp., both belonging to ␤-proteobacteria. decreasing the temperature by • c (in two steps of • c each) resulted in an increase in the population of ␥-proteobacteria and a decrease in the population of ␤-proteobacteria in both reactors. application of genus specific probes showed an increase in the pseudomonas population from % of the ␥-proteobacteria at • c to % at • c. the pollutant removal capacity decreased to and mg l − day − in ra ( • c) and rb ( • c), respectively. the ␣and ␦-proteobacteria, cytophaga-flavobacteria and actinobacteria the survey was carried out in urban and rural areas of two cities (ankara and isparta). this paper is only analysing urban people by excluding villagers. urban sample is consisted of urban consumers and professionals. professionals were selected amongst pharmacists, doctors, agricultural engineering's and industrialists that is thought are affective in the process of developing new technologies and also the development of biotechnology in the society. basic data was gathered by a questionnaire including both structured and open-ended questions besides deep interviewed. workforce development for life sciences-the scottish experience carol booth scottish entreprise, uk the presentation will look at, the background and definition of workforce development for scottish enterprise, examine information available to support scottish enterprises economic intervention and where and when to intervene. conclusions emerging from the evidence base will be used to outline scottish enterprises approach to workforce development and look at which actions might be required to address identified issues. moving on to reasons for integrating workforce development into business development and how the life sciences cluster team at scottish enterprise, stakeholders and partners in scotland have approached their current and future contributions to workforce development for life sciences using a variety of projects. the national institute for bioprocessing research and training (nibrt) in ireland is a proposal that will be a state-of-the-art training, research and pilot plant service facility that brings together institutions with complementary expertise and state-of-the-art research technology, and industry partners. these include university college dublin, trinity college dublin, institute of technology, sligo and dublin city university. nibrt is an innovative collaboration between academic institutions at the forefront of biotechnology, cell biology, engineering and pharmacy and industry. for training, two separate training labs, for upstream and downstream training, in addition to research labs are planted. it will also include a state-of-the-art pilot plant fermentation facility for fermentation optimisation, fermentation scale-up, product separation and purification, regulatory aspects and automation. by aligning with industrial demands, the new institute will tailor its training programmes while remaining on the cutting edge of biotechnology research and technologies. the fermentation facility will offer hands-on training workshops and educational modules for outside researchers and companies. these workshops cover the fundamentals of small-scale fermenta-tion, scale-up considerations, and fermentor design and set-up. the training and educational philosophy underpinning the nibrt will focus on the needs of industry with an emphasis on providing training for accreditation of existing industry staff and prepare technicians and graduates for the technical, business, regulatory and professional aspects of the industry. the strategy is to provide specialised modules in nibrt in support of courses established in the higher education institutions, which will provide the certificates, diplomas and degrees. modules will be offered for all categories of students and will be given credits respected by other third level institutions in ireland. the role of professional graduate degrees in meeting current and future biotechnology industry workforce needs a. stephen dahms san diego state university, usa the presentation will review the status of new graduate training models designed to meet the unique needs of the biotechnology industry as it transitions to commercialization. emphasis will be on professional master's degree programs in biotechnology and their various versions, with a focus on operational and funding strategies and industry acceptance. discussion will also centre upon the creation and operation of industry-validated, specialized and highly targeted professional masters degrees in various refined aspects of the drug development process, including regulatory affairs, biomedical quality systems, clinical affairs, management of drug development, management of reimbursement affairs, bioinformatics, etc. the eurodoctorate in biotechnology, new combined mba/phd combined degrees in the molecular life sciences, the u.s. professional doctorate in chemistry and the proposed u.s. professional doctorate in biotechnology will be also discussed. data will also be presented on the current workforce and the industry's projected needs. genetic studies show that mankind is a rapidly expanded population of closely related individuals with very similar disease sensitivity. bad nutrition and infections dominate among the main health problems in the world. apart from malnutrition, the overeating habits of the developed world are now creating problems in the developing world as well. infectious diseases are also a global problem since new contagious agents like hiv, sars and avian flew do not recognize borders. thus, the global responsibilities of modern health care are obvious. many research scientists from and in developing countries find it nearly impossible to use their talents for the benefit of their own countries. some struggle to develop research and education programmes with poor facilities, some leave science completely, and others migrate to more developed countries. the talents of such people are either being wasted or lost completely to their home countries just at the time they are most needed to combat the great humanitarian challenges of hunger, illness and lack of knowledge. europe must strengthen programmes which allow third world scientists to work to their full potential in their home countries or regions. yang beijing genomics institute, chine academy of sciences, beijing, china europe has all the reasons to be proud of being the cradle of modern science and of its achievements and resources in life sciences. as a model of having solved many of the problems that many other counties are now facing, europe is expected by the whole world to make its further contribution to a better future of mankind and to play a more important role in the international community of life sciences. tropical diseases and public health basilio valladares director of the university institute of tropical diseases and, public health, university of la laguna, la laguna, tenerife, spain. e-mail: bvallada@ull.es the presentation will look at the main research interests of the university institute of tropical diseases. these are the following: . immunology and molecular biology of parasites. we express and purify recombinant proteins from leishmania sp. which have been shown to act as immunomodulators and protect against disease such as l , hsp , hsp . the study of acanthamoeba pathogenic factors has also resulted in the isolation and silencing of extracellular proteins related to their pathogenecity, which has a great potential in the development of novel chemotherapeutics. . diagnosis of parasites. the immunological diagnosis of leishmaniasis has been one of the main research interests in our laboratory for several years. as a result, we have identified peptides which could be used to develop kits for the immunological diagnosis of leishmaniasis such as hsp c-end, l n-end, etc. we have also developed some dna based methods for the identification of acanthamoeba species from biological and environmental sources. . water quality. biological parameters. our water research group has the expertise to identify and characterise bacterial, viral and parasitic indicators of faecal contamination in diverse water sources including tap water, rivers, reservoirs, sea, etc. this research area has been developed in collaboration with the local sewage treatment plant and reservoir managing authorities. currently, we are establishing a conjoined project with the center for disease control and prevention (cdc) in atlanta, usa for the identification of water-borne emerging pathogens. . development and formulation of chemotherapeutic antiparasitic agents. in this field, we evaluate the leishmanicidal activity both in vivo and in vitro of natural and synthetic drugs and synthetic peptides. in a later stage, the drugs which have shown the highest antiparasitic activity have been subjected to cytotoxicity assays and their molecular targets dissected. some of the drugs tested in the last few years have been submitted to patent due to their outstanding activity. finally, in order to allow the commercialization of these drugs, both in vivo and in vitro assays are being carried out to predict their chemical stability and degradation pathways. this will be followed by the use of liofilization and controlled crystallisation strategies for the development of efficient and safe treatments. . human and population genetics. tachykinins and their receptors in different tissues and groups of patients and their association with molecular polymorphisms is another one of our research interests. the knowledge of ligand and receptor sequences and their similarities will allow the rational development of drugs with specific activity against these receptors. furthermore, we are also interested in the interspecific variation along the evolutionary scale of these markers. . nitrate assimilation group. research in our group is focused on understanding nitrate assimilation in the yeast hansenula polymorpha. several biotechnological companies use this yeast to produce heterologous proteins (hepatitis b vaccine). genetic manipulation techniques for h. polymorpha are available in our laboratory. head of the department of biotechnology, technological institute of canary islands, pozo izquierdo santa lucía, las palmas, spain single cell analysis by flow cytometry has proved to be a tool to perform simultaneous and rapid measurements related to cell morphology and physiological state. previous studies showed the possibility of quantifying neutral and polar lipids spectrofluorometrically using a lipid specific fluorescent dye, nile red (nr), however the existence of inter and intraspecific variations in the fluorescent response had not been clearly established. in this work, two strains of marine microalgae: crypthecodinium cohnii and tetraselmis suecica, characterized both by high contents of polyunsaturated long chain fatty acids (dha and epa, respectively) and an hypersaline microalgae: dunaliella salina, characterized by a high ␤-carotene production, were grown under different conditions and collected at different growth phases to be used for in vivo lipid quantification with nr by flow cytometry. our results showed a high correlation between the mean fluorescence signal of nr stained cells and the neutral and polar lipid content measured by gravimetry for each strain. in this respect, these data make feasible the development of a rapid method for lipid quantification in monoalgal cultures. however, differences in the dye uptake related to specificity were detected. in this communication we also assess the possibility of use this cytometric technique to select microalgal strains with high lipid and polyunsaturated long chain fatty acids content from mixed samples. performance of such technique would be a good alternative to the time-consuming traditional screening protocols based on gravimetry and gas chromatography and would optimise the search of new commercial strains of microalgae. claverie-martín head of research unit, biomedical research institute, hospital universitario, de n.s. de candelaria, santa cruz de tenerife, spain our group is involved in the cloning and production of proteins of interest to the food and pharmaceutical industry. we have recently expressed in yeast the cdna that encodes the precursor of caprine chymosin. chymosin is the enzyme responsible for the coagulation of milk in the abomasum of unweaned calves. this enzyme is secreted by gastric mucosa cells as an inactive precursor, known as prochymosin. in the acidic conditions of the lumen, prochymosin is converted into the active form by autocatalytic cleavage of the n-terminal prosequence. chymosin is used extensively in cheese production because it cleaves -casein in a specific manner with low proteolytic activity. several biotechnology companies are producing the bovine recombinant enzyme for commercial use in the process of cheese making. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. it is well known that the activity of these types of extracts varies depending on the age of the animal and the type of food ingested. these difficulties should be overcome using a recombinant caprine chymosin. the caprine mrna used for the synthesis of the cdna was obtained from the abomasum of milkfed kid goats. the cdna fragment encoding the mature portion of caprine prochymosin was fused in frame to a signal sequence in yeast expression vectors. culture supernatants of yeast cells transformed with the recombinant plasmids showed milk-clotting activity after activation at acid ph. proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed -casein (patent ). the recombinant caprine chymosin could be an alternative milk coagulant in cheese making. work is underway to optimise the expression of the new recombinant prochymosin for further purification and characterization. smart molecules for health victor martín head of research, university institute of bio-organics "antonio gonzález", avda. astrofísico francisco sánchez , la laguna, tenerife, spain the instituto universitario de bio-orgánica "antonio gonzález" (iubo) is a multidisciplinary research centre that belongs to the university of la laguna. the iubo is located at the town of la laguna, inscribed on unesco's world heritage list in , and former capital of the canary island of tenerife. the geographical location in addition to its mild climate has made the canary islands to posses a variety of ecosystems with unique plants and animals. the institute was started up in the s with the need to study the natural products and secondary metabolites produced by those marine and terrestrial organisms, thus providing a new source of bioactive products. the main research lines that are being developed at iubo are summarised in the following paragraphs: anticancer agents from natural sources: several natural products and their semisynthetic derivatives are produced at the iubo in diverse joint projects for the development of new antitumour drugs with novel mechanisms of action. as an example we can mention natural products from the mevalonic, shikimic or polyketide pathways. some products have recently shown in vitro reversion of the resistance in multidrug resistant (mdr) tumour cell lines. genetic engineering: in vitro cultures of the plants atropa baetica, maytenus amazonica and m. macrocarpa are developed in order to manipulate their biosynthetic pathways and induce the production in large scale of the secondary metabolites for diverse applications, including arthritis, rheumatism, and back pain. marine organisms and toxins: dinoflagellates are marine organisms responsible for the red tides and food poisoning episodes. among others, okadaic acid and yessotoxin are the most common toxins present in european shellfish. the isolation of these products is best done from the microorganism cultures, since they are present in very low amounts in the natural sources. at the iubo we develop culture systems to provide us with amounts of toxins large enough to perform biological, metabolic and bioactive studies. insecticide and repellents: natural products are being isolated for their use against plagues, specially those affecting agriculture. these projects are run in collaboration with a number of public institutions and agrochemical companies throughout europe and latin america. fine chemicals and pharmachemicals: our institute possesses large expertise in the field of organic synthesis devoted to the synthesis of medicinal substances, with special focus on asymmetric processes. of particular interest is the development of new methodologies for the total synthesis of biologically active substances like polyether toxins, (un)natural amino acids, sphingosine analogs, alkaloids, etc. with an annual source of s of new compounds, the fine chemicals and medicinal chemistry branch at iubo have recently started and anticancer screening program in collaboration with the biomedical research unit at the hospital universitario n.s. de la candelaria. the program is committed to the discovery of novel drugs for application in cancer treatment. the outcome of this project in its first year has been outstanding, leading to the finding of several leads that form the basis for current and future projects. institute of canary islands, biotechnological department, playa de pozo izquierdo s/n, santa lucía, gran canaria, spain the presentation will look at the main research interests of the biotechnological department of the technological institute of canary islands. these are the following: nutrition and feeding in aquaculture: we conduct studies on digestion, absorption, transport, and utilization of the different nutrients applying also different techniques such as histology, enzymology, genetic or immunology among others. aquaculture feeding is the main important cost in fish farms, being higher that the prize of fries, personnel cost or energetic costs. thus, studies conducted on the improvement of diet formulation and the use of different dietary ingredients is one of our main research lines (such as vegetable oils and meals to be used as alternative to fish meal and oil, or carotenoid sources to improve fish colour). not only the use of the different ingredient is studied, but also the effect of these ingredients on fish health, flesh quality, flesh healthy aspects related with human consumption, are being studied. different formulae are being developed and patents of different diets are being obtained. studies on nutritional requirements are also conducted allowing us to patent different formulae in larvae studies, developing microdiets to substitute the high-cost processes associated with live prey in larval nutrition. development of immunostimulants, anti-stress diets, immune techniques to be applied as bio-indicators of fish health and welfare, as well as use of dietary ingredients derived from bio-reactors are other research lines in our group. genetics: genetic techniques applied to aquaculture are being an important tool. microsatellites are being used to determine genealogy of the fish, allowing to decreases important problems in aquaculture such as fish deformities. genetic techniques such as micro-arrays and gene expression are being applied to obtain indicators of stress and health in fish. these technologies also permit to make different selective breeding programs, increasing the accuracy to estimate genetic parameters and evaluating brood-stock. furthermore, this technology allows to obtain a procedure to increase the productivity and quality of fish hatcheries. new species for aquaculture. development of new culture techniques: the diversification of species cultured is one of the main objectives of european aquaculture, since nowadays only four marine species are commercially produced: gilthead sea bream. european sea bass, turbot and salmon. we have been developed rearing techniques for new species such as red porgy or canarian abalone and also we are conducting studies on different new species, such as different sparids species, octopus or yellowtail. new rearing technology: the election of adequate systems for fish growth for each species and site of production and the localization of more appropriate sites for farms, using gis technology are also of special importance in our research team. besides, new larval rearing techniques such as semi-extensive hatchery (mesocoms) were development and nowadays is being used to increase fry quality (survival, no-deformities, better growth). this technology is being exported to other countries in order to offer new technology easy to manage to be implanted in developing countries. alejandro cañeque project officer, canary islands special zone, ministry of finance, c/leon y castillo - a planta, edificio urbis, las palmas, spain. e-mail: acaneque@zec.org the canary islands special zone is the newest tax instrument within the canary islands economic and fiscal regime (ref). it offers a reduced tax rate of between and % of corporate income tax for companies setting up a business. the companies must cover the following minimum requirements: create employment and make a minimum investment. the zec offers other tax advantages such as the exemption from paying transfer tax and stamp duty and the canary islands general indirect tax (igic). this tax scheme particularly fosters the biotechnology and pharmaceutical sectors. references fahnert references bechor the antimicrobial drugs high-level production of human collagen prolyl -hydroxylase in sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates microbial processes and products press. biotech. bioeng. . van hijum microbial xylitol production from corn cobs using candida magnoliae the role of bacillus methanolicus citrate synthase gene, city, in regulatiing the secretion of glutamate in lysine-secreting mutants plasmid-dependent methylotrophy in thermotolerant bacillus methanolicus , -anhydrod-fructose; a versatile chiral building block: biochemistry and chemistry detailed dissection of a new mechanism for glycoside cleavage: the ␣- , -glucan lyase ␣- , -glucan lyase, a new class of starch and glycogen degrading enzyme ␣- , -glucan lyase, a new starch processing enzyme for production of , -anhydro-d-fructose efficient purification, characterization and partial amino acid sequencing of two ␣- , -glucan lyases from fungi ␣- , -glucan lyases producing , -anhydro-d-fructose from starch and glycogen have sequence similarity to alpha-glucosidases enzymatic description of the anhydrofructose pathway of glycogen degradation i. identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and ␣- , -glucan lyase in the fungus anthracobia melaloma a systems approach to dissecting immunity and inflammation integrative biological analysis of the apoe* -leiden transgenic mouse innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble cd soluble forms of toll-like receptor (tlr) capable of modulating tlr signaling are present in human plasma and breast milk proteomics of the rat gut: analysis of the myenteric plexuslongitudinal muscle preparation an integrative metabolism approach identified stearoyl-coa desaturase as a target for an arachidonate-enriched diet the challenges of modeling mammalian biocomplexity rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by mudpit technology post-genomics of lactic acid and other food-grade bacteria to discover gut functionality genetics, metabolism and application of lactic acid bacteria. fems microbiol complete genome sequence of lactobacillus plantarum wcfs functional ingredient production: application of global and metabolic models metabolic engineering of lactic acid bacteria for the production of nutraceuticals reduction in antinutritional and toxic components in plant foods by fermentation the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no k - ). the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no. k - ) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aÇ ikel at gazi university. the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no k - ) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aÇ ikel at gazi university. mrs. lynnette fernandez, johanna mäkeläinen, m.sc. and olli rämö, m.sc. are gratefully acknowledged for their help. this work was supported by the health science council, the academy of finland and the tekes-neobio program. supported by the mec of spain (ppq - -c - ) and the canary islands government. jmp thanks icic for a postdoctoral fellowship. frpc thanks cajacanarias for a fpi fellowship. tm thanks the spanish mcyt-fse for a ramón y cajal contract. this work was supported by the korean systems biology research grant from the ministry of science and technology, lg chem chair professorship, ibm sur program and bk program. this study was supported by ankara university biotechnology institute (project no: ). this work was partially supported by mec and feder (bio - ), and fundación séneca carm ( /pi/ ). this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. keto sugars have long been implicated as attractive intermediates or substrates for further chemical or enzymatic reactions, to generate a number of synthetic sugar derivatives and fine chemicals. the quinone-dependent pyranose dehydrogenase (pdh) purified of the basidiomycete fungus agaricus meleagris catalyzes with high specificity the oxidation of the c- of glycosidically bound d-glucose, whereas in contrast it oxidizes simultaneously the c- and c- of free d-glucose. considering the broad substrate tolerance, pdh provides a new convenient tool for high yield production of -keto- this work was partially financed by the scientific and technological research council (cicyt, spain), grant ren - , by the iii pla de recerca de catalunya (generalitat de catalunya), grant sgr- , and by the generalitat de catalunya to the "centre de referència en biotecnologia" (cerba). this work was supported by mega a.s. (czech republic) (www.mega.cz) and following vega grants: / / and / / . this work was partially supported by mec and feder (bio - ), and fundación séneca carm ( /pi/ ). financed by a marie curie re-integration grant merg-ct- - and consejería de educación y ciencia de la junta de andalucía. this project is part of the collaborative research centre sfb "development of biotechnological processes by integrating genetic and engineering methods", which is supported by the german research foundation dfg. this research was supported in part by grants from the hungarian scientific research fund (otka t , f and d ) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb / ). this research was supported in part by grants from the hungarian scientific research fund (otka t , f and d ) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb / ). this research was supported in part by grants from the hungarian scientific research fund (otka t , f , d ) and gvop- . . .- - - . the authors thank dr. hamid narjiss (director of morocco inra) for the instruction to identify nadorcott mandarin by molecular markers and helpful discussions regarding this paper. this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). this work has been financed by xunta de galicia (pgidt pxib pr). sevgil sadettin, gönül dönmez department of biology, faculty of science, ankara university beşevler, ankara, turkey this study was supported by ankara university biotechnology institute. demet Ç etin , sedat dönmez , gönül dönmez : department of biology, faculty of science, ankara university, beşevler, ankara, turkey; department of food engineering, faculty of engineering, ankara university, dışkapı, ankara, turkey sulfate-reducing bacteria (srb) that could grow on modified postgate c medium (pc) containing chromium(vi) were isolated from industrial wastewater and their chromium(vi) reduction capacities were investigated as a function of changes in the initial ph values, chromium, sulfate, nacl concentrations and carbon source. the optimum ph value at mg l − initial chromium(vi) concentrations was determined as . chromium(vi) reduction by srb was investigated at . - . mg l − initial chromium(vi) concentrations. at the end of the experiments, the mixed cultures of srb were found to reduce more than % of the initial chromium(vi) levels which ranged from . to . mg l − within - days period. the effects of initial - . g l − concentrations of sulfate and - % (w/v) concentrations of nacl to chromium reduction were showed that, the lowest concentrations of sulfate and nacl, were the best for chromium reduction in the pc media including mg l − chromium(vi). when the % whey was used as carbon source in the pc medium, . % of the . mg l − initial chromium(vi) concentration was reduced within this study was supported by ankara university biotechnology institute. this study was carried out as a part of the project for analyzing and controlling the mechanism of biodegrading and processing entrusted by the new energy and industrial technology development organization (nedo). this work has been financed by xunta de galicia (pgidt pxib pr). lead ions are considered a high pollutant of different waters. in our previous work were selected seven potential sorbent-strains rhodotorula mucilaginosa , rh. aurantiaca , rhodotorula sp. , williopsis californica , candida krusei t, cryptococcus sp. wt of lead ions. it was determined their stability to high concentration (up to mg/l) of lead ions in medium. the ph changes and yeast physiologies of growth were studied in medium with these heavy metal ions. the influences of environmental factors such as ph of solution, age of microbial culture, biosorbent concentration in suspension, alive or living state of biosorbent, time of contact on sorption were investigated. the levels of maximal sorption ability and biomass affinity to heavy metal ions were established by experimentally received sorption isotherms with mathematical modeling of biosorption process separately for everyone researched yeast strain. the sorption isotherms obtained in these experiments for non-living yeast biomass showed that the maximal sorption capacity was and × − mol (g sorbent) − for rh. aurantiaca and s. cerevisiae , respectively. in the case of living biomass, the high- this work has been financed by the spanish ministry of science and technology and european feder (project ctm - ). the authors wish to thank dra. m.j. martínez (cib, csic, madrid, spain) for providing coriolopsis rigida. this work was supported principally by embo and the mrc. fed-batch cultivation of haematococcus pluvialis under illumination with leds for production of astaxanthin abdolmajid lababpour, tomohisa katsuda, shigeo katoh department of molecular science and material engineering, kobe university, kobe, hyogo - , japan photosynthetic microalga haematococcus pluvialis is a most promising microorganism for production of astaxanthin, which has powerful antioxidant activity and is used both for human being as a food supplement and cosmetic; as well as animal farming such as salmon and poultry. the deficiency of nutrients in batch culture of h. pluvialis decreases the growth of cells and increases the induction of astaxanthin as an induction factor. therefore, it is impossible to reach to high cell concentrations in batch cultures. in previous experiments, the medium replacement increased the cell concentration, while accumulation of astaxanthin was not induced without other factors. in this work, the effects of fed-batch addition of culture medium on the cell growth and astaxanthin production in h. pluvialis cultures were studied. h. pluvialis was cultivated in cm culture medium containing sodium acetate, yeast extract, l-asparagines, mgcl · h o, feso · h o and cacl · h o (ph . ). light was supplied by panels of blue or red led lamps. the temperature was kept at • c and the culture was mixed with a magnetic stirrer. in fed-batch cultures of h. pluvialis, the cell concentration and production of astaxanthin increased in comparison with those in batch culture. in addition, the operation in feb-batch manner is easier than medium replacement from industrial viewpoints for production of astaxanthin. simvastatin and similar compounds are wide used as antihypercholesterolemic agents. simvastatin is obtained by c-methylation of side chain of lovastatin. this process is not perfect and some unreacted lovastatin is present in the reaction mixture. simvastatin is separated from lovastatin using fungi clonostachys compactiuscula. using of living microbials has some disadvantages such as high cost, difficult product separation from the reaction mixture. we work on overcome these difficulties by separation and immobilization of enzyme that hydrolyses lovastatin ammonium salt in presence of simvastatin ammonium salt. our results of purification and immobilization lovastatin hydrolase will be presented. investigation of peptide antibiotics produced by trichoderma strains isolated from winter wheat rhizosphere a. szekeres , l. kredics , l. hatvani , z. antal , l. manczinger , a. nagy , c. vágvölgyi : department of microbiology, university of szeged, p.o. box , h- szeged, hungry; hungarian academy of sciences, university of szeged, microbiological research group, hungry; pilze-nagy ltd. , kecskemét, p.o. box , species of the imperfect filamentous fungal genus trichoderma with teleomorphs belonging to the hypocreales order of the ascomycota division are of great economic importance as sources of enzymes, antibiotics, as plant growth promoters, decomposers of xenobiotics, and as commercial biofungicides. peptaibols and related peptaibiotics (prps) are secondary metabolites constituting a family of fungal peptide antibiotics which is constantly growing since alamethicin was isolated from cultures of trichoderma viride. these compounds are linear, amphipathic polypeptides composed of - amino acids and usually containing several non-proteinogenic amino acid residues, which are representing characteristic building blocks of the structure. one hundred and twenty trichoderma strains were isolated from roots of winter wheat grown in agricultural fields of southern hungary. the identity of species was examined based on morphological and molecular characters. the presence of prps-producing strains among the isolated trichoderma strains was detected by biological tests and the antibiotics were partially purified using a multistep chromatography procedure involving exclusion chromatography, adsorption chromatography and thin-layer chromatography. about % of the isolates proved to be able to produce prps. the antibacterial activity of the compounds was tested against staphylococcus aureus, bacillus subtilis, micrococcus luteus and escherichia coli, while the antifungal effect was recorded against fusarium oxysporum, f. culmorum, rhizoctonia solani and pythium debaryanum. ergezinger, m., bohnet, m., berensmeier, s., buchholz, k., . integrierte enzymatische synthese und adsorption von isomaltose in einem mehrphasenbioreaktionsreaktor. cit , - . glucansucrases from family of glycoside-hydrolases are transglucosidases that produce ␣-glucans from sucrose, a very cheap substrate, without any use of nucleotide activated sugars. based on sequence analyses, these enzymes have been classified in two families, the family and the family of glycoside hydrolases. among the natural diversity existing in family in which are found the glucansucrases produced by lactic acid bacteria, three enzymes have been selected for their distinctive specificities: dextransucrase from l. mesenteroides nrrl b- f (dsr-s), which catalyses almost essentially the synthesis of ␣- , -linkages, alternansucrase from l. mesenteroides nrrl b- (asr), which produces alternan polymer formed of ␣- , and ␣- , -alternated linkages and finally dextransucrase from l. mesenteroides nrrl b- (dsr-e), which is responsible for the synthesis of a branched dextran composed of about % of ␣- , -linkages in the main chain and % of ␣- , -branched linkages. for all these enzymes, the natural polymerase activity can be shifted towards oligosaccharide production or gluco-conjugate syntheses by introducing acceptors in the reaction medium. a number of sugar acceptors have been successfully glucosylated with the view of developing new functional food products. acceptor glucosylation yield as well as acceptor reaction product structures were shown to be highly dependant on the enzyme specificity. consequently, using glucansucrases of distinctive specificities and varying the acceptors give access to a large variety of applications. amylosucrase, the sole glucansucrase found in family of glycoside-hydrolases is also of great interest for functional food applications. this enzyme is able to synthesize highly resistant amylose from sucrose. again the reaction conditions can be used to modulate the yield and the size of amylose. the aim of our work is to further develop the applications of these enzymes via rational and combinatorial engineering. the most recent results obtained in this field will be discussed. novel food structure engineering concepts with enzymes johanna buchert vtt biotechnology, espoo, finland food structure is a very important quality attribute in food choice, since it affects not only the sensory perception of texture, but also release of flavour. enzymes offer specific means to engineer food structure by creating cross-links to food biopolymers, i.e. to proteins and/or carbohydrates. enzymatic cross-linking of food biopolymers can be exploited to create novel types of structures to foods without any need of added food ingredients. laccases and peroxidases can be used to crosslink ferulic acid containing carbohydrates, such as sugar beet pectin or arabinoxylan. proteins can be crosslinked by different oxidative or transferase type of enzymes. transglutaminases can crosslink protein via formation of isopeptide bond between glutamine and lysine residues. laccase and peroxidase can oxidize tyrosine residues to corresponding radicals, which in turn can further react with different groups in proteins. tyrosinases, on the other hand, oxidize tyrosine to a quinone, which can further react with aromatic ring, amine and thiol groups present in proteins. the biopolymer networks formed can be further engineered by combining adequate processing to the enzyme treatment. in this work the potential of enzymatic food structure engineering is reviewed. asparaginase-mediated reduction of acrylamide formation in baked, fried, and roasted products hanne vang hendriksen, beate kornbrust, steffen ernst, mary stringer, hans peter heldt-hansen, peter Østergaard novozymes a/s, dk- bagsvaerd, denmark. e-mail: hvhe@novozymes.com (h.v. hendriksen) in , it was discovered that acrylamide is formed in several potato and grain-based foods (e.g. chips, french fries, toasted bread, biscuits, cereals) and in coffee, all of which have been prepared at high temperatures. the level of this potential carcinogen in the final food appears to range from to ppb. later that year, the mechanism of acrylamide formation was unraveled, demonstrating that asparagine and reducing sugars are the precursors for acrylamide. this pointed to several potential enzymatic approaches to remove the root cause of the problem by degrading the precursors in situ. here, we demonstrate that asparaginase treatment leads to a more efficient reduction in acrylamide than alternative enzymatic treatments. asparaginase from aspergillus oryzae is used to reduce acrylamide formation significantly in laboratory models of a range of common food products. examples are french fries, biscuits, crisp bread, and fabricated chips. the sensory qualities appear to be constant. the implications for scaling up the processes for industrial food production are discussed. effect of cultivation conditions on folate content in yeast: exploring the potential of yeast as a bio-enrichment vehicle for folate in foods sofia hjortmo , johan patring , jelena jastrebova , thomas andlid : department of chemical and biological engineering, chalmers university of technology, po box , gothenburg, sweden; department of food science, swedish university of agricultural sciences, po box , uppsala, sweden. e-mail: sh@fsc.chalmers.se (s. hjortmo) over the past years, the interest in health benefits of the b vitamin folate has increased considerably. a good folate status may hinder progression of several diseases such as neural tube defects and downs syndrome in foetus, as well as cancer, dementia, alzheimer's disease and cardiovascular disease in adults. it is however not easy to reach the recommended intake and new strategies have to be developed to increase the folate status. in this project we explore the use folate producing microorganisms for this purpose. many yeasts have the ability to synthesise folate de novo and can thus serve as a source for humans. folate enrichment in fermented foods could be much improved by using starter cultures better at producing folate compared to traditional strains. this is, e.g. applicable to bread making fb over-expression of isoprene biosynthetic enzymes in the ␤-carotene producer zygomycete mucor circinelloides tamás mucor circinelloides has been involved to study the carotene biosynthesis genesis of fungi. this fungus is more amenable to molecular techniques than the others traditionally used in carotenogenic studies (e.g. blakeslea trispora and phycomyces blakesleeanus). moreover, mucor has a great advantage: it is a dimorphic organism. this type of morphology is preferred by the fermentation industry because yeast-like growth allows the submerged culture, when usually higher biomass production can be achieved and cells can be more easily separated from the media. β-carotene is a terpenoid-type chemical compound likewise to sterols, quinones or chlorophylls. the production can be increased by improving the non-carotene specific terpenoid biosynthesis. this can be carried out by the overexpression of the genes responsible for the ratelimiting steps of these pathways. in this study, polyethylene glycol mediated transformations of m. circinelloides protoplasts were performed with autoreplicative expression vectors containing the known terpenoid genes of m. circinelloides (e.g. isoa encoding farnesyl pyrophosphate synthase and carg encoding geranylgeranyl pyrophosphate synthase). carotene production of the transformants and the wild-type strains were analysed by high-performance liquid chromatography (hplc). transformants harbouring plasmids with isoa or carg produce about . times more carotene than the recipient strain, while carotene production increased about two times in the co-transformants containing both type of plasmids. members of the genus rhizopus are important from biotechnological aspects in consequence of their effective extracellular enzyme, alcohol and organic acid production. moreover, rhizopus strains are used for fermentation of various foods, because they are capable of transforming soybeans into edible products. the high affinity iron permease (ftr ) contains both highly conservative and variable regions applicable for phylogenetic comparisons. the aim of this study was the comparative analysis of this gene of different rhizopus species in order to elaborate a simple and fast method to identify these fungi at a species and subspecies level. conserved regions of candida albicans and rhizopus oryzae ftr genes (fu et al., ) have been analysed to design degenerate primers for polymerase chain reaction. they were used to amplify the homologous regions from different strains of r. oryzae, r. microsporus, r. stolonifer and r. niveus. isolates of the similarly thermophilic rhizomucor miehei and r. pusillus, as well as a strain of m. rouxii were involved in the study as outgroups. deduced protein sequences were aligned and phylogenetic analysis was performed. surprisingly the r. oryzae isolates formed a group completely different with a significant distance from the r. microsporus isolate. r. niveus is currently not distinguished from r. stolonifer var. stolonifer because of morphological considerations. however, phylogeny of ftr gene sequences, in agreement with earlier results based on rapd data (vágvölgyi et al., ) , raise the need to handle r. niveus as a separate species. sequences and pcr primers useful for identification of all tested rhizopus strains were elaborated. potential application in a lactose conversion process. the prebiotics market is at high demand therefore the development of the process to produce 'prebiotic' galacto-oligosacharides efficiently and inexpensively is our particular interest. citrus, particularly mandarins and clementines, are among the most economically important fruit crops in morocco. besides morphological traits multiple molecular markers have been used for the caracterisation of citrus germplasm. the main aim of this study was to evaluate the moroccan mandarin germplasm and to identify specific polymorphisms among accessions sharing identical name. eighty mandarin and two sweet orange varieties were analyzed by dna markers. issr markers were amplified using anchored primers and analysed by agarose gel electrophoresis. aflp markers analyses were performed using three primer combinations. the dice coefficient was used to estimate genetic similarities and the upgma algorithm was utilised to generate a phenogram depicting the genetic relationships among the acessions. the selection of primers out of the issr primers primarily assayed, allowed us to maximize the average number of amplified fragments analyzed per reaction ( . ), and the percentage of informative polymorphisms ( %). the three combinations of ecori/msei primers revealed reliable aflp markers, ( %) of witch were polymorphic. the range of fragment sizes varied from to bp. contrasting with the phenotypic diversity for agronomic and fruit quality traits, very low variability at the dna level has found among mandarins, which always showed a high (s > . ) coefficient of genetic similarity. the molecular marker analyses allowed the clarification of ambiguous denominations and the establishment of phenological relationships. the mandarin cultivars have been clustered into several different sub groups. this study allowed the identification of one issr marker, distinct and specific for the clementine sidi aissa and some aflp markers specific to maroc late and w. navel. many hybrids used in this study presented high coefficient of the similarity with one of their parents, such as siamelo and king of siam (s = . ), fortuna and clementine (s = . ), kara and king of siam (s = . ). this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). the new morocco mandarin variety nadorcott became very important in the international market because of high quality, good size, easy peeling and absence of seeds. also known as afourer or w. murcott, this variety was selected in - at the afourer experimental station, inra, located near to beni mellal city, as an original tree among several -year-old murcott honney (c. reticulata × c. sinensis) seedling trees. in order to shed additional light on the genetic origin of this variety, we have carried out isozyme and dna fingerprinting analyses. for better interpretation of the nadorcott molecular profiles, others mandarin cultivars, among which murcott honney, were also analyzed by molecular markers. three enzymatic systems (idh, pgm and pgi) permitted the discrimination between nadorcott and his female parent murcott honney. the molecular patterns displayed by these cultivars point out the sexual origin of nadorcott and discard the previously assumed hypothesis for its origin as a mutation of a nucellar zygote. the issr and rapd markers analyses allowed the identification of kinnow; du japon, vietnam; and swett lime as the genetically most closely related mandarins (s ∼ . ) to nadorcott. strong genetic similarity was also found with the clementine group, a possible male parent of nadorcott. the analysis by aflp markers confirmed the hybrid origin of nadorcott and the high genetic relatedness (s = . ) of this mandarin to its putative female parent murcott honey, and other cultivars as kinnow (s = . ) and clementine (s = . ). the possibility of an accurate molecular identification of nadorcott by specific molecular markers is of paramount importance for the protection and management of this original moroccan citrus variety. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions g/l from white distilled lees and . g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. thermophilic cyanobacterial strains that could grow in the bg media was isolated from hot springs and their reactive dye bioaccumulation was studied under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye accumulation. in the experiments performed with newly isolated synechocystis sp. and phormidium sp., the optimum ph values at about mg l − initial reactive dye concentrations was determined as . lipases are extremely versatile enzymes, that catalyze both hydrolysis and synthesis reactions. they have a wide range of industrial applications, among which the manufacture of detergents, pharmaceuticals and fine chemicals are outstanding. the fungus rhizopus oryzae has been reported to synthesize a number of commercially interesting enzymes. in this work, its ability to produce extracellular lipases when grown in solid state culture has been assessed. cultures were carried out in erlenmeyer flasks, using a complex medium and several supports, both synthetic (nylon sponge) and natural (barley bran, ground walnut or peanut). the latter appeared to be more suitable for lipase production. since the best results were initially obtained with lipid-containing supports, barley bran cultures were supplemented with a vegetable oil, in an attempt to optimise lipase production and design an efficient procedure for reusing this agroindustrial waste. surprisingly, this strategy did not improve enzyme synthesis. however, when a surfactant (triton x- ) was added to the basal medium, a dramatic increase in extracellular activity was detected (up to -fold). the results agreed with those previously obtained in submerged cultures of r. oryzae, in which addition of olive oil did not increase lipase production, while the presence of triton x- had a remarkably beneficial effect. also, enzyme concentration in solid state cultures was up to two-fold that of the submerged ones. est maximal sorption capacity was found for yeasts cryptococcus sp. wt and rh. aurantiaca . these cultures also demonstrated the high sorption affinity, which makes them especially efficient biosorbents at low concentrations of lead ions. the high efficiency of lead elution was shown with . n edta. isolation and identification of marine bacteria from deep-sea sediments els maas , cara brosnahan , vicky webb , helen neil , phil sutton : marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand; oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand marine sediments were obtained using a piston corer with associated trigger core ( . m, . m diameter). cores were collected from depths of to m, along norfolk ridge and across challenger plateau. sediments ranged from coarse carbonate sands in the north to sandy and silty hemipelagic mud with increasing depth and latitude. all samples sites underlie subtropical surface water masses associated with, and south of, the tasman front. sediment samples were aseptically taken from the triggers cores upon recovery. samples were stored in sterile tubes at • c on board the vessel for - days. the core samples were plated on several different agar types and incubated aerobically for weeks at • c. individual colonies were sub-cultured and purified using standard microbiological techniques. morphological and molecular taxonomy revealed that the bacteria isolated from the sediments were closely related to novosphingomonas, halomonas, stappia, glaciecola, pseudoalteromonas and leeuwenhoekiella. phylogenetic trees constructed using s rrna gene sequence data showed that two other isolates were unrelated to known genera. the bacterial isolates are currently being investigated for their biotechnological potential. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by % was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were and %, respectively. while the color and aromatocity decreased by and %, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was . h − while the monod constant based on the consumed tp and cod (mg/l) were and , respectively. the control of water pollution has become of increasing importance in recent years. the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g − ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph. the maximum adsorption at ppm was . % equal to . mg of dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of - ). the effect of contact time was studied at initial concentration ( ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. flocculation of saccharomyces cerevisiae (diastaticus) ifo was studied. cells of ifo did not flocculate even in the stationary phase without mg + ("mg + -deficient cells") although they began to flocculate strongly h after inoculation in the presence of mg + ("complete cells"). cycloheximide completely inhibited induction of floc-forming ability of "mg + -deficient cells". co-flocculation between "complete cells" and "mg + -deficient cells" was investigated by chemical modification. treatment of "mg + -deficient cells" by proteolytic enzymes did not affect the co-flocculation with "complete cells". photo-oxidation or mercaptoethanol-reduction of "mg + -deficient cells" failed to weaken the co-flocculation with "complete cells" while treatment of "mg + -deficient cells" by periodate brought about a significant loss of the co-flocculation. on the contrary, "complete cells" deflocculated by proteolysis or chemical modification of proteinaceous component failed to co-flocculate with "mg + -deficient cells". these findings suggest that "mg + -deficient cells" are non-flocculent because of lack of proteinaceous component essential for flocculation of cells of ifo . the industrial toscano cigar production starts with the dark firecured kentucky tobacco fermentation process. during this phase the present work is a trial to study the portal serum factors which stimulate the cell proliferation of the schistosomules, aiming to find ways to block or inhibit their effects. our previous studies showed that portal serum of human and hamster (highly susceptible hosts) and a - kd fraction separated from human portal sera by ultrafiltration stimulate cell proliferation in immature schistosomules ( days old) in vitro. for further identification of the portal serum factors in the range of - kd that stimulate cell proliferation, schistosomules were incubated in vitro in medium containing % fetal calf serum, % portal human serum or % peripheral human serum or their fractions separated by native electrophoresis followed by electroelution, incubations were performed in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. the results showed that human portal sera enhanced cell proliferation of schistosomules compared to the peripheral serum. this stimulatory effect was substantially reproduced by fraction separated from human portal serum with molecular weight . kd. these results may help in designing a drug or antibody therapy to block the stimulating effect of the portal serum fraction and subsequently to disturb the life cycle of the parasite at early stage of development. center of molecular biosciences, university of the ryukyus, okinawa - , japan. e-mail: naoya-s@comb.u-ryukyu.ac.jp (n. shinzato)oil strage tank sludge, mainly composed of water and solid hydrocarbons (waxes) needs to be treated when harvesting the stored oil. although the sludge treatment by microbial surfactant or microbial cracking are considered as the feasible method, microbial degradation of the waxes (ca. solid n-alkanes) have been reported in a very limited species such as acinetobacter. in addition, long-chain n-alkanes (so called paraffin waxes) are one of the major components of oil, and their resistant properties to biological attack hold up the recovery of oil-polluted environments. in this report, we have screened n-tetracosane (c ) degrading bacteria from soils in okinawan island, an unique sub-tropical area in japan, to know the bacterial diversity and their degrading mechanism. srdna phylogenetic analysis of the isolates, totally ca , showed they were not only acinetobacter and pseudomonas, but also other proteobacteria (alcaligenes), actinomycetes (gordonia, nocardia, and leifsonia), bacillus, staphylococcus, and unidentified ones. they also grew not only the solid n-alkanes but also iso-alkanes, mid-chain n-alkanes as the sole carbon source. results for the biosurfactant production will also be shown. the properties of environmental enterococci were studied. the strains were isolated mainly from surface and waste waters and several strains from sheep manure were also included. species identification was provided by combination of phenotypic (micronaut system, merlin) and molecular detection methods (automated its-pcr, ddl-pcr). several discrepancies were observed when comparing molecular and biochemical identification. six enterococcal species were overall identified; e. faecium and e. hirae were the most abundant ones, almost % of isolates belonged to these two species. the distribution of selected genes conferring virulence to enterococci (cyla, gele and esp) was investigated, the positive signal was obtained mainly for e. faecalis strains. the strains were also characterized for the possession of enterocin genes (enta, entb, entp, ent , entl ab) and high frequency of enterocins was observed. biosorption of three different dyes (reactive black , cibacron brilliant yellow, cibacron brilliant red) onto immobilized scenedesmus obliquus a microalga was investigated in a batch sys-tem. the immobilized alga exihibited the highest dye uptake capacity at the initial ph value of . for all dyes. the effect of temperature on equilibrium sorption capacity indicated that maximum was obtained at • c for rb , cby and cbr biosorption. the freundlich, and langmuir adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants were evaluated. biocontrol properties of microbially-treated sugar beet wastes in presence of rock phosphate n. vassilev, i. nikolaeva, m. vassileva department of chemical engineering, faculty of sciences, university of granada, c/fuentenueva s/n, granada- , spain. e-mail: nbvass@yahoo.com (n. vassilev) the effect of soil application of sugar beet wastes (sb) treated with aspergillus niger in the presence of rock phosphate (rp) on the control of fusarium wilt of tomato were studied. two treatments and a control were used: inoculation with glomus intraradices (am), further inoculation with a. niger grown on sb + rp medium, and the control (c). application of the am fungus increased plant growth, p and n uptake and reduced disease caused by fusarium oxusporum f. sp. licopersici (fol) as compared to non-mycorrhizal control plants. soil amendment with sb + rp + a. niger resulted in % and % (versus c) higher plant shoot biomass in plant-soil experiments contaminated or not with fol, respectively. in this case, disease severity and number of fol cfu reached the lowest levels while soil phosphatase and beta-glucosidase activities increased compared to all other treatments. fol negatively affected plant root mycorrhization determined in the am treatment while the difference between the mycorrhization of plants grown in the presence and absence of f. oxysporum in sb + rp + a. niger-amended soil was insignificant ( % versus %, respectively). in conclusion, the fermentation mixture containing mineralized organic matter, partially solubilized rp, and a. niger biomass could be efficiently used not only in improving plant growth, nutrient uptake and properties of degraded and polluted soils, as previously reported (vassilev and vassileva, ) , but also in environmentally-mild management of fusarium wilt. vassilev, n., vassileva, m., . appl. microbiol. biotechnol. , - . biological treatment processes allow for the effective elimination of charged inorganic micropollutants, e.g. a number of oxyanions, heavy metals, etc. from contaminated drinking water supplies. however, dedicated technologies have to be implemented in order to eliminate the target pollutants without changing the quality of treated water, avoiding its secondary pollution by cells, nutrients and metabolic by-products. some innovative technologies, which combine the use of membranes with the bioconversion of charged micropollutants in order to deal with the secondary water contamination problem, will be presented and critically compared. a special on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by % was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were and %, respectively. while the color and aromaticity decreased by and %, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was . h − while the monod constant based on the consumed tp and cod (mg/l) were and , respectively. advanced start-up strategy of an anaerobic three-phase turbulent bed reactor treating winery wastewaters r. cresson, h. carrère, n. bernet, j.p. delgenès laboratoire de biotechnologie de l'environnement, institut national de la recherche agronomique (inra), avenue des etangs, narbonne, france. e-mail: cresson@ensam.inra.fr (r. cresson)the objective of our study was to compare two start-up strategies for an anaerobic biofilm process, to create an effective biofilm and increase the organic loading rate (olr) as quickly as possible. two methanogenic three-phase biofilm reactors have been started, using the same operational parameters (solid hold-up ratio, gas velocity of mm s − ), in order to test two different strategies:• maximal load strategy (reactor a): the olr is increased as long as the global amount of removed cod (biogas production) increased. • maximal removal strategy (reactor b): the olr is increased stepwise as soon as the cod removal rate reaches %.both reactors have been operated for days, until a volumetric olr of g cod l − j − , with more than % of carbon removal. the total amount of cod removed and methane produced were higher in reactor b ( . and . %, respectively). in both reactors, the short hydraulic retention time (hrt) applied during all the experiment caused a rapid wash-out of planktonic bacteria and an exclusive use of the substrate by the attached micro-organisms, which accelerates the biofilm growth. the lag-phase was reduced to approximately days. the reactor submitted to repetitive disturbance by the maximal removal strategy appeared to be more robust when confronted to perturbation like organic overload or nutritional deficiency. experiments have demonstrated capability and the efficiency of the aggressive strategy for controlling anaerobic bioreactor start-up. sustainability is the generally accepted paradigm for future industrial development. the re-integration of waste products into production processes is a major aspect of environmental sustainability. in this study the use of sugar cane molasses is being investigated for the production of bioplastics by mixed microbial cultures, with the added possibility of parallel biohydrogen production. polyhydroxyalkanoates (phas) are polyesters synthesized by bacteria and accumulated as granules in the cytoplasm. studies conducted by this group have shown that mixed microbial cultures subjected to dynamic feeding conditions may accumulate phas up to % cell dry weight, a value close to that obtained for pure cultures. volatile fatty acids are good substrates for the production of phas by mixed cultures. on the other hand, sugar molasses, with a very high sugar content (about % dry weight), can produce organic acids by fermentation. the two-stage process being implemented in this study includes a molasses fermentation step, in which the high sugar content of the molasses is converted into volatile fatty acids (vfas), and a pha production step, in which the vfas serve as the precursors to the formation of phas under dynamic feeding conditions. moreover, hydrogen can be produced by anaerobic bacteria from carbohydrate-rich substrates giving organic fermentation end products, h and co . to optimize the production of both high-value products, design of experiments (doe) is being used to elaborate a set of experiments to study the effect of ph, hydraulic retention time and organic loading on both the organic acids distribution (which will serve as precursors for pha production in the second step) and h production in the acidogenic fermentation reactor (a l cstr). preliminary results show that the effluent of the acidogenic reactor fed with g/l total sugars and operated at ph and d = . h − (composed mainly of acetate and propionate) can be successfully fed to a polymer-accumulating mixed culture. under these conditions, the h production yield has been estimated at . mol h /mol sucrose. vanillin is a flavour compound used in food industry, fragrances and pharmaceutical preparations, which is nowadays mainly produced by chemical synthesis. the increased demand of natural products for the food industry as well as the high cost of natural vanillin extracted from vanilla pods has recently stimulated the research for alternatives to produce this compound by a natural way. the microbial transformation of ferulic acid, a phenolic compounds from lignin degradation, is recognized as being the most interesting alternative to produce natural vanillin. the combined effects of initial ferulic acid concentration (s ) and biomass concentration (x ) on vanillin production by resting cells of escherichia coli strain were investigated using response surface methodology. e. coli jm /pbb a recombinant strain producing key enzymes of ferulate catabolic pathway from p. fluorescens bf (feruloyl-coa synthetase and feruloyl-coa hydratase/aldolase) was utilized in this work. a full-factorial design was employed for experimental design. the results shown a possible inhibition phenomena at a vanillin concentration of about . g l − leading to the accumulation in the fermentation media of secondary compounds like vanillic acid and vanillin alcohol. removal of dissolved nutrients from wastewater using a microalgae biofilter line christensen, suvina sooknandan, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, odense, a microalgae biofilter can be used for treatment of wastewater from landbased fish farms in order to remove excess amounts of dissolved nutrients such as nitrate, ammonium and phosphate. a bubble column bioreactor has been developed for cultivation and characterization of microalgae. this type of bioreactor is equipped with a control system that enables online determination of the photosynthetic quotient and optimization of light intensity. furthermore the bioreactor has a dualsparging system simultaneously allowing adequate mixing and high gas-liquid mass transfer coefficients. different species of microalgae have been cultivated in batch and fed batch cultures to characterize growth and ability to take up the different dissolved nutrients. the specific growth rate and substrate uptake rate have been determined to compare and select the algal species most suited for use in a biofilter. additionally the composition of lipid, protein and carbohydrates has been measured to determine the nutritional quality of the algae when used as animal feed. at present, biological nitrogen-removal is mostly carried out through several complicated steps. to simplify the present systems for nitrogen-removal, we have investigated a new nitrogenremoval bioreactor using packed gel envelopes capable of simultaneous nitrification and denitrification. the envelope consists of two plate polymeric gels with a spacer in between. ammonia oxidizer, nitrosomonas europaea and denitrifier, paracoccus denitrificans are co-immobilized in the plate gels. when the envelopes are exposed to wastewater containing ammonia, the immobilized n. europaea oxidizes ammonia to nitrite in the outer aerobic surfaces of envelopes. at the same time, as ethanol solution is injected into the internal anaerobic spaces of envelopes, the immobilized p. denitrificans reduces the nitrite to nitrogen gas using the ethanol solution as an electron donor for denitrification. in this way, the envelopes can remove ammonia from wastewater in a single step. we have already reported advantages of our bioreactor in laboratory-scale experiments. in this study, we show our large-scale bioreactor (water volume . m ) could treat three kinds of wastewater derived from coal power plants. ammoniacontaining wastewater that occurred regularly in a coal power plant was continuously treated with the bioreactor using thirty envelopes for over a year. the bioreactor could remove more than % of total nitrogen at hydraulic retention time (hrt) of h. at hrt of h, the bioreactor accomplished a maximum rate (the transformation of nh + to n ) of . g n/day m of the envelopes' surface. the performance was equivalent to that obtained in the laboratory-scale experiments. furthermore, our bioreactor showed similar nitrogen-removal performances when it treated nitrate-containing wastewater occurring regularly and condensed ammonia-containing wastewater occurring at irregular intervals in coal power plants. these results show that our bioreactor can treat various wastewater containing nitrogen in coal power plants. thus, our concept is effective to simplify the large-scale systems in coal power plants and the other plants. in order to establish an environmentally friendly process for the treatment of metal containing waste, in a portuguese refinery a process involving sulphur oxidizing acidophilic microbes is being considered. bioleaching of metal containing bottom ash, from fluidised bed incineration of sludge resulting from the refinery water treatment station, was performed using a sulphur oxidising acidophilic culture isolated from an acid pool resulting from the weathering of sulphur piles from the claus plant. this sample served as inoculum for liquid medium cultures with % sterile sulphur flowers as source of energy. application of monod kinetics to adapted culture growth of free cells presented a value of µ = . day − . yield of sulphur conversion to sulfate after days was η = %. in the presence of bottom ash from the incineration of refinery sludges µ = . day − and the yield of sulphur conversion was η = . %. a η fe = % removal of iron is obtained from the treated ash. x-ray fluorescence spectroscopy of the solid residue revealed a total removal of metals namely, v, cu, ni, zn and most of the fe after days of bioleaching. the present of heavy metals in the environment is a serious problem. they are commonly present in effluents from mining and industrial activities. usually, chemical conventional methods are very expensive and they have limitations when heavy metals are in low concentrations. at the moment the interest increases for processes that involve microorganisms as alternative method. some effluents present heavy metal sulphates which are soluble compounds. sulphate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulphate as an electron acceptor and generate hydrogen sulphide. hydrogen sulphide reacts with heavy metal ions to form insoluble metal sulphides that can be easily separated from a solution. the purpose of this work was to evaluate the ability of srb to reduce cr(iii), ni(ii) and zn(ii) in artificial contaminated solution. desulfovibrio vulgaris and desulfovibrio sp. strains has been tested in this study. batch cultures was carried out in ml sealed bottles with different concentrations of studied metals ( - mg/l), with % of inoculum bacterial and adapted to postgate's medium c. a gaseous nitrogen current was employed to purge oxygen and obtain anaerobic conditions. the assays were incubated statically represented very low portion (less than - %) of the total bacterial community at all temperatures tested. schistosomules of schistosoma mansoni ( days old) were incubated in rpmi medium containing % fetal calf serum, % hamster portal venous or % hamster peripheral venous serum (highly susceptible host) or % rat portal venous or % rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. also the rate of cell proliferation of s. mansoni were assessed in vivo in hamster to study the cell proliferation in the natural ontogeny of the organism. the rates of cell proliferation as expressed by brdu labeling indices (blis) were determined as a function of time of incubation by immunohistochemistry using monoclonal antibody to brdu. compared to schistosomules cultured in presence of rpmi plus % fetal calf serum, blis were increased by % in the presence of hamster portal, but not in peripheral serum. while in case of rat, no significant changes were observed in the blis in both portal and peripheral sera. the experiment was repeated using hamster portal and peripheral sera containing different schistosomal igg antibody titres. the results showed decreased values of blis compared to sera which did not contain the schistosomal antibody(ies). the in vivo results revealed that there was no cell proliferation of s. mansoni schistosomules ( days old) in the lungs. cell proliferation was detected in schistosomules of days old and the results revealed a significant decrement in the brdu labeling indices (blis) with the increase of the age of schistosomules in vivo. the results indicated that hamster portal venous serum (highly susceptible host) could have stimulating factor(s) for schistosomule cell proliferation which is not found in rat (poorly susceptible host) and the presence of antibody(ies) greatly inhibit the cell proliferation. this could be due to the blocking of some portal serum factors, which stimulate the cell proliferation by the antibody(ies). the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g − ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph, the maximum adsorption at ppm was . % equal to . mg dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of - ). the effect of contact time was studied at initial concentration ( ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. compared to other european nations, the austrian population shows a low level of knowledge in biosciences and a strong denial of gene technology ( ). the austrian non-profit organisation dialog<>gentechnik, a scientific society, organizes various activities to raise awareness for the "hot topics" in the life sciences. according to its principle of independence, all activities are funded publicly. projects on behalf of the austrian authorities and international cooperations demonstrate credibility and trust in dialog<>gentechnik ( ). a few examples will be presented. dialogue with the public: on the occasion of the first anniversary that the gmo labelling rules became effective, the action "gene technology on my plate" is performed austrian wide in shopping centres. here, consumers are informed about health and labelling aspects of gm food. two days of open discussions were organized in the context of the austrian genome research program gen-au ( ): topics were "gene diagnosis" ( ) and "genome research-what is in it for me?" ( ) . an open lab is currently set up in vienna to offer "hands on" experience in life sciences for everybody. motivating students: in the very successful gen-au summer school ( ), high school students spend - weeks in the lab and work with scientists. the best documentations are awarded. in an innovative project, student groups (age - ) work on the topics stem cells and cloning and develop units of an elearning course which will be accessible for all austrian schools in the near future. engaging stakeholders: dialog<>gentechnik manages interdisciplinary wor king groups that develop leaflets, brochures and questionnaires on various aspects of gene diagnosis. four products are currently distributed to the public and to health services. ( ) european commission. eurobarometer . , december ; ( ) www.dialog-gentechnik.at; ( ) www.genau.at. the aim of this paper is to compare the attitudes of the urban consumers with professionals towards to new technologies, especially to biotechnology. it was tried to find out in which area, medical, agriculture or industry, people can accept biotechnological developments and in which area not accept. key: cord- - wnmdvg authors: nan title: p – p date: - - journal: clin microbiol infect doi: . /j. - . . _ _ .x sha: doc_id: cord_uid: wnmdvg nan resistance rates (rr) over a period of years were generally better than those reported for a total of icus. the mean mrsa rr was . %, for all the sari icus, whereas it was only . % in the study icu. by the end of duration of treatment for pneumonia had been reduced to - days and written guidelines on empiric antibiotic treatment and prophylaxis were revised with respect to the resistance situation of the study icu. the significant decrease between and in total antimicrobial ad from , to in the study icu resulted mainly from the reduced consumption of nd generation cephalosporins, carbapenems and imidazoles. ni did not change significantly over time. compared to the year , the costs for antibiotics were halved from € , to , , which corresponds to € . /pd and € . /pd, respectively. the percentage of antibiotics in the total icu budget for pharmaceuticals decreased from . % to . %. conclusion: surveillance and feedback of antibiotic use and resistance can serve as a valuable quality control instrument and can have an impact on antibiotic treatment. from to , antibiotic use was reduced by % and costs for antibiotics/pd were cut by two third in the icu study without any increase in device associated nosocomial infection rates. the resistance situation was generally better than in all sari icus, but showed heavy fluctuations. similar illness burden but different antibiotic prescription to children: a population-based study k. hedin, m. andre, a. håkansson, n. rodhe, s. mö lstad, c. petersson (växjö, falun, malmö, linköping, se) objectives: respiratory tract infections are the most common reason for antibiotic prescription in sweden as in other countries. the prescription rates vary markedly in different countries, counties and municipalities. the reasons for these variations in prescription rates are not obvious. the aim of the study was to find possible explanations for different antibiotic prescription rates in children. therefore a prospective population based log book study was conducted in four municipalities which, according to official statistics, had high and three municipalities which had low antibiotic prescription rates. methods: during one month, parents recorded all infectious symptoms, physician consultations and antibiotic treatments, from -month-old children in a log book. the children's parents also answered a questionnaire about socioeconomic factors and concern about infectious illness. results: antibiotics were prescribed to . % of the children in the high prescription area and . % in the low prescription area (crude or . ( % ). after multiple logistic regression analyses taking account of socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations, differences in antibiotic prescription rates remained (adjusted or . ( %ci . - . )). the variable that impacted most on antibiotic prescription rates although it was not relevant to the geographical differences was a high level of concern about infectious illness in the family. conclusion: the differences in antibiotic prescription rates could not be explained by socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations. the differences may be attributable to different prescription customs, in which case physicianś prescription patterns are not always rational. decreasing outpatient antibiotic prescribing in germany, germany, - , does not include newer macrolides, fluoroquinolones and extendedspectrum beta-lactams w.v. kern, k. de with, k. nink, h. schrö der (freiburg, bonn, de) objective: the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project has shown that outpatient antibiotic prescribing in germany has been comparatively low among european countries. we assessed trends over time and regional variation of outpatient antibiotic use in germany, and wondered if the observable decreasing trend included all drug classes to a similar extent. methods: prescription data (compulsory health insurance covering > % of the population, sample of . % until the year , all prescriptions thereafter) were analysed using the atc/who methodology and current ddd definitions. we specifically defined the following drug groups: ''basic'' penicillins (bpens, oral penicillin or aminopenicillins), extended-spectrum betalactams (esbls, oral cephalosporins, staphylococcal penicillins, aminopenicillin/betalactamse inhibitor combinations, parenteral cephalosporins and broadspectrum betalactams), newer macrolides (nmls, roxithromycin, clarithromycin, azithromycin) versus older macrolides (omls). quinolones (fqs), folate synthesis inhibitors (t/ss) and tetracyclines (tets) were also assessed. data were expressed in yearly ddd/ persons covered by the insurance (ddd/ ). findings: outpatient prescribing in was ddd/ (corresponding to . did = ddd/ and day) and decreased to ddd/ in the year and to ddd/ in . the decreasing trend over the last years was observed in all regions. the decrease was most significant for omls () %), t/ss () %), tets () %), and bpens () %) while there was no decreasing use of esbls (± %) and increases in the rate of prescribing nmls (+ %) and fqs (+ %). tets and bpens, however remained the most prescribed antibiotics in . regional variations in remained large for bpens (> -fold) with very low prescribing rates in the eastern region, but were small for t/ss, nmls and fqs (< -fold). conclusions: over a decade we observed a % decreasing outpatient antibiotic prescribing that included relevant antibiotic drug classes except esbls, nmls and fqs. the relative increase was most significant for fqs. severe community-acquired pneumonia admitted to the intensive care unit: impact of antibiotic therapy delay on hospital mortality antibiotic therapy were enrolled in the study. pts were divided in groups according to time to treatment (< h gi, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . baseline severity scores (apache ii, sofa, psi, curb- ), microbiological documentation, and hospital outcome were compared for all groups. results: pts were included in the study. microbiological documentation was achieved in % of all pts, positive blood cultures in / ( . %), s. pneumoniae in / ( %). mean age was ± , apache ii . ± . , sofa . ± . , psi ± , curb- . ± , mechanical ventilation in . % and vasopressors use in . %. overall icu and hospital all-cause mortality were . % and . %, respectively. baseline severity scores were comparable in all groups and their respective hospital mortality is provided in table . conclusions: in severe cap, treated with a combination therapy, time to treatment seems to have an impact on hospital all-cause mortality. based on our results, antibiotic treatment should be initiated within the first hours after hospital admission. (period ii) -cefoperazone/ sulbactam ( g a day) as monotherapy were used as the empirical antibacterial therapy of vap. the rotation from cefepime to cefoperazone/sulbactam was performed due to our previous study demonstrated high frequency of esbl producers among enterobacteriaceae. the samples from the lower respiratory tract were obtained by mini-bal. the sensitivity of microorganisms to the antibiotics studied (ceftazidime, cefepime, cefoperazone/sulbactam and carbapenems) was determined by the disk diffusion method. results: the main pathogens of vap were s. aureus ( %), p. aeruginosa ( %), enterobacteriaceae ( %) and this structure did not changed during both periods. the antibiotic sensitivity of p. aeruginosa and enterobacteriaceae (k. pneumoniae and e. coli), was studied separately. a high level of resistance of enterobacteriaceae to cefepime can be explained by the strains prevailing in the given icu, which produced extended spectrum beta-lactamases (ctx-m). the resistance of enterobacteriaceae to cefepime was . % in i period and . % in ii period, to ceftazidime - . and %, meropenem - and %, imipenem - . and . %, cefoperazone/sulbactam - . and . %, respectively. a change of cefepime for cefoperazone/sulbactam was not followed by any decrease of enterobacteriaceae resistance level to cefepime during ii period. the resistance level of p. aeruginosa to cefepime was . % in i period and . % in ii period, to ceftazidime - . and . %, meropenem - . and . %, imipenem - . and . %, cefoperazone/ sulbactam - . and . %, respectively. conclusion: the exclusion of cefepime for months didn't improved the sensitivity of enterobacteriaceae to this medication. the level of resistance of p. aeruginosa and enterobacteriaceae to cefoperazone/sulbactam did not increased despite a wide use of this antibiotic during months. antibiotic consumption in german acute care hospitals m. steib-bauert, k. de with, e. meyer, p. straach, w.v. kern (freiburg, frankfurt, de) objective: outpatient antibiotic use in germany differs substantially between eastern and southern parts of the country (relatively low use) and western part (relatively high use). there is no nationwide estimate of hospital antibiotic use and its geographic variation if any. the aim of the present study was to provide an estimate of recent hospital antibiotic use density in germany and to identify basic unit/hospital characteristics associated with excess use. methods: data on hospital consumption of systemic antibiotics in anatomical therapeutic chemical (atc) class j were obtained from a convenience sample of acute care hospitals in germany that participated in an ims survey in the year and had complete data (dispensed drugs and patient-days per year) for at least one non-paediatric, non-psychiatric department or ward. a total of non-icu surgical departments/wards, non-icu non-surgical (general medicine, oncologyhaematology, neurology/stroke) departments/wards, and icus covering > million patient-days were analysed. data were expressed in ddd (who/atc definition version ) or ''prescribed/recommended daily doses'' (pdd, better reflecting [high] dosages given to hospitalized patients) per patient days (ddd/ and pdd/ ). findings: the weighted mean over all departments/wards incl. icus was . ddd/ ( . pdd/ ). as expected, icu antibiotic use density was much higher than use in non-icu areas, and use in haematology-oncology was higher than in other non-surgical departments/wards. in univariate analyses, bed-size category and university affiliation (icus, surgical wards), region (icu, surgical and non-surgical wards) and haematology-oncology as specialty (non-surgical wards) were associated with use density, but these associations were only partly confirmed in multivariate logistic regression analyses of factors associated with excess ( ‡ %) use density which showed university affiliation and haematology-oncology to be independently associated with high use. conclusions: based on this hospital sample, antibiotic use in german hospitals shows little, non-significant regional variation and appears to be similar to what has been described from other european countries. adjustment of the data at least for university affiliation and haematology-oncology is important in comparative analyses of hospital antibiotic consumption. impact of formulary change in medical intensive care unit on outcome of infection and antimicrobial resistance sought to evaluate a formulary change and impact it has on infection and resistant. methods: prospectively, all patients in a -bed icu were followed for a period of months in phase i ( patients per patient days) and to collect baseline data after a decrease in the use of piperacillin-tazobactam (pt) when substituted by cefepime for a period of months in phase ii ( patients per patient days). results: total infections in phase i vs. phase ii were lower respiratory tract (lrti) patients ( %) vs. patients ( %); urinary tract infection (uti) patients ( %) vs. patients ( %); and sepsis of undetermined aetiology patients ( %) vs. patients ( %), respectively. there were no significant differences in death ( % vs. %) , cure or improvement of infection ( % vs. %), readmission to the unit ( . % vs. . %), hospital risk of death ( . % vs. . %), mean length of icu stay ( . days vs. . days), or rates of nosocomial infection ( . % vs. . % for lrti; . % vs. . % for uti; . % vs. . % for soft tissue infection; . % vs. . % for bacteremia; . vs. . per patient days for intravenous catheter infection) in phase i and ii respectively. the cost of antimicrobial acquisition in phase i and ii were $ and $ per patient respectively (p < . ). the mean antimicrobial treatment costs per patient for pt were $ vs. $ and cefepime were $ vs. $ in phase i and ii respectively (p < . ). the in vitro susceptibility and rate of infection and colonization with escherichia coli were unchanged in both study periods. there were vs. staphylococcus aureus (p < . ); of these percnt vs. percnt were methicillinresistant s. aureus and vs. enterococcus faecium ( % vs. % vancomycin-resistant enterococci) in phase i and ii respectively. there were % vs. % pseudomonas aeruginosa and % vs. % klebsiella pneumoniae extended spectrum beta lactamases in phase i and ii respectively. conclusion: the implementation of formulary substitution of pt to cefepime in the medical icu had resulted in a decrease in the use of pt. in addition, there were decreased costs and less s. aureus infections without adversely affecting the outcome of infection or antimicrobial resistance. intravenous antibiotic use in scottish hospitals; evaluation of the glasgow antimicrobial audit tool r.a. seaton, d. nathwani, p. burton, e. douglas (glasgow, dundee, uk) introduction: there are few data on antibiotic prescribing within scottish hospitals and a coordinated multisite point prevalence survey had not been performed before. there is concern that antimicrobials are overused in hospitals. methods: antibiotic use in acute medical and surgical units in scottish hospitals across trusts, was investigated using a point prevalence survey. data were collected by pharmacists. appropriateness of the iv route of administration was determined by review of data by an infectious diseases physician (idp) and compared with a specifically designed computerised algorithm. the idp also judged the appropriateness of the chosen iv agent against local guidelines. patients from hospitals in regions were surveyed on a single day. ( . %) were receiving an antibiotic, ( . %) intravenously. receiving oral antibiotics had received an iv previously. median duration of iv therapy was days (iqr - days) and time from iv to oral switch was . ( ) ( ) ( ) ( ) ( ) . the idp judged appropriate iv route in % patients compared with . % by the algorithm. the sensitivity of the algorithm was . % and specificity . %. the positive predictive value was . % and the negative predictive value was . %. the idp judged iv agents to have been chosen and administered appropriately in %. most frequently prescribed iv agents were rd generation cephalosporins ( gc) ( . %), co-amoxiclav ( . %), metronidazole ( . %), glycopeptides ( . %). significant regional differences were seen for most antibiotic groups including gcs ( . % (site ) vs . % (sites , , , ) , p < . ) and glycopeptides [ . % (site ) vs . % (site , , , ) , p < . ]. it is possible to coordinate, collect and compare data from scottish hospitals. the gaat gives a good estimate of the appropriateness of iv therapy. significant differences in prescribing patterns between similar patient groups across different hospital sites were demonstrated. such data may usefully inform local and national audit and support prescribing initiatives. associations between continuous variables were tested in univariate analysis with the spearman correlation test (r). multiple linear regression analysis was performed in a backward stepwise approach. results: the median rate of total hospital glycopeptides use was . (range . to . ) ddds per , pd with higher consumption in large public hospitals. consumption was higher in intensive care areas (median . ; range . to ) than in surgery areas (median . ; range . to . ) and in medicine (median . ; range to ). glycopeptides use correlated with number of central line per , pd (r: . ; p: . ) and with size of the various areas in the hospital (for intensive care, r: . ; for medicine areas, r: . and for surgery areas, r: . ; p < . ). median incidence of mrsa was . per , pd. incidence of mrsa explained a small proportion of the variation in hospital glycopeptides consumption (r : . ). in a multivariate linear regression model, incidence of mrsa and number of beds in surgery areas were independent predictors of total glycopeptides use in the hospital (r adjusted: . ). after controlling for these factors, number of central-line per , pd was no more associated with glycopeptides use. conclusion: in our hospitals, total glycopeptides use was not heavily determined by incidence of mrsa. although glycopeptides use in surgery areas was not the highest, the total number of surgery beds in the hospital explained a large variation of the total hospital glycopeptides use. therefore we had to take it into account to interpret these consumption and to decide further evaluation. antibiotic management of acute lower respiratory tract infections among dutch elderly patients in primary care j. bont, c. birkhoff, t. verheij, e. hak on behalf of esprit objectives: acute lower respiratory tract infection (lrti) can cause various complications leading to morbidity as well as mortality notably among elderly patients. antibiotic treatment of lrti is common, despite dutch clinical guidelines recommending antibiotics only in case of pneumonia or high risk of serious complications. we assessed the course of illness and outcome of pneumonia, acute bronchitis and exacerbations of copd or asthma among dutch elderly patients in primary care and assessed whether gps were inclined to prescribe antibiotics more readily to patients with potential risk factors for complications in acute bronchitis or exacerbations of copd/ asthma. methods: we retrospectively analysed medical data from , episodes of lrti among patients ‡ years of age presenting in primary care to describe the course of illness and outcome. the relation between prescriptions of antibiotics and patients with risk factors for a complicated course was assessed by means of multivariate logistic regression. risk factors for a complicated course included heart failure, history of myocardial infarction, angina pectoris, diabetes, history of stroke, dementia, malignancy, and history of pneumonia or hospitalisation in preceding year. results: one or more complications arose in % of episodes of lrti. among these, % suffered from pulmonary complications, % had cardiovascular complications (heart failure, myocardial infarction etc.), % had a protracted course and . % had a diabetes event. in . % of the patients complications led to hospital admission and in . % lrti were fatal. antibiotics were more readily prescribed to patients aged ‡ years, when heart failure was present and in patients with diabetes. no significant association was observed in patients with other co-morbid conditions. patients diagnosed with an exacerbation of copd or acute bronchitis with a history of pneumonia or hospitalisation in the preceding year were not more likely to receive antibiotics. conclusions: a considerable part of elderly patients with a lrti suffers from a severely complicated course in primary care. although gps are inclined to prescribe more readily antibiotics in the very old and those with heart failure or diabetes, other potential risk factors are not taken into account. objectives: in this study it was aimed to analyse the infectious diseases (id) trainees' night/weekend shift consultation process in terms of patient and consultant characteristics, types of recommendations, and compliance with recommendations. methods: all consultations performed by id trainees in night shift and at the weekends between june th-august th were analysed in terms of consultation type [treatment continuation (tc), consultation for surgical antibiotic prophylaxis (pa), and consultation with or without a request of a specific antibiotic (others)]. appropriateness of recommendations was assessed the day after the consultation by infectious diseases specialists (ids). adherence to recommendations was assessed days after the consultation by idss. recommendations including antibiotics were considered appropriate, if they were appropriate according to national and international guidelines. recommendations were considered complied, if they were done in up to hours after the consultation (except the consultations in the emergency medicine and the consultations in which antibiotics were started by the counselling idss). results: of consultations was for tc, was for pa and was for others. the clinic where id consultations were requested mostly was general surgery clinic ( / , . %) . in % of all consultations trainees consulted the specialists. overall consultations ( for sp, for a clinical infectious disease diagnosed clinically, for an infectious disease diagnosed microbiologically) were for requesting spesific antibiotic(s). pa were approved in of consultations. antibiotic was not recommended in of other consultations. in six of consultations for pa antibiotic was changed for a clinically diagnosed infectious disease. in one of consultations for tc antibiotic was changed due to lack of response to the given antibiotic, in others tc was approved. inappropriate antibiotic recommendation rate was . % ( / , inappropriate choice, inappropriate dosage, one antibiotic unnecessary). overall compliance to id recommendations was . % ( / ). rate of compliance to antibiotic recommendations was evaluated in consultations and was found . % ( / ) and was higher than compliance to other (microbiology etc.) recommendations ( . %, / , chi square p < . ). conclusion: methodologies to improve the compliance to nontreatment based recommendations and optimizing antibiotic selection is necessary. study of the influence of online practice guidelines on the appropriateness of antibiotic prescribing in a university-affiliated psychiatric hospital j.f. westphal, c. nonnenmacher, d. gregoire, m. hittinger, c. oulerich, f. jehl (brumath, strasbourg, fr) background: problems with the dissemination of guidelines are frequently cited as a major reason for failure to impact practice. reviews of the effectiveness of various methods of guideline dissemination show that the most predictable impact is achieved when the guideline is made accessible through computer-based reminders that are integrated into the clinician's workflow. we report a time-series prospective investigation aimed at comparing the appropriateness of antibiotic (ab) orders for pneumonia at the treatment initiation level after vs. before having embedded our current ab guidelines for pneumonia in the computerized physician drug-order entry system of our teaching psychiatric hospital comprising adult beds. methods: in total, consecutive ab orders for pneumonia were evaluated by the pharmacy department, including orders just before and orders just after implementation of online ab guidelines. appropriateness of ab orders relative to the guidelines was assessed according to criteria: ( ) the choice of ab with respect to the mode of acquisition (community-or hospital-acquired) of pneumonia or the presence of clinical risk factors for involvement of gramnegative bacilli, ( ) the daily dosage, ( ) the planned duration of treatment. data were extracted from the computerized infection declaration system that recorded all ab-requiring infections in our hospital. results: the number of ab orders with at least criterion of inappropriateness tended to decrease, yet not significantly (p = . ), after vs. before implementation of online guidelines: / ( . %) and / ( . %), respectively. the number of criteria of inappropriateness relative to all ab orders for pneumonia was significantly lower in the post-implementation period: . % vs. . % before implementation (difference . %, % ci . - . , p < . ) , with a trend to a decreased number of orders containing more than criterion of inappropriateness. analyzed separately, the numbers of inappropriate orders for the choice of the ab, or the daily dosage, or the planned duration of treatment decreased, yet not significantly (p > . for each criterion), in the post-vs. preimplementation period : vs. , vs. , vs. , respectively. conclusion: in this study, the moderate impact on ab prescribing practices of online guidelines available at the time of drug order shows that additional types of intervention are needed to improve further the quality of ab prescribing. material: the pilot hospitals had a median capacity of (range, to ) beds; their regional distribution was representative of population size; were general hospitals, teaching hospitals and general hospitals with teaching beds. results: ams were internists ( ), microbiologists ( ) and pharmacists ( ). amts included a mean of members who met every weeks on average. all hospitals irrespective of size or affiliation had undertaken a wide range of antibiotic management interventions in , which increased in ; these included (in and , respectively) : major review of formulary (in and hospitals), development of clinical guidelines ( and topics), restricted access to selected antibiotics (carbapenems, glycopeptides, quinolones, new drugs; in and hospitals). in , antibiotic consumption databases were established in hospitals and antibacterial susceptibility databases in hospitals. in , cross-analysis of these databases was performed in hospitals. in , prescribing assistance, antibiotic stop orders, treatment streamlining and iv/po therapy switch were implemented in , , and hospitals, respectively. in , hospitals reported a better use of target antibiotics, hospitals a decrease in consumption of restricted antibiotics, hospitals a decrease of total antibiotic consumption, hospitals a decrease in high consumer departments. conclusion: all hospitals participating in the amt pilot scheme have developed multiple antibiotic policy interventions and established monitoring and guidance of antibiotic prescription. preliminary data from some hospitals indicated success in meeting self-defined targets of appropriate use and reducing the consumption of selected antimicrobial agents. more systematic evaluation using standard quantitative and qualitative indicators is planned. antibiotic prescribing practices at two linked london teaching hospitals p comparison of different antibiotic consumption measurement methods in large multidisciplinary hospital e. pujate, i. apine, u. dumpis (riga, lv) objectives: antibiotic selection pressure is determined by the total amount of antibiotics, number and density of patients treated with antibiotics in the particular geographical area. several antibiotic consumption detection methods should be combined in the hospital setting. our objective was to evaluate efficacy of different approaches in large multidisciplinary hospital. methods: point prevalence studies were repeated annually at [ ] [ ] [ ] in stradins university hospital ( beds) in latvia. all patients receiving antibiotics on the day of the survey were identified and their medical records were reviewed. data on antibiotics, dose and route of administration were collected. in addition, annual data on antibiotics dispensed to the departments were collected from pharmacy. total used grams for each antibiotic were expressed into defined daily doses (ddd-who). bed days (bd) and admission days (ad) were used as denominators. results: table total use of antibiotics in stradins university hospital hospital - the most commonly used antibiotic groups in the pharmacy study were st generation cephalosporins ( . ddd/ bd in , . in , . in ) and penicillin's with extended spectrum ( . , . , . ) followed by fluoroquinolones ( . , . , . ) and metronidazole ( . , . , . ). there was no significant difference between distribution of different antibiotics from prevalence and pharmacy studies if calculated in ddds. in contrast, distribution of antibiotics calculated per patient in the prevalence study was quite different; st generation cephalosporins ( . %, . %, . % in , , respectively) and fluoroquinolones ( . %, . %, . %) with smaller proportion of extended spectrum penicillins ( . %, . %, . %) and metronidazole ( . %, . %, . %). conclusions: there were no differences in the distribution of antibiotics calculated in ddds per bed days and admissions. distribution of antibiotics in annual pharmacy studies and point prevalence studies if calculated in ddds were also similar. in contrast, the prevalence data expressed as a proportion of patients with selected antibiotics showed quite different distribution. studies using only ddds may overestimate use of certain antibiotic groups in our setting where who ddds are significantly different from actual pdds used. a study of prescribing patterns and errors of antibiotics in a saudi hospital m. al-jamal, m. al-barrak (riyadh, sa) background: the term ''prescribing patterns'' has been used extensively in studies to describe different aspects of the prescribing process. antibiotics as well as other drugs are prescribed for the purpose of achieving definite therapeutic outcomes that improve a patient's quality of life while minimizing risk. in the clinical literature, the incidence of antibiotics prescribing errors ranges between . % and . %. objective: in this study we will address antibiotics prescribing patterns and the incidence of prescribing errors in a tertiary hospital and the potential relationship between them. methods: a prospective study of all prescriptions in a -month period (june to august ) in a tertiary hospital has been analysed. the hospital provides both primary and secondary levels of care. criteria used include frequency of selected prescribed drugs, average number of items per prescription, compliance to the hospital formulary, frequency of prescriptions for antibiotics, generic prescribing and diagnosis. the prescribing patterns and the incidence of prescribing errors were performed. results: total number of prescriptions for the -month study was , . emergency room (er) and primary care have the highest number of prescriptions ( . %). the average number of items per prescription is . . the most prescribed drugs by primary care ( . % errors), emergency are antibiotics ( . %), medicine ( . ), ophthalmology ( . ), gynaecology ( . ), and paediatrics ( . ). the prescription errors were . % in primary care and . % in emergency department. discussions and conclusions: over prescriptions were included in this study. the incidence of prescribing errors was . % the average number of items per prescription was . . total prescription errors are also related to frequency of prescribing antibiotics. there was a relation between prescribing of antibiotics and prescribing of trade names (p < . ), and compliance to the hospital formulary (p < . ). several factors influence prescribing patterns and variations in prescribing rates has been identified. these include general physician behavior, differences in morbidity and mortality patterns, social perception toward illness, and physician clinical skills, experience and qualification, as well as physician continuing education and training. special antibiotic prescribing guidelines and restrictions should target primary care and emergency department physicians. effect of a policy for restriction of selected classes of antibiotics on antimicrobial drug cost and resistance of the non-restricted antibiotics. the logistic regression model we performed showed that the new policy had an independent positive effect on the in vitro antimicrobial susceptibility of pseudomonas aeruginosa (p = . ) but not of acinetobacter baumannii and escherichia coli isolates. conclusion: our data suggest that there are considerable limitations of the programs aiming to reduce the consumption of restricted antibiotics through the approval of their use by specialists, at least in a proportion of settings. education programs that aim to involve the medical staff directly responsible for the care of patients in voluntary decisions regarding the appropriate use of antimicrobial agents may have more profound and sustainable success, and thus, deserve to be studied. estimating hospital versus ambulatory care consumption of antibiotics in southwestern germany k. de with, m. steib-bauert, h. schrö der, k. nink, w.v. kern (freiburg, bonn, de) objective: preliminary data from the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project indicated that the proportion of hospital care (hc) antibiotic use on total antibiotic use in several european countries ranges between and %. only few countries, however, have so far been able to report representative countrywide information on both hc and ambulatory care (hc) antibiotic consumption. we estimated ac versus hc consumption of antibiotics for one of the german federal states located in the southwestern part of the country with a . million population. methods: data on hc consumption (atc class j ) were obtained from a convenience sample of acute care general hospitals (n = ), extrapolated to state-wide consumption (using official statistics for the total state-wide general plus special non-psychiatric/non-paediatric/non-radiotherapy hospitals), expressed in defined daily doses per inhabitants and day (did), and finally compared to ambulatory care antibiotic use density in the same region and period of time (years and ) . findings: the estimated state-wide hc consumption of antibiotics was . did ( % confidence interval, . to . did) in both years. state-wide antibiotic consumption in the ac setting during the same time was did (~ % of total consumption). ac consumption of fluoroquinolones ( . - . did, %) and macrolides/clindamycin ( . did, %) made up a major proportion of total use of that drug classes. conclusions: hospital antibiotic use in the southwestern part of germany can be estimated to contribute~ % to overall antibiotic consumption in the general population. antibiotic use profile and temporal trends during a -year period at a greek university hospital: implications for antibiotic policy changes e.i. kritsotakis, p. assithianakis, p. kanellos, n. tzagarakis, m.c. ioannides, a. gikas (heraklion, gr) objectives: to investigate the profile and temporal trends of inpatient antimicrobial use over a -year period at the university hospital of heraklion crete, greece. further, to examine the way in which frequency of data collection and stratification by different patient-care areas provides guidance to antibiotic policy changes. methods: retrospective monitoring of antimicrobial consumption was carried out according to the who anatomic therapeutic chemical classification (atc) and defined daily dose (ddd) measurement methodology. pharmacy records were used to obtain aggregate data of drug deliveries to individual wards. results were expressed as usage density rates in ddds per bed-days (ddd/ bd). linear regression was used in order to assess the statistical significance of a temporal trend in usage densities. results: during - , hospital-wide antimicrobial use (atc group j ) significantly increased by %, from . to . ddd/ bd. the annual average increase rate was . ddd/ bd. stratification by clinical service demonstrated differences in the intensity and profile of class use, as well as varying temporal trends (figures , ) . pooled usage rates in ddd/ bd, overall percentage increases and annual average increase rates were respectively . , . %, . for medical wards; . , . %, . for icu's; and . , . %, . for haemato-oncology wards. surgical wards had a fairly constant usage rate ( . ). a shift towards the newer broad-spectrum antibiotics to the detriment of the older penicillins and cephalosporins was noted in all hospital areas. conclusion: surveillance of aggregate data on the consumption of antimicrobials using the atc/ddd system provided a clear picture of the profile of hospital usage. monthly data over a sufficient surveillance period allowed the assessment of temporal trends. stratification of usage rates by clinical service allowed areas of concern to be specified. thus, surveillance of monthly antimicrobial consumption rates stratified by patientcare area can provide a simple, rapid and efficient tool for triggering antibiotic policy changes in the hospital and targeting more detailed quality-of-use audits. appropriate use of aminoglycosides: the impact of an antibiotic control team c. rioux, p. lesprit, j.r. zahar, a. hulin, a. bernier-combes, c. brun-buisson, e. girou (créteil, paris, fr) objectives: many factors are involved in the appropriate use of aminoglycosides (ag), such as modalities of administration, serum monitoring and duration of treatment. we assessed prospectively the risk factors and the impact of an antibiotic control team on the appropriateness of ag prescriptions. methods: in a setting of a restricted delivery system of ag in our hospital, we first performed an observational audit (oa) to assess the appropriateness of prescriptions including justification of prescribing, adequacy of drug choice, adequacy of administration modalities, modalities of serum monitoring and duration of treatment. after implementation of specific guidelines hospital wide, we then performed an interventional audit (ia) where an antibiotic control team could interfere when ag prescriptions were considered inappropriate. appropriateness of ag prescriptions between the audits was then compared. results: prescriptions were analysed. during the ia, % of prescriptions were modified by the control team. as compared to the oa, prescriptions in the ia were significantly more appropriate with regard to treatment duration ( vs %, p = . ) and serum monitoring ( vs %, p = . ). median treatment duration was shorter in the ia ( d) than in the oa ( d) (p < . ). a logistic regression model showed that risk factors for appropriate treatment duration were (adjusted or, % ci, p value): hospitalization in intensive care unit ( . , . - . , . ) , polymicrobial infection ( . , . - . , . ) and antibiotic control team intervention ( . , . - . , . ) . table: conclusions: despite a restricted delivery system, ag use was frequently associated with excessive treatment duration and errors in monitoring modalities. reinforcing practice guidelines through direct counselling improved appropriateness of prescriptions. hospital antibiotic consumption in southern and eastern mediterranean countries: preliminary results from the armed project p. zarb, m.a. borg, h. goossens, m. ferech for the armed participants introduction: armed is an international research project investigating antimicrobial resistance and consumption in southern and eastern mediterranean countries through the collection of comparable and validated antimicrobial resistance data as well as information about antibiotic consumption patterns and infection control initiatives. objectives: the consumption part of the study aims to collect data on antimicrobial use within participating hospitals in the region, which information is currently unavailable. methods: data collection is planned over a -month period using anatomical therapeutic chemical (atc) classification, a validated methodology adopted by the european surveillance of antimicrobial consumption (esac -www.ua.ac.be/esac). hospitals are participating: cyprus ( hospitals); egypt ( ); jordan ( ); malta ( ); tunisia ( ); turkey ( ). results are expressed in ddd/ bed-days. results: data from , the first year of data collection, indicates that turkish hospitals seem to show the lowest overall consumption [ - ddd/ bed days], whilst the cypriot hospitals show highest values [ - ddd/ bed days]. the most common antibiotics used are the beta-lactams, especially the penicillins although in jordan and turkey cephalosporin consumption is very close to the penicillins. broad-spectrum penicillins [j ca] are the mostly utilised penicillins in cyprus, jordan and tunisia whereas in malta and turkey the combination penicillins [j cr] are the most widely used. there is more variability where cephalosporin consumption is concerned. cyprus utilises mainly first generation, jordan and malta the second generation. in egypt, tunisia and turkey there is significant variability between hospitals; nevertheless use of third generation cephalosporins appears to be significant. conclusion: a significant variability was evident between countries. this is likely to be multifactorial depending on the antibiotics licensed in a country, the national and/or hospital formulary, the type of hospital as well as any antibiotic donations that are relevant in some of the study hospitals. nevertheless, the preliminary results suggest that trends within hospitals of the same country tend to be similar. furthermore, the region as a whole seems to utilise a considerable quantity of broad-spectrum antimicrobials. this can be a factor in the high prevalence of resistance already documented in the study. russian pharmacoepidemiology study of the antibiotic prescription during pregnancy results: mean age of the patients was . ± . (min - , max - ) years, mean gestational ages at admission to hospital was . ± . ( to ) weeks. most often ( . %) infection was community acquired and . % -nosocomial, in % patients there was not to estimate origin of the infection. the most prevalent infections during pregnancy in russia was urinary tract infections - . %, std - . %, candidiasis - . %, rti - . % therefore the most interest was analysing the antibiotic prescription for uti in pregnancy (table) . in % cases were used topical (intravaginal) antimicrobial administration. most often of topically administrated antimicrobials ( . % of all prescriptions) were prescribed combined drugs included antibacterials and amtimycotics. in . % cases antimicrobials were prescribed systemically. mostly prescribed antimicrobials were beta-lactams ( . % for outpatients and . % for inpatients), ampicillin was prescribed more often ( . % for outpatients and . % for inpatients). amoxicillin + clavulanic acid was prescribed in . % of outpatients and . % inpatients pregnant women with uti. cephalosporins were prescribed in . % and . % for outpatient and inpatient uti (mainly iii-and ist generations). mitroimidazoles - . - . % (in general metronidasole), nitro-furanes - . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . %, aminolglycosides - . - . % were prescribed quite often but unjustified. other antimicrobials (fluoroquinolones, doxicicline, antiviral drugs, antifungals) were prescribed relatively rarely. despite the fact that most prescribed drugs were class b by fda, . % all antimicrobials prescribed to pregnancy were class c, . % class d and . % were unclassified. conclusions: most often prescribed antimicrobials for uti (the most prevalent infections during pregnancy in russia) are betalactams and combined topical antibacterials. in . % cases were prescribed antimicrobials of class c, d or unclassified by fda. in % outpatient and . % inpatient were used antibiotics with low in vitro activity for uropathogens. objectives: to study the dynamics of the antibiotic usage in children from orphanages located in different russian cities as the result of interventions with the increased use of the most active antimicrobials and restrictions on use of the least active. methods: the study was performed in orphanages ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) from cities of european russia (moscow, saint-petersburg, smolensk, karachev, bryansk) . use of antimicrobials during the previous months was analysed upon reviews of medical records of children < years in . appropriate recommendations on predominant use of selected beta-lactams (e.g. amoxicillin/clavulanate -amc) with restriction of antimicrobials of other classes (e.g. co-trimoxazole -sxt) were made where applicable on the basis of the expert analysis of antibiotic usage and pneumococcal nasopharyngeal resistance rates. repeated antibiotic usage analysis was performed months later in upon reviews of medical records of children < years. results: total usage of antimicrobials increased . increase of resistance to pen and aminopenicillins. enhanced use of cephalosporins led to increase of resistance to these drugs. in spite of recommendations to restrict usage of am/ox, aminoglycosides and sxt, the analysis showed that these antimicrobials still accounted for . %, . % and . % of all prescriptions, respectively, thus dictating the need for further enforcement measures. antibiotic consumption in ambulatory care in latvia, s. berzina, m. ferech, g. ozolins, h. goosens (riga, lv; antwerp, be) objectives: to collect data on antibiotic consumption in ambulatory care (ac) in latvia according to the esac data collection protocol. esac (european surveillance of antimicrobial consumption, granted by dg sanco of the ec) is an international network of national surveillance systems, aiming to collect reliable and comparable data on antibiotic consumption in europe. methods: the data on ac antibiotic consumption for have been collected using atc/ddd classification (who, version ) and expressed in defined daily doses per inhabitants per day (did). data were obtained from the state medicinal agencybased on the reports of the wholesalers for ac. results: the overall use of antibiotics in ac was . did in , which positions latvia to countries with comparatively low antibiotic consumption in europe. the mostly used class of antibiotics in ac were penicillins with extended spectrum (mainly amoxicillin) - . did ( . %). other frequently used antibiotics were tetracyclines (mainly doxycycline), representing . did ( . %), combinations of penicillins/with betalactamase inhibitors (essentially co-amoxiclav) - . did ( . %), macrolides (mainly clarithromycin) . did ( . %), fluoroquinolones (essentially ciprofloxacin) - . did ( . %) and combinations of sulphonamides and trimethroprim, incl.derivatives - . did ( . %). the most frequently used antibiotics in ac in latvia, in , were amoxicillin ( . did) , doxycycline ( . did) , and co-amoxiclav ( . did) . conclusions: valid data on outpatient antibiotic use in latvia has been for the first time collected and delivered to european surveillance of antimicrobial consumption. this allows international comparison of the pattern of antibiotic consumption in latvia with other european countries. trends in glycopeptide antibiotics consumption over a -year period in a general hospital, athens, greece introduction: glycopeptide use is under restriction in hellenic hospitals since late ' s. the aim of our study was to record trends in their consumption over the last years in our hospital (''a. fleming'' general hospital - beds) and to correlate these data with the numbers of important gram (+) strains isolated in our hospital during the same time period. methods: we measured glycopeptide use for the period - by using data from the pharmacy computer. consumption was expressed as ddds/ patient days (abc calc . ). furthermore we correlated these data with data from the microbiology department concerning numbers of mrsa, mrse and enterococci isolated during the same period. results: glycopeptide consumption was . , . , . , . , . and ddds/ patient days for the years , , , , , ( % increase) . at the same time the cumulative number of mrsa, mrse and enterococci isolated were , , , , , respectively ( % increase) . when both types of data were put on the same graph, glycopeptide consumption correlated well with the number of important gram(+) strains isolated (figure). furthermore vre percentage among enterococci was , , . , . , . , for the study years respectively. it is worth noting that % of our mrsa strains were sensitive to rifampin, % to clindamycin, % to cotrimoxazole, % to clindamycin + rifampin and % to cotrimoxazole + rifampin. linezolid has not been introduced in our hospital yet. conclusions: (a) despite the restriction policy, a tremendous increase in glycopeptide use was recorded in our hospital during the study period and this correlated to the number of the important gram(+) strains isolated; (b) nevertheless, vre is not a significant problem for our hospital yet; and c. the huge increase in glycopeptide use could be avoided at least in part, since other, older and simpler antibiotics could substitute for glycopeptides in many cases. an audit of linezolid use in a university teaching hospital, galway, ireland objective: to audit linezolid use over a six-month period among the in-patient population of a -bed teaching hospital that includes most medical and surgical specialties with the exception of nephrology, rheumatology and orthopaedics. methods: a prospective audit was carried out of the prescribing of linezolid to in-patients from october to april . the ward pharmacist recorded the details of all patients who were prescribed linezolid. a chart review was performed to assess the profile of patients prescribed linezolid, clinical and microbiological indications for treatment, adherence to treatment guidelines and documented adverse events. results: over the -month period courses of linezolid were prescribed. fifty two percent of the patients for whom linezolid was prescribed were from surgical specialties; half of these patients were under the care of one surgeon. pneumonia was the clinical indication for use in % of cases and soft tissue infection in % of cases. the microbiological indication was clear in % of cases where mrsa or vre had been isolated. in % of cases therapy was either ( ) empiric with no significant organisms isolated prior to prescription of linezolid or ( ) therapy was directed against an organism that could have been treated with an alternative agent. duration of treatment exceeded to days in % of courses. an adverse event was recorded in the case of only one course of linezolid. conclusion: in more than a third of cases linezolid use was prescribed without clear justification. avoidable use of linezolid is associated with increased costs and risks of acquired resistance. participants prior to on an oral interview during which the interviewer filled in the answers. results: a total of cm specialists completed the inquiry. this represents approximately % of the national quorum. mean age was years ( - ). of the interviewed cm specialists worked full-time with more full-time employment in hospital labs (hl). next to routine microbiology, other activities performed by cm specialists are mainly the other domains of clinical biology, hospital hygiene and to a lesser extent quality control and lab management. almost two thirds of the interviewed cm specialists believes that their training hasn't prepared them properly for the tasks they are performing now. most desired changes include more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. cm does not exist as a separate speciality in belgium but is included in the 'clinical biology' speciality training. the majority of the respondents thinks that cm should become a sub-speciality (still part of clinical biology) but with a specific minimal training that needs to be defined. the majority of the cm specialists also believes that cm can share lab infrastructure with other disciplines and that the essential aspect of cm lies predominantly in the medical expertise. conclusion: cm training should put more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. the majority of the respondents feels that cm should become a sub-specialty (still part of clinical biology), with a well defined training curriculum. objectives: since more than years, all infectious disease consultations have been recorded in a computerized database (epi info . d, cdc). here we report on consultations of a fellow, conducted during year, compared with consultations conducted by two veteran board-certified infectious disease consultants during the same period. methods: we analysed computerized consultation records, including demographic details of patients; referring department; initiative for, route and purpose of the consultation; and recommendations; and compared between the different consultants. results: a larger percentage of veterans' compared to the fellow's consultations, were requested by attending physicians ( % vs. %, p < . ), while follow-up ( % vs. %, p < . ), laboratory results ( % vs. %, p < . ) or prescription for a restricted antimicrobial agent ( . % vs. . %, p < . ) were more prevalent in fellow consultations. the fellow had a higher rate of additional consultations (in which the patient was seen more than once) ( % vs. %, p < . ), and performed more bedside consultations ( % vs. %, p < . ) or consultation by curb side discussion ( % vs. . %, p < . ), and less consultations by telephone ( . % vs. . %, p < . ). diagnosis and prophylaxis were more often the purposes of the veterans' consultations ( . % vs. . %, p < . , . % vs. . %, p < . , respectively), and they also offered new diagnoses more frequently (p < . ). the veteran consultants more often conducted consultation for communityacquired infections ( % vs. %, p < . ), and more often started antibiotic treatment ( % vs. . %, p < . ). conclusions: significant differences were detected between consultations conducted during the first year of a fellow compared to those of veteran infectious disease consultants. these differences reflect the changing demands and activities in the consultant's work as experience and knowledge accumulate. periodic analysis of computerized data of consultations facilitates supervision as well as direction of consultants' work, addressing issues such as antibiotic use and patterns of microbial resistance. bridging the gap between health care and public health; capacity building in infectious disease control objectives: in recent years, the european union (eu) has developed and supported many activities in the field of communicable diseases. these activities not only concern surveillance networks of specific infectious diseases (e.g. enter-net for salmonella and escherichia coli infections, ewgli for legionella infections, eiss for influenza infections), but also eu training programmes like epiet (european programme for intervention epidemiology training) and a eu communicable disease bulletin. even more recent are the eu's initiative bichat to improve preparedness and response to bioterrorism, and the development of a eu cdc. moreover, a major part of the new programme of community action in the field of public health (ph) ( ) ( ) ( ) ( ) ( ) ( ) concerns id, with not only a commitment to improve information and information exchange, but particularly to strengthen the international rapid response capacity.all this, to illustrate the importance of idc on the eu agenda. methods: the european public health association (eupha) is an umbrella organisation for ph associations in eu. in eupha has created an eupha section idc bringing together eupha members with expertise in this field and representatives of the various above mentioned eu initiatives in order to: promote and strengthen research in the field of idc; provide a platform for the exchange of information, experience and research in idc; bring together researchers, ph practitioners and policymakers active in idc; encourage joint activities in idc; and improve idc training. results: by now the section has members from different countries. as of the section is represented in the ecdc advisory forum. different section activities: organising workshops, a breakfast meeting and a pre-conference meeting on timely idc topics during eupha conference. objectives: to establish a cross-border dutch-german network (www.mrsa-net.org) providing a user-friendly knowledge centre for hospitals, public health authorities, gps, nursing homes and laboratories. primary purpose is to aid in the reduction of mrsa-rates and limit the cross-border transmission of mrsa. guidelines and their implementation play a significant role in reaching these aims. cross-border (ca-) mrsa guidelines will be redesigned according to international standards and socio-cultural differences between the nations. methods: based on quality standards for safety and healthcare documentation used in high risk chemical organizations, a framework for a systematic content analysis of national mrsaguidelines was developed. national guidelines were analysed on the basis of this framework. results: a content analysis of the current national mrsaguidelines showed five dominating mrsa-perspectives: rule-, expert-, risk-, demand-and community-driven. german guidelines are mainly dominated by the rule-and expertdriven perspectives (guidelines are literally derived from law and follow the infection transmission route), in contrast to the dutch which focus on the demand of the user and the community (addressed to public health and acceptability of guidelines by users). conclusion: the analysis showed that the fact that there are different guideline-perspectives results in an enormous, confusing set of guidelines. the management and use of guidelines becomes uncontrollable and leads to an illusory organisation where healthcare workers don't act in accordance with the guidelines and start applying their own insights. this might lead to cost-increasing and contrasting situations. to implement guidelines successfully in a cross-border situation, a cultural and technical synchronisation alongside an integrated approach of the different perspectives of guidelines is necessary, inline with the current disease management models. further research about the redesign and the evaluation of those guidelines in practice will help achieving this. r. tsiklauri (tbilisi, ge) background: animal bites are a common but under recognized public health problem. it has been estimated that there are - bites each year in georgia, and based on an average visit and post-exposure treatments cost at list $ per year. despite the frequency and expense of these injuries, there is little information about the incidence of animal bites because of a lack systematic reporting and a lack of measurement of the quality and completeness of reported data. objectives: to investigate animal bites and rabies reported cases, revealed unreported cases, analyse and based on study results find more effective epidemiological measures of animal bites and deaths (due to rabies) prevention in georgia. methods: the capture-recapture method was used, along with log-linear modelling. for sources were used to identify victims: policlinic/ambulatory reports, hospital reports, animal control reports and victim reports. results: in - years dog and other animal bites were reported. the capture-recapture method estimated that there were unreported bites. during these period deaths due to rabies was registered in georgia and ( %) cases among them have been registered during the last years. the reasons of fatal cases were untreated ( %), uncompleted treated ( %) and late began post-exposure treated ( %) cases of bites (mostly dog bites). about % of bitted persons did not know about rabies and it's prevention measures. about % had incorrect information about prevention and only % of them knew epidemiological and clinical aspects of disease. about % of physicians who were responsible on quality post-exposure treatment had not an adequate knowledge. conclusion: dog and other animal bites are common but preventable injuries. to improve surveillance and prevention of rabies in georgia, the focus should be on educating the general public about the serious consequences of animal bite injuries and developing the animal's vaccination strategy. pharmacoeconomics and electronic resources p the expected economic burden of methicillinresistant staphylococcus aureus in complicated skin and skin structure infections: a modelling approach a. kuznik, r. mallick, d. weber (collegeville, chapel hill, us) objective: to model the expected rate of clinical failure of initial empiric therapy and economic burden likely to be associated with the increasing prevalence of methicillinresistant staphylococcus aureus (mrsa) in patients hospitalised with complicated skin and skin structure infections (csssi) in the united states. methods: using published data on ( ) the prevalence of mrsa and other bacterial pathogens causing csssi in the us, ( ) the in-vitro susceptibility rates of commonly used regimens in csssi in the us in relation to the most pervasive pathogens identified above, and ( ) estimated costs of failure of initial, empiric treatment from a recent study of a large us multi-hospital database, we developed a model to predict the expected clinical and economic impact of increasing prevalence of mrsa. specifically, clinical failure of of the more commonly used initial regimens in csssi was modeled in terms of their in-vitro susceptibility rates with respect to mrsa, weighted by mrsa prevalence. varying the rate of mrsa further yielded projected clinical failure rates and costs attributable to increasing levels of methicillin resistance over time. results: given current % prevalence of s. aureus pathogens in csssi, half of them methicillin-resistant (base case mrsa = %), the model projected an overall clinical failure rate of . % for of the more commonly used initial regimens, with an expected overall treatment cost (in us dollars) of $ , per patient (range, $ , - , ) . if none of the s. aureus pathogens were resistant (mrsa = %), clinical failure rate was projected to be . % and treatment cost to be $ , per patient. the differences in the two scenarios translated to an expected clinical failure rate of . %, an incremental cost of $ per patient, and for the , patients hospitalised for csssi annually in the us, an expected health care system burden of $ million attributable to mrsa. under a "worst-case" scenario in which mrsa was the only causative pathogen (mrsa = %) in csssi, clinical failure rate was projected to be . %, and treatment cost per patient was expected to be $ , . conclusions: going beyond existing estimates, our model generated a substantial expected clinical failure rate and economic impact attributable to current mrsa levels, as well as simulations of the expected impact of increasing mrsa prevalence over time, varying levels of mrsa across regions and choice of initial empiric regimens. treatment of complicated skin and skin structure infections in the us: expected cost differences between tigecycline and vancomycin/aztreonam r. mallick, a. kuznik, d. weber (collegeville, chapel hill, us) objective: to compare tigecycline and vancomycin/aztreonam in terms of treatment-related costs for patients hospitalised in the united states with complicated skin and skin structure infections (csssi). methods: we conducted a retrospective analysis of pooled data from us centres in two randomized, double-blind clinical studies comparing tigecycline and vancomycin/aztreonam in the treatment of csssi. using regression analysis, we estimated the effect of tigecycline treatment on hospital length of stay (los), controlling for other significant predictors. using published estimates of daily hospitalisation cost of csssi in the us from a multi-hospital audit, we then translated the estimated impact on los into economic terms. this analysis was repeated for the subgroup of patients in which the primary pathogen was methicillin-resistant staphylococcus aureus (mrsa). clinical efficacy (tigecycline %, vancomycin/aztreonam . %; p = . ) was similar across treatments and was not included as a model parameter. results: our retrospective analysis of the pooled clinical data from us centres found that tigecycline was associated with a shorter los [) . days (p = . )] compared with the combination of vancomycin/aztreonam in the treatment of patients with csssi. at a mean daily hospitalisation cost (in us $) of $ , excluding antibiotic costs, this translated into expected medical cost savings of $ , per patient for tigecycline compared with vancomycin/aztreonam. in the mrsa subgroup, comprising % of the clinical study sample, tigecycline was associated with a greater reduction in los [) . days (p = . )] compared with vancomycin/ aztreonam, translating to expected medical cost savings of $ , per patient treated with tigecycline. these expected medical cost savings more than offset the higher average daily drug acquisition costs of tigecycline ($ /day) relative to the vancomycin/aztreonam combination ($ /day). conclusion: in a retrospective analysis of pooled clinical data of patients with csssi treated at us centres, tigecycline was associated with a significantly reduced length of hospital stay relative to vancomycin/aztreonam; this translated into substantial cost savings, especially in the subset of csssi patients with mrsa. the economic impact of linezolid in the treatment of skin and soft tissue mrsa infections in italy m. eandi, p. dale, s. sorensen, t. baker, m. procaccini, s. duttagupta (turin, it; london, uk; bethesda, us; rome, it; new york, us) objective: linezolid has been shown to be highly effective against infections caused by methicillin-resistant staphylococcus aureus (mrsa) in patients with complicated skin and soft tissue infections (cssti). the objective of this study was to evaluate the clinical and economic consequences of using linezolid for the empiric treatment of cssti from the italian hospital perspective. methods: a decision-analytic model was developed to calculate the clinical and cost outcomes of empiric treatment of hospitalized patients with cssti in italy prescribed linezolid, vancomycin or teicoplanin. efficacy data were derived from clinical trials. costs from published sources were applied to tests, adverse events, and days of intravenous and oral (linezolid only) treatment and hospitalization by ward type (general, intensive-care). resource use and utilization patterns were obtained from a combination of clinical trial data and expert opinion. outcomes included total costs per patient, cost per cure and cost per death avoided. uncertainty surrounding the ce ratio was tested using one-way sensitivity analysis. results: starting empiric treatment with linezolid resulted in . % of patients cured from mrsa compared to . % with vancomycin. the average cost per patient treated with linezolid was € , versus € , for patients treated with vancomycin. this resulted in a cost per cure of € , . in a separate analysis more patients were cured using linezolid ( . %) compared to teicoplanin ( . %). the average total cost per episode was € , for linezolid treated patients versus € , for teicoplanin treated patients, resulting in a cost per cure of € , . the most sensitive parameters included hospital los and mrsa resistance rate. conclusions: in the treatment of cssti due to suspected mrsa in italy, the empiric use of linezolid is cost-effective when compared to vancomycin and teicoplanin p outpatient and home parenteral antimicrobial therapy for the treatment of cellulitis: evaluation of efficacy and cost h. ziglam, r. tilley, c. wootton, j. morrison, d. nathwani (dundee, uk) objective: outpatient and home parenteral antibiotic therapy (ohpat) programmes are effective, well tolerated and economically advantageous in carefully selected patient populations. skin and soft tissue infections represent a high burden disease which in amenable to treatment by ophat programmes. we retrospectively analysed our outcomes registry to evaluate the clinical and health economic impact of treating cellulitis in this setting. methods: we have reviewed patients with cellulitis and erysipelas who were treated with ohpat. each patient treatment has a full integrated care pathway (icp). the icp documents the microbiological outcome, drug and vascular access complication rates, impact on drug costs and in-patient bed days on the number of patients treated from april to march are presented here. we also reviewed using the smr o inpatient discharge diagnosis data from the information statistics division scotland (isd) and the dundee infectious diseases units (didu) outcomes registry database. the key diagnosis (icd codes) groups considered were cellulitis (lo , , , , , ) and erysipelas (a x) over eight consecutive years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: the patients received intravenous antibiotic therapy for a mean duration of . days. the two primary agents administered were once-daily ceftriaxone in %of patients and teicoplanin in . % of patients. of the patients, ( . %) were cured or improved; worsened and required surgery. tinea pedis was found in % of patients treated for cellulitis. economic benefits were realized despite use of more expensive agents. data from the dundee outcomes registry revealed a mean reduction in length of hospitalization from . days ( / ) to . in - -a reduction of % compared to scottish data from isd which did not show any changes in length of hospitalization between year / ( . days) and year - ( . days) . conclusions: we have found that ohpat is clinically effective and can be administered safely and successfully in an outpatient setting. the majority of complications were minor, and % of patients were cured. tinea pedis and were found to be significant risk factors for acute cellulitis and indicate that improved awareness and management of toe web intertrigo might reduce the incidence of cellulitis. this analysis also supports the premise that an adult ohpat programme can substantially reduce healthcare resource use in the european healthcare setting. cost-effectiveness analysis of intravenous moxifloxacin compared to levofloxacin in hospitalised elderly patients with communityacquired pneumonia objective: to evaluate the cost-effectiveness of moxifloxacin compared to levofloxacin in hospitalised patients aged ‡ with community acquired pneumonia (cap). methods: a randomised double-blind parallel group study was conducted in us hospitals. patients had radiological evidence of bacterial pneumonia confirmed by at least other signs, were aged ‡ years and were managed as inpatients on initiation of treatment. patients initially received moxifloxacin mg iv o.d. or levofloxacin mg iv o.d., and once stabilised were switched to oral therapy with the same agent. the effectiveness endpoint for the economic analysis was the percentage of patients successfully treated, defined as patients with marked improvement, resolution or clinical cure at test of cure visit after - days of therapy who did not experience a serious cardiac adverse event. total costs were estimated from the perspective of the treating hospital and included antibiotic drugs, hospital stay, hospital re-admission within days and cost of managing treatment failures. results: patients were included in this analysis, randomised to moxifloxacin and to levofloxacin. % ( % ci: - %) of moxifloxacin and % ( - %) of levofloxacin treated patients were successfully treated (resolution, clinical cure at toc and no serious drug related aes). in the moxifloxacin group patients reported a mean of . days of iv antibiotic treatment and . days inpatient stay with . days iv antibiotic treatment and . days inpatient stay in the levofloxacin group. mean per patient drug cost was $ in the moxifloxacin group, $ in the levofloxacin group. mean total cost was $ , in the moxifloxacin group and $ , in the levofloxacin group. findings were consistent across a range of patient subgroups. costs were sensitive to length of hospital stay. conclusions: patients in the moxifloxacin group had higher rates of successful treatment at slightly lower average costs than the levofloxacin group. this confirms the results of the target study where moxifloxacin showed superior clinical efficacy in comparison to co-amoxiclav with or without clarithromycin in hospitalised cap patients. antibiotic costs were slightly higher in the moxifloxacin group than the levofloxacin group but total costs were slightly lower, due to reduced hospital stay. economic impact of invasive fungal infections in icu patients in a tertiary care hospital in switzerland a. imhof, w. zingg, r. laffer, c. ruef (zurich, ch) objectives: invasive fungal infections (ifi) cause significant morbidity and mortality. the management of invasive fungal infections is currently undergoing important changes due to the availability of new therapeutic agents with improved safety profiles but the acquisition costs of these new agents are high. we evaluated the average overall cost of management (microbiological diagnosis and treatment) of invasive fungal infection in critically ill patients at a large university hospital. methods: a retrospective ( ) ( ) , pairwise-matched cohort study was performed on surgical icus and one medical icu at our university hospital. icu patients with documented ifi (n = ) were matched with control subjects (n = ) on the basis of disease severity, sex and age (± years). clinical outcome was principally evaluated by in-hospital mortality. the economic impact of microbiological studies and antibiotic treatment was assessed. calculations were based on the period between admission and diagnosis of ifi in cases and the duration of hospital stay in controls, respectively. results: the median length of hospital and icu stay differed significantly between cases and controls ( vs days, vs days, p < . , respectively). ifi occurred after a median hospital stay of (range - ) days. the mortality rate for patients with ifi and matched control subjects were . % and . %, respectively (p = . ). there was no significant difference between cases and controls for charlson index, mccabe and saps ii score. median number of antibiotic treatment courses was for cases and for controls (p = . ), with a median duration of therapy of days vs day (p < . ), respectively. microbiological studies (mis) were conducted times/ patient-days (pd) in cases and times/ pd in the control group (p = . ). the most frequent samples were bloodcultures in both groups. swabs were ordered significantly more frequently in cases (median (range: - ) vs. ( - ); p = . ). the cost for mis was ' euro/ pd in cases vs. euro/ pd in controls, and the costs for antifungal therapy ' euro/ pd vs. euro/ pd, respectively. conclusions: ifi is associated with excess length of icu and hospital stay, increased use of antibiotics and microbiological diagnostics. the microbiological studies have a significant economic impact on the treatment of ifi. cost-effectiveness of voriconazole to amphotericin b deoxycholate in early and late treatment of invasive aspergillosis r. greene, j. mauskopf, c. roberts, t. zyczynski, h. schlamm (boston, research triangle park, new york, us) objective: we estimate the cost-effectiveness of alternative initial drug treatments of invasive pulmonary aspergillosis (ipa) in suspected earlier and later lung involvement, based on the presence or absence of the halo sign on thoracic computed tomography (ct). methods: we constructed a decision analysis model comparing -week treatment outcomes for a subset of patients enrolled in a clinical trial of initial treatment of ipa with amphotericin b dexoycholate (ambd) vs voriconazole (vor). patients included those with suspected lung involvement who underwent a baseline thoracic ct. the subset was subdivided into two groups based on the presence or absence of a characteristic ct halo sign, a perimeter of ground glass ct opacity surrounding a solid lung nodule ‡ cm diameter, known as an early indicator of ipa. healthcare resource use and survival data were obtained directly from the clinical trial. us unit costs for drugs and health care services were applied from standard data sources. cost and survival at -weeks were estimated for those with and without a halo sign at baseline. incremental cost-effectiveness ratios comparing vor to ambd were calculated for both patient subgroups. sensitivity of results to uncertainty in health care use and cost estimates was tested. results: patients in the halo subgroup had better survival than those in the no-halo subgroup ( . % vs . %), with lower total treatment cost ($ , vs $ , ) . survival was higher for vor than for ambd in both patient subgroups (halo: . % vs . %; no-halo: . % vs . %). in the halo subgroup, total costs were lower for those treated with vor than for those treated with ambd ($ , vs $ , ) . in the no-halo subgroup, total cost per patient was slightly higher for those treated with vor ($ , vs $ , ) . accounting for the difference in survival, the incremental cost-effectiveness ratio for vor compared to ambd was $ , per additional -week survivor in this subgroup. conclusions: earlier identification and treatment of ipa appears to result in better survival and potentially lower costs than later treatment. initial treatment of ipa with vor improves survival in patients with early or late disease compared with ambd, is cost saving in the halo sub-group, and is cost-effective in the no-halo subgroup, within the constraints of our analysis. objective: to describe and compare the nursing labor time required for preparation and administration of liposomal amphotericin b (l-amb), amphotericin b deoxycholate (ambd), and voriconazole (vor). methods: activities associated with nurse preparation and administration of the three study drugs were timed by trained observers at five hospitals (one in italy, three in france, and one in the united kingdom). target tasks were classified as those likely to be affected by the difference between the drugs and excluded those tasks likely to differ because of site-specific factors (e.g., travel time to a patient room in different hospitals). target tasks included: obtain supplies and medications; prepare medications; educate patient; administer medications; monitor for adverse events; and prepare follow-up medications. the mean times for administration of a single day of study drug were summarised and compared, accounting for a single daily dose of l-amb and ambd and daily doses of vor iv or oral. results: sixty-nine patients were observed receiving doses of study medications at the five hospitals. time of administration in minutes per day was , , , and for l-amb, ambd, vor iv, and vor oral, respectively. administration time was significantly lower for vor iv compared with l-amb (p < . ) and for vor oral compared to all iv regimens (p < . ). the task of preparation of medications required the most time for iv formulations, and was longer in the l-amb group than the others (l-amb: mins vs ambd: mins; vor iv: mins). ambd required more time for patient monitoring and administration of followup drugs than other formulations (ambd: mins vs l-amb: mins; vor iv: mins). conclusion: vor iv required significantly less time to prepare and administer on a daily basis compared to l-amb. measurements of iv antifungal versus oral vor administration suggest the opportunity to save - minutes per day by switching to oral therapy when possible. need of cost-effectiveness investigation focused on diagnosis, management and prevention of osteopenia and osteoporosis in the setting of hiv disease treated with haart: when to act, how to act, which patients are the first target of intervention r. manfredi, l. calza, f. chiodo (bologna, it) background: osteopenia/osteoporosis are emerging untoward effects of hiv infection/haart. the pathogenesis is multifactorial, involving all classes of anti-hiv drugs, although protease inhibitor use, overall haart duration, and the male sex, seem related to a greater risk.epidemiologicalclinical data. in an ongoing study at our centre where > hiv-infected patients (p) are followed, bone mineral density was assessed in lumbar spine/femural head by a dual energy xray absorptiometry (dexa) exam to estimate the prevalence of osteopenia/osteoporosis. in a screening of~ p, the frequency of osteopenia and osteoporosis (based on lumbar t-score) was % and~ %, respectively. an increased risk was found in p treated with protease inhibitors versus those receiving nonnucleoside reverse transcriptase inhibitors or triple nucleoside/ nucleotide combinations. discussion and future insights: prospective studies of extensive p samples are needed, to elucidate the epidemiology, pathogenesis, clinical issues and evolution of hiv-associated bone metabolism anomalies. when planning strategies for their early diagnosis, prevention and management also cost-effectiveness issues should be considered, since no pharmacoeconomic data still exist in this setting. although severe consequences (e.g. pathological fractures, prosthetic implants) are expected to be infrequent their consequences in terms of length and intensity of hospitalization, related costs, and especially severe consequences on the p's quality of life, play a notable role. anyway, the most reliable diagnostic procedure (dexa) has affordable costs (around eur . for a total-body scan which also offers a body composition assessment), as well as the first-line drugs for osteopenia, e.g. supplementation with calcium (eur - . /month), and vitamin d (eur /month). these costs cannot be compared with the costs of a standard care of an asymptomatic haart-treated p (eur to /month) and the immunological, virologic, laboratory and clinical controls made at least quarterly. like postmenopausal osteopenia/osteoporosis (burdened by a greater risk of bone mass anomalies) also hiv disease should be investigated from multiple cost-effectiveness points of view to establish which p are the preferred candidates for a dexa screening when this examination is more useful during hiv disease course and therapy, when the exam should be repeated and when and how to intervene pharmacologically to prevent serious and potentially invalidating complications. a comparative study on the cost of new drugs in different therapeutic categories m. falagas, k. fragoulis, g. zouglakis, i. karydis (athens, gr) objectives: drug treatment is becoming more expensive due to the increased cost for the introduction of new drugs and there seems to be an uneven distribution of medication cost for different therapeutic categories. we hypothesized that the cost of new antimicrobial agents may differ from that of other therapeutic categories and this may play a role in the stagnation of development of new antibiotics. methods: we performed a pharmaco-economical comparative analysis of the drug cost of treatment for new agents introduced in the united states drug market in various therapeutic categories. we calculated the drug cost [in us dollars (usd)] of a -day treatment of all new drugs approved by the fda during the period between january and july , according to the red book pharmacy's fundamental reference. results: new anti-neoplastic agents were found to be the most expensive drugs in comparison to all other therapeutic categories with a median -day drug-treatment cost of usd compared to the median -day drug-treatment costs of all other categories ranging from to usd (table) . on the other hand, new antimicrobial drugs were found to be much less expensive with a median -day drug-treatment cost of and usd for all anti-microbial agents and for anti-microbial agents excluding anti-hiv medications, respectively. conclusion: the drug-treatment cost of new medications varies considerably by different therapeutic categories. this fact may influence industry decisions regarding the development of new drugs and may play a role in the shortage of new anti-microbial agents in the fight against the serious problem of anti-microbial resistance. usage and expenditure of f-quinolones in a tertiary hospital in t.a. peppas, k. malengou, n. zachos, d. voutsinas, o. kosmopoulou, n. galanakis (piraeus, athens, gr) objective: our aim was to assess -f-quinolone ( fq) usage, distribution and expenditure over years. objective: to analyse antiobiotic utilization in croatia using anatomical-therapeutic-chemical (atc) drug classification system and number of defined daily doses (ddd). methods: data on the number of packages and purchase price were collected for each individual drug. these data were used to calculate the number of defined daily doses (ddd) and ddd per inhabitants per day (ddd/tid). data obtained from % of pharmacies and % of hospitals were extrapolated to the total number of pharmacies and hospitals in croatia. drug utilization % (du %) segment was used as a prescribing quality indicator. results: in , the overall utilization of antibiotics in croatia amounted to . ddd/tid. according to drug groups, penicillins (j c) showed highest utilization ( . ddd/tid), predominated by the subgroup of penicillin combinations (including beta-lactamase inhibitors, j cr) with . ddd/ tid, within which the combination of amoxicillin + clavulanic acid accounted for . % with . ddd/tid. broad-spectrum penicillins (j ca) accounted for . %( . ddd/tid) of total penicillin utilization, with a . % predominance of amoxicillin ( . ddd/tid). cephalosporins (j d) ranked second with . ddd/tid, followed by macrolides and lincosamides (j f) with . ddd/tid, with an . %predominance of macrolides (j fa) with . ddd/tid. among the latter, azithromycin showed highest utilization with . ddd/tid, accounting for . % of total macrolide utilization. tetracyclines (j a) ranked fourth with . ddd/tid, accounting for . % of overall antibiotic utilization, followed by quinolones with . ddd/tid, other antimicrobials with . ddd/tid, and aminoglycosides with . ddd/tid. sulfonamides (j e) accounted for a negligible proportion of overall utilization. du % segment included of antibiotics registered in croatia, with amoxicillin + clavulanic acid as the leading one, followed by cephalexin with . , cefuroxime with . , azithromycin and norfloxacin with . each, nitrofurantoin with . and clarithromycin with . ddd/tid. hospital utilization accounted for . % of overall antibiotic utilization expressed in ddd/tid and . % of the respective financial cost, predominated by aminoglycosides (j g) with % and . %, and lowest proportion of tetracyclines (j a) with . %, and . %, respectively. conclusion: the utilization of antibiotics in croatia is among the highest in europe, mostly due to overuse of amoxicillin + clavulanic acid, which has no rational ground in professional guidelines. objective: the objective of the research was to analyse antibiotics prescripsion behavior by family doctors and specialists treatments prior to and after the introduction of the health care reform in poland. materials and methods: prescriptions from the first six months of and were compared. the data was collected from two randomly chosen pharmacies in the city of zabrze that supply the citizens of the silesian agglomeration from various social backgrounds. taking into account the value of a single antibiotics package and the price a patient has to pay for it an average price of medications prescribed by family and specialist doctors was calculated. results: a total of prescriptions were analysed out of which dated from and from .in the first half-year of the percentage of prescriptions for antibiotics reached . % on average, and in the year -the average was . %. in the first half-year of family doctors mostly prescribed: penicyllins ( %), makrolids ( %), cephalosporins ( %), tetracyclins ( %), chinolons ( %). in the same period spcialist doctors prescribed: penicyllins ( %), cephalosporins ( %), makrolids ( %), tetracyclins ( %), lincozamids ( %), chinolons ( %). in the first half-year of family doctors most often prescribed penicyllins -( . %), makrolids -( . %), cephalosporins -( . %), tetracyclins -( . %) and lincozamidsbased ( . %) treatments specialist doctors, on the other hand, prescribed penicllins ( . %), makrolids ( . %), cephalosporins ( . %), tetracyclins ( . %), lincozamids ( . %) and chinolons ( %). the average prices of the prescribed medications in the years and were, respectively: for family doctors-eu . and . , for specialist doctors eu . and . . conclusions: there has been a considerable increase in the percentage of prescriptions for antibiotics from . % (in ) to . % (in ). the tendency towards prescribing antibiotics in the specific groups of doctors has not changed significantly. in both years prescriptions for antibiotics were in line with the recommendations. also, prices of medications prescribed by family doctors have risen. internet guide on antimicrobial resistance a. sosa, f. traub, s. valovic, p. chea (boston, us) objectives: ( ) to organize the plethora of information available, providing clinicians the tools to easily access available online resources that include academic institutions, professional societies as well as sites maintained by private individuals; ( ) to inform clinicians of new advances in the epidemiology, diagnosis, treatment, and prevention of most common infections; ( ) to inform on subjects such as clinical trials in antimicrobial resistance, information about specific pathogens and their infections, genomic resources, culture collections, electronic images of pathogens and antimicrobial agents, antimicrobial resistance lecture and teaching materials, environmental health and safety information, and a listing of websites of infectious disease and clinical microbiology professional societies. methods: we defined four inclusion criteria after extensive consulting with apua staff and scientific advisory members: ( ) recognized/reputable source; ( ) high quality of information presented; ( ) potential usefulness to medical professionals and the general public; and ( ) ease of navigation. ideal parameters were determined for the guide's scope and the appropriate sources identified online were subsequently reviewed. a set of broad categories was established to organize the topics and the online resources. sites reviewed included those maintained by the federal government, academic institutions, nonprofit organizations, and commercial entities. some personal websites were included because of their quality and their association with academic institutions. this review is intended as an introduction to amr websites. results: with use of popular search engines, such as google, yahoo!, and altavista, we initially identified a great number of websites. using broad search terms, such as "antibiotic resistance," we identified , , web addresses. the term "antimicrobial resistance" generated , hits, and the term "drug resistance" generated , , hits. conclusions: websites found were classified following a systematic topic structure. each website listed describes: ( ) full citation of the resource: author/editor and title of website; ( ) date of publication or last revision; ( ) methods and results: the portal is free to use but requires registration and has more than registered users as of november . of these, % are in-training positions, % hold a faculty position in infectious diseases (id), microbiology or hemato-oncology, % are specialists in a non-university, but a teaching setting. the remaining are a mixed group of primary physicians, other speciality doctors and pharmaceutical company workers. running costs of the portal are partially covered by educational grants from pharmaceutical sponsors who have no role in organization of the site, but their names are acknowledged. articles chosen by the two faculty members, one id physician and one haematologist, are sent to registered users by daily e-mail postings. these are selected from toc alerts of various core clinical journals and well known educational web sites (e.g. cdc, who, medscape). a short turkish summary is provided and the reader is referred to the abstract and if available, to the free full text of original article with a link. other materials included guidelines, free slide sets, study protocols and updates from the group, cme activities and meeting announcements. registrants may also use the site for expert opinion. during the trial period, the site has been visited times with hits. approximately . gb material was downloaded. the frequency of readings are related with the time (highest between . - . am during weekdays and lowest during weekends), the type of documents (i.e. educational materials and guidelines), popularity of the news (e.g. peaked during an epidemic of avian influenza when related news and articles announced). the pages are most frequently visited by id specialists followed by clinical microbiology, haematology and pharmaceutical company workers ( %, %, % and % respectively). conclusion: timely published medical data have high attraction rates among physicians. our results also indicated that, a web page gets ''old'' after about a month of publishing, emphasizing the importance of well-timed announcements of the portal material. neli and nric survey: information needs of infectious disease professionals p. kostkova, s. d'souza, g. madle, j. mani-saada, s. wiseman, a. roy, j. weinberg (london, uk) healthcare professionals are increasingly facing the problem of information overflow. it is getting impossible to keep up-to-date with the latest research findings, care guidelines and pathways, government strategies and national and local policies. internet enables an instant information dissemination enabling access to the latest results at any time as well as informal knowledge exchange by using chat rooms and discussion forums. however, it is getting increasingly difficult for busy professionals to find reliable quality-assured information on the internet when they need it. national internet libraries in the uk are addressing this problem: the umbrella portal national electronic library of infection neli (http://www.neli.org.uk) providing a single access portal to quality-assured information on treatment, diagnosis, prevention and management of infection diseases, and the national resource for infection control nric (http:// www.nric.org.uk) -a single-stop shop for policies, guidelines and research around infection control, hosted by neli. to better meet the information needs of these internet portals, accessed by unique users per month, we are conducting an information needs study to explore clinical questions, user needs and disease priorities of users seeking answers on neli and nric. a pilot qualitative online questionnaire-based study revealed that our users come from the variety of professionals: clinical scientist, consultant, registrar, psychotherapist, lecturer, gp, medical librarian, information scientist, health protection. these have questions mainly around hiv, tinea, molluscum contagio, meningitis, cold, mrsa, lyme, toxoplasma, chicken pox, influenza, diarrhoea and vomiting, rash, staph. aureus, traveller infections antibiotics resistance, malaria, mmr, meningitis, viral myocarditis, anthrax, smallpox, and tb. this is in line with our quantitative weblog-based evaluation of the most commonly access topics on neli by nhs-based users: antimicrobial resistance and hae ( . %), tb ( . %), meningitis ( . %), hiv ( . %), chlamydia ( . %), e. coli ( . %), staph. aureus ( . %), adenovirus ( . %), blood borne infections ( . %). the results of the ongoing analysis of google search keywords that brought users to neli and nric will be discussed. further results identifying the needs specific to the infection disease professions will be discussed in relation to differences in the national variations in information needs and priorities. training in infection s. d'souza, p. kostkova, f. cooke, a. holmes (london, uk) specialists today require prompt access to quality information in order to work effectively. the diversity of specialist interests in the field of infection has led to the formation of a large number of professional and scientific societies. these play an increasingly important role in ensuring that the trainee is effectively supported, not only during the period of training, but also in longer-term personal development. details of relevant societies, conferences, grants etc are, on most society websites, confined to those for either that society or others in that specialty only, and knowledge of the numerous places in which to look for this information is necessary to find out the latest information. training in infection (tii -www.trainingininfection.org.uk) is an online resource, primarily aimed at infection specialist trainees but useful throughout the career path, which brings together this information into one central access point, so that users from all infection specialties can find the appropriate information for their specialty quickly and easily. it identifies and links to the key relevant resources covering a broad range of infection related disciplines in a dynamic database structure. information on societies, conferences, grants, journals, textbooks and more are available on the site, and have been put together to create a one-stop infection training portal. online discussion forums to be implemented will allow trainees' to share ideas and make the most of their combined expertise, and users will be able to receive alerts on new information in their specialty as well as be reminded of conference deadlines, journal submission deadlines etc. the ability to discuss regional issues online within specialties also aims to promote greater local and international collaboration. training in infection is endorsed by the national electronic library of infection (neli -www.neli.org.uk), an established digital library bringing together the best available online evidence-based resources on the investigation, treatment, prevention and control of infectious diseases. research designs and statistical methods in medical abstracts m. kompoti, m. matsagoura, a. koutsovasilis, a. koutsovasili, s. drimis (athens, gr) statistical methods used in biomedical research articles are being increasingly scrutinized in medical journals. however, no such strict policy is generally applied in abstracts presented in medical congresses. objective: this study aimed at assessing the frequency of research designs and statistical methods reported in abstracts presented in two successive years of the european congress of clinical microbiology and infectious diseases (eccmid). material and methods: we reviewed all abstracts included in the abstract book of the th eccmid (prague ) (pg) and the th eccmid (copenhagen ) (cp). all abstracts of original research studies but no abstracts of lectures were included in our study. two independent investigators read all abstracts and extracted information concerning origin, type (clinical, laboratory, animal model), research design, sample size and statistical methods used in the study. data analysis was performed with logistic regression and pearson's chi-square test for categorical variables and student's t-test for continuous variables. statistical significance level was set at p < . . results: a total of abstracts were included in the analysis according to eligibility criteria ( from pg and from cp). laboratory studies prevailed ( %) followed by clinical studies ( %) and experimental studies with animal models ( %). the majority ( . %) of the studies were observational (retrospective, prospective, cross-sectional) of which . % concerned diagnostic accuracy testing of laboratory methods and . % were pharmacological studies, . % were randomized controlled trials. statistical evaluation was clearly described in . % of abstracts ( . % in pg and . % in cp, p < . ), while the rest of abstracts included only descriptive statistics or no statistics at all. the proportion of statistical methods reporting varied according to the type of the study (animal model studies . %, clinical studies . % and laboratory studies . %, p < . ). multicentre research studies reported statistics more frequently than single-center studies ( . % vs. . %, respectively, p = . ). conclusions: statistical analysis is an inseparable part of original research. research design as well as the implemented statistical methods should always be reported in an adequate manner, thus improving the scientific quality of abstracts. antimicrobial pk/pd p nxl -oral streptogramin: a phase i, doubleblind, single escalating oral dose study to evaluate safety, tolerability and pharmacokinetics in healthy adult male volunteers m. rangaraju, j. rey, j. hodgson (romainville, vitry sur seine, fr) background: nxl (formerly xrp ) is a novel semisynthetic oral streptogramin that consists of a / (w/w ratio) association of a pristinamycin ia (pi) derivative and a pristinamycin iib (pii) derivative. nxl is being developed for the treatment of respiratory tract and skin and skin structure infections. methods: healthy male subjects were enrolled in this study. subjects in each of cohorts ( mg, mg, mg, mg, mg and mg) received either nxl ( ) or placebo ( ) . an additional cohort of subjects received a single dose of mg nxl in fasting and fed conditions. blood and urine samples for pk analysis were collected at multiple time points. safety was assessed via adverse events, physical examination, clinical laboratory data, ecg and cardiac monitoring. results: nxl administered as mg capsules at single doses from mg to mg was well tolerated and safe. there was no serious or severe adverse event, no dosedependency in the number of aes or their severity, no significant variation in blood pressure or heart rate, no abnormality on ecg recording, and no clinically significant changes compared to baseline for laboratory parameters. both components were rapidly absorbed; pi being slightly more rapidly absorbed than pii. the cmax and auc ( -t) increased approximately in proportion with dose. the proportion of pi and pii components estimated on mean exposure values was approximately comparable to that administered ( / ), indicating that the relative bioavailabilities of pi and pii are simila. elimination half-life ranged from between to hours for pi to to hours for pii. food increased the bioavailability of pi and pii by approximately %. conclusions: nxl is safe, well tolerated and exhibits predictable pk properties in healthy volunteers in doses up to mg administered as a single dose. correlation of vancomycin and daptomycin susceptibility in staphylococcus aureus in reference to accessory gene regulator polymorphism and function w. rose, m. rybak, b. tsuji, g. kaatz, g. sakoulas (detroit, new york, us) objective: polymorphism at the accessory gene regulator (agr) locus in s. aureus (sa) defines groups (i-iv). agr group ii sa have been associated with glycopeptide treatment failure in patients. sa with loss of agr function appear to have a higher tendency to become vancomycin (v) resistant. it is unknown whether this association only pertains to glycopeptides. we examined the effect of varying v and daptomycin (d) against agr+ and agr null pairs in an in vitro pharmacodynamic model (ivpm). methods: agr group i and ii wild-type prototype and knockout (tetm::agr) pairs were evaluated. mic values were determined according to clinical laboratory standards institute. ivpm glass and hollow fibre models were used to simulate dosages and auc/mic exposures for v ranging from . mg- g q h fauc/mic range - mg/l*h, and d . mg/kg- mg/ kg/day fauc/mic range . - . the dosage regimen and auc/mic breakpoints that produced resistance was then evaluated in the hollow fibre ivpm. all ivpm simulations were performed in duplicate over h. resistance was evaluated using and x mic screening plates at , , , , and h. results: pre-exposure mic values for agr i± and agr ii± were . / and for v and . lg/ml for d respectively. vintermediate resistance (mic = mg/l) was detected in both agr i and ii null strains at a simulated v dosage of . mg q h (auc/mic ), representing an mic increase of - fold. this breakpoint for resistance was verified in the hollow fibre model. although significant regrowth was noted with suboptimal dosing of d, no resistance was detected on d screening plates for any daptomycin regimen evaluated. conclusions: exposure of sa to v approximating / of optimal serum concentrations resulted in the development of heteroresistance in the agr null group i and ii. loss of agr function did not correlate with the development of d resistance despite suboptimal simulations of d exposures. these results implicate loss of agr function important to the development of glycopeptide resistance but not to loss of susceptibility to d. teicoplanin efficiently penetrates into the rabbit infected vitreous but may enhance expression of virulence factors at sub-inhibitory concentrations e. forestier, f. jehl, c. gallion, r. andres, s. bronner, l. leininger, g. prévost (strasbourg, fr) objectives: fluoroquinolones are the antibiotics that most efficiently penetrate inside vitreous. however, alternative treatments for endophtalmitis may be required in some cases for example resistant bacteria. we used a rabbit experimental model of endophtalmitis to evaluate the penetration of teicoplanin in different conditions. the influence of subinhibitory concentrations of teicoplanin was also evaluated on the expression of s. aureus virulence factors. methods: new zealand rabbits (> kgs) received one or repeated doses of intra-venous (iv) teicoplanin ( mg/kg) every hours for days plus one dose a day for more days. another group of rabbits was infected by cfu of a methicillin resistant s. aureus ibs (cmi = . mg/l) producing enterotoxin a, panton-valentine leucocidin and luke-lukd. they were administrated - hours later with mg/kg teicoplanin, as a single dose or as to reach the steady state. vitreous ( ll) was sampled before new injections of teicoplanin or at indicated time as well as blood before or min after teicoplanin injection. teicoplanin concentrations were measured by hplc. bacterial counts were recorded and expression of virulence factors was semi-quantified by dedicated competitive rt-pcr tests. results: in safe eyes, teicoplanin penetration remains moderate reaching about mg/l within about - h after one iv injection. the half-life of teicoplanin in the rabbit vitreous is about hours. after days of repeated injections, intra-ocular concentration stabilises around mg/l while residual blood concentrations were comprised between - mg/l. in infected eyes, teicoplanin, when repeatedly administrated after the beginning of clinical signs, i.e. h postinfection = cfu/ml, reaches intra-vitreal concentrations of . ± . mg/l h post-infection, and increases to . ± . mg/l h post-infection and h after a fourth injection. however, at sub-inhibitory concentrations (~ mg/l), it may be responsible for a significant increase of agr, gammahemolysin hlga, luked and panton-valentine leucocidin luk-pv expressions with ratio ranging from to folds. conclusion: these preliminary results strongly suggest that teicoplanin iv administration constitutes an interesting alternative therapy for endophtalmitis provided high intraocular concentrations are rapidly obtained. investigations now concern optimisation of teicoplanin dosage regimen. pharmacokinetics of temocillin in intensive care patients and monte carlo simulations to evaluate susceptible breakpoints j.w. mouton, r. dejongh, v. basma, p. tulkens, s. carryn (nijmegen, nl; genk, brussels, be) background: temocillin (tmo) is a narrow spectrum penicillin with good activity against gram negative micro-organisms including esbl and ampc producers. little pharmacokinetic data are available however. we performed a pharmacokinetic study in icu patients receiving tmo g q h. parameter estimates were used to predict concentrations during continuous infusion (coinf) and compared with data obtained from other icu patients receiving coinf to validate the model. the model was then used to perform monte carlo simulations (mcs) to determine probabilities of target attainment (ptas) for pharmacodynamic indices (pdi) in order to evaluate and suggest clinical breakpoints. methods: blood samples were taken from icu patients prior to (t = ) and after (t = , , , , h) a m infusion of g tmo (n = ) or after h during coinf with g/ h (n = ), and then cooled, centrifuged and stored at ) °c until analysis by hplc. protein binding was determined using an ultrafiltration method. results were used to estimate population pharmacokinetic parameters by winnonmix including the covariance matrix. miclab was used to perform simulations for coinf as well as to perform mcs ( cycles) and obtain ptas for the unbound fraction including % confidence intervals (ci) for the target concentrations. ft>mic was chosen as the pdi because of the pharmacodynamic properties of tmo. results: protein binding was %. a one-compartment model best fitted to the data, with estimates (se) of vc = . ( . ) l and k = . ( . ) /h corresponding to a mean half-life of . h. using these estimates, the predicted unbound concentration during coinf was . mg/l, while the mean concentration in the other patients was . mg/l, a bias of less then %. the breakpoint mic for a mean ft>mic of % was mg/l. however, mcs -taking the variation in the population into account -indicated that % ptas of a g q h dose were obtained at , , and mg/l for , and % ft>mic, respectively. the % ci at % ft>mic indicated a clinical breakpoint of mg/l. the % ci was relatively large, as expected from data obtained in patients rather than volunteers. conclusion: the population pharmacokinetic estimates from icu patients were very well in agreement with the validation study, with a bias of < %. the mcs indicate a susceptible breakpoint for temocillin of £ mg/l provided an administration of g q h is used. tissue penetration and pharmacokinetics of moxifloxacin in diabetic foot infections:an interim analysis j. majcher-peszynska, k. karrasch, m. saß, r. mundkowski, a. gussmann, p. kujath, b. ruf, w. schareck, h. koch, b. drewelow (rostock, bad saarow, lubeck, leipzig, beeskow, de) objectives: with its broad spectrum of activity against grampositive, gramnegative and anaerobic organisms moxifloxacin covers the pathogens of the mainly polymicrobial infections associated with the diabetic foot. inflammatory and fibrotic processes in diabetic foot infections (dfi) contribute to impaired tissue penetration of antibiotics. in addition, diabetic patients represent a pharmacological risk population, physiological changes in diabetic patients may alter the pharmacokinetics of antibiotics. the study was designed to investigate the penetration of moxifloxacin into perinecrotic tissue in patients with dfi and the pharmacokinetic properties of moxifloxacin in diabetic patients. methods: the interim analysis of this open, multicentre study included adult, hospitalized male and female patients (mean age: . years) with type diabetes mellitus and dfi. the pharmacokinetic parameters of moxifloxacin and penetration into dfi tissue at steady state (day to ) following once daily administration of mg iv or po were evaluated. correlations between penetration of moxifloxacin and clinical and laboratory parameters were examinated. results: in all patients the moxifloxacin concentrations measured in infected diabetic foot wounds hours after administration exceeded the in vitro mic values of susceptible staphylococci ( . mg/l). the moxifloxacin concentrations achieved in dfi tissue correlated more strongly with the auc - (r = . ; p < . ) than with the corresponding plasma concentrations (r = . , p < . ), but not with the extent of the systemic inflammation and the blood glucose level. taking into account the predictive pk/pd parameters for moxifloxacin (based on an in vitro mic value of . mg/l for staphylococcus aureus) a therapeutic success can be expected (auc /mic: . ; cmax/mic: . ). significant differences between the routes of administration (iv vs po) were only observed for tmax (p < . ) and t / (p < . ), but not for other clinically relevant parameters (auc - , cmax, moxifloxacin tissue concentration). this allows sequential therapy i.v./p.o. in this indication. conclusion: based on adequate plasma concentrations in diabetic patients, the sufficient penetration into dfi tissue and the possibility of a sequential therapy, moxifloxacin representsfrom a pharmacological point of view -a valuable therapeutic option in the treatment of diabetic foot infections caused by susceptible organisms. fluoquinolones effects on patient lymphocytes during prolonged treatments e. bertazzoni minelli, a. benini, d. doria, p. franceschetti, m.e. fracass (verona, it) fluoroquinolones (fq), widely used in clinical practice, are well tolerated. the most common adverse reactions are those affecting gastrointestinal tract, phototoxicity and allergy. the aim of the study is to evaluate the possible cellular damage in lymphocytes of patients treated with different fqs according to pharmacokinetic data. blood samples obtained from thirty-six patients treated with ciprofloxacin (cpx, pts), levofloxacin (lvx, pts.) and moxifloxacin (mfx, pts) at different doses were analysed. patients treated with cpx and lvx were in therapy with other drugs (diuretics, cardiovascular drugs, omeprazole, antiinflammatory drugs, etc.). mxf treated patients were not in therapy with other drugs. samples were collected at time (before fqs administration) and after and days of treatment. serum levels of fqs were determined with microbiological method and hplc. comet test was performed on lymphocytes, to evaluate dna damage. gsh levels were determined as efficiency marker in metabolic process of detoxification. cpx showed good serum concentrations; its levels increases proportionally with administered doses (from to mg). lvx concentrations resulted in good inhibitory levels after treatments ( mg) both per os and i.v. patients orally treated with mg showed similar serum levels (from . to . mg/l). mfx levels were between . and . mg/l after and days. repeated cpx administration induced a dose-dependent increase in all dna damage parameters, with statistical differences after treatments. mfx ( mg) and lvx administration didn't induce dna damage after and days. intracellular levels of gsh were similar in all treated groups, even if cpx treated patients showed the lowest concentrations. no statistical correlations were found between all parameters studied. these data indicate that cpx induce dna damage in lymphocytes in combination with a reduced efficiency in detoxification system. this effect does not seems to depend on high intersubject variability for fqs administered doses, co-administration of other drugs, different ages of patients and low samples numbers. effects of single fqs molecules seems to be structurespecific and selective. objectives: ertapenem is a carbapenem commonly used to treat intra-abdominal infections. the antibacterial spectrum includes the major causative pathogens. clinical trials proved excellent clinical and microbiological efficacy in peritonitis. on the other hand in inflammatory pancreatic diseases sufficient antibiotic concentration in the inflammatory tissue is vital for the outcome of the disease. we therefore investigated ertapenem concentrations in pancreatic tissue and juice in comparison to the plasma levels measured at the same time. methods: in a prospective clinical trial ertapenem was given in a dosage of g i.v. minutes prior to operation in patients ( - years) suffering from chronic pancreatitis or pancreas carcinoma undergoing pancreas resection. blood samples were collected every minutes during the operation. moreover we collected pancreatic tissue and pancreatic juice shortly before resection and shortly before finalisation of the anastomosis. the samples of ertapenem (blood, juice, tissue) were determined by hplc. results: in patients ( female, male, mean age . ± . years) ertapenem blood concentrations were determined and demonstrated intraoperatively high concentration ( ± mg/l) above mic values for major expected pathogens. concomitantly in of these patients ertapenem concentration was determined also in pancreas tissue and pancreas secretion (in further patients in pancreas secretion only). in / patients sufficiently high ertapenem levels were detected in pancreatic tissue. in patients with chronic pancreatitis no accumulation was seen. mean pancreas tissue concentration was . ± . lg/g tissue. of patients with pancreas carcinoma had increased ertapenem levels in pancreas secretion but only of patients with chronic pancreatitis. conclusion: in patients with pancreas carcinoma, ertapenem levels were measured in pancreatic tissue as well as in pancreatic secretion and penetration seems to be similar to imipenem. due to chronic inflammation and possibly altered microcirculation only in one half to one third of chronic pancreatitis patients ertapenem levels were detected. bacterial strain-independent pharmacodynamics of linezolid/doxycycline combinations with staphylococcus aureus: -day simulations using an in vitro dynamic model m. smirnova, i. alferova, i. lubenko, y. portnoy, s. zinner, a. firsov (moscow, ru; cambridge, us) objective: to delineate the possible advantages of linezolid (l)/doxycycline (d) combinations over monotherapy, the pharmacodynamics of l, d and l+d were studied with s. aureus. methods: s. aureus atcc and a clinical isolate s. aureus were exposed to twice-daily l (half-life h) and once-daily d (half-life h), alone and in combination ( : ratio based on -h auc/mics), for five consecutive days. to provide simultaneous mono-exponential elimination of l and d with different half-lives, a previously described dynamic model was modified according to blaser and zinner. the mics of l were . and . mg/l and mics of d were . and . mg/l for s. aureus atcc and s. aureus , respectively. nine dosing regimens were simulated with each organism exposed to different auc/mics (in hours): l , l and l ; d , d and d ; l + d , l + d and l + d . the cumulative antimicrobial effect was expressed by its intensity (ie) measured from the start of treatment to the time after the last antibiotic dose when numbers of antibiotic-exposed bacteria reached at least cfu/ml. emergence of resistance was monitored daily by quantitating surviving organisms on agar plates containing x and xmic of l or d. results: with both s. aureus atcc and s. aureus exposed to l or d, ie increased with increasing simulated auc/ mic ratios, although significantly higher ies were produced with l , l and l treatments relative to d , d and d treatments. each of the combined treatments, i.e., l + d , l + d and l + d , produced much greater ies than the sum of l and d ies observed in the respective mono-treatments with both s. aureus strains. based on population data, a pronounced selection of s. aureus resistant to d occurred in all mono-treatments with d. it was also observed with l + d and, to a lesser extent, with l + d but not with l + d . no resistance to l was observed with l mono-or combination treatments. conclusions: these data predict a synergistic interaction of l with d against s. aureus. anti-staphylococcal effects of telavancin in an in vitro dynamic model: impact of different half-lives and initial concentrations that simulates normal (nek) and impaired elimination kinetics (iek). materials and methods: a glycopeptide intermediately susceptible strain of s. aureus (gisa) mu- with a telavancin mic of . mg/l was selected for the study. with both nek and iek simulations at a starting inoculum of log cfu/ml, gisa mu- was exposed to different ratios of the peak concentration (cmax) to the mic of telavancin (as a single dose), i.e., . , . and . based on time-kill data, the intensity of the antimicrobial effect (ie -the area between control growth and time-kill curves) was determined from time zero to the time when the effect no longer could be detected, i.e. the time after the last dosing at which the number of antibiotic-exposed bacteria reached log cfu/ml. results: in each treatment, bacterial regrowth followed gradual reduction in the starting inoculum during the first h (similar in nek and iek simulations) that led to significantly lower minimal numbers of surviving organisms in iek simulations compared to nek simulations. despite similar rates of initial killing, times to regrowth were much longer in iek than nek simulations. at a given cmax/mic ratio, the ies observed in iek were greater than in nek simulations (figure). conclusions: these findings demonstrate pharmacokineticdependent pharmacodynamics of telavancin with staphylococci. pharmacokinetics of amoxicillin in pregnant women with pre-term premature rupture of the membranes objectives: amoxicillin is widely used during pregnancy, in particular to treat group b streptococcus. insufficient knowledge on the pharmacokinetics just before and during delivery, could pose patients with preterm premature rupture of the membranes (pprom) at serious risk for under dosing. we investigated the pharmacokinetics in patients with pprom in this critical situation. methods: seven healthy women at - weeks of gestation were included. they received g (first dose g) amoxicillin for pprom according to local guidelines. from each patient - blood samples were taken. antibiotic serum concentrations were determined by a validated hplc method. pharmacokinetic parameters were estimated by population pk modeling using nonmem. to discriminate between various models the minimum value of the objective function (mvof) was used. a reduction of > in mvof was considered significant. results: a three-compartment pharmacokinetic model best described the time course of amoxicillin. the clearance and volume of distribution of the central compartment (vc) were estimated at respectively ± . l/h and ± . l (mean ± se). estimates of the parameters and model discrimination improved when we assumed the size of the third compartment to be equal to the first compartment. the residual error was found to be proportional to the serum concentrations. most of the inter-individual variability could be explained by variation of clearance. the mean volume of distribution at steady state (vss) and terminal half-life were . l and . h respectively. estimated values of elimination and distribution rate constants were: k = . h- , k = . h- , k = . h- , k = . h- and k = . h- . as was to be expected due to the small population size, no significant relationship was observed between the individual posthoc estimates for clearance and patient characteristics. conclusion: here we describe the pharmacokinetics of amoxicillin in pregnant women with pprom. it was found that the pharmacokinetics clearly differs from that in nonpregnant individuals. clearance and vss were significantly higher and the terminal half-life was shorter. furthermore, a -compartment model was found to describe the data better than a -compartment model. it is an intriguing question whether this rd compartment is a unique feature associated with pregnancy. these data offer a theoretical basis to make proper dose-adjustments in a particular patient group in a critical condition. penetration of piperacillin and tazobactam in severe acute pancreatitis objectives: acute necrotizing pancreatitis is still related to an extremely high mortality rate, based on local infectious complications, particularly in necrotizing areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotizing pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of betalactamases. on that score, the penetration of co-administrated pip and bli into inflamed or necrotic pancreatic tissue has not been investigated yet. methods: adressing the penetration capability of bsp and bli a clinical trial was designed to investigate the penetration of piperacillin (pip) and tazobactam (taz) in patients with severe necrotizing pancreatitis undergoing pancreas surgery. samples (n = ) were taken from plasma (pl), necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of . g pip and . g taz. concentrations of pip/ taz were determined by hplc/ uv. results: mean plasma concentrations at . h after application were . ± . mg/l (pip) and . ± . mg/l (taz). exceeded in pl and bs, nearly reached in pn but not in pft. the concentration of pip in combination with taz exceeded or reached the mic in pl, pn and bs against e. coli, klebs. spp., enterobacter, proteus spp. and clostr. spp., in pl and bs even against pseudomonas and bacteroides. conclusion: given in combination both -pip and taz -have been demonstrated to reach rapidly effective inhibitory concentrations in inflamed and necrotic compartments of pancreatic and peripancreatic tissue. co-administration of piperacillin and tazobactam may have a potential clinical benefit in prevention and treatment of local infectious complications of severe necrotizing pancreatitis. pk/pd challenges of in vitro time-kill curves -a new modelling approach s. schmidt, o. burkhardt, w. treyaprasert, h. derendorf (gainesville, us; bangkok, th) objective: in vitro pk/pd models, based on time-kill curve data, have become a powerful tool to predict the in vivo situation. up to date, several modelling approaches have been undertaken to develop suitable pk/pd models that fit in vitro data sufficiently well. widely used simple sigmoid emax models meet these criteria only partly. a further approach was undertaken to address the weak points of currently used models and applied to model the effects of ceftriaxone against escherichia coli. methods: constant concentration time-kill curves were performed in mueller-hinton broth (mhb, difco) with and without bovine serum albumin (bsa) g/l. using concentrations of ceftriaxone, ranging from . mic to mic, the change in number of bacteria (cfu/ml) versus time was linked to its effect. escherichia coli atcc was employed as the test organism. samples were taken at , . , , . , , , , and hours. the data were modelled simultaneously, using a modified sigmoid emax model and the software scientist Ò for windows tm . results: a differential equation, characterized by growth rate constant (k ) times the starting number (n) of bacteria represent the simplest case. barging from log-growth phase to stationary phase can be described by an additional nmax term. however, bacteria do not necessarily start growing in the loggrowth phase. this delay in onset of growth can be modeled by an exponential term, characterized by a factor beta (b) and time (t). to describe the overall change in number of bacteria not only growth but also concentration (c) dependent kill has to be taken into account. from certain drug specific concentrations on, a maximum effect is reached, described by the maximum kill rate constant kmax. however, it may be necessary to model a delay in the onset of kill with an additional exponential term, characterized by a factor alpha (a) and time (t). finally, a hill factor/shape factor (h) is necessary to smooth the predicting curves out. as shown in figure this new model meets the in vitro time-kill curve data sufficiently well. the final equation including all parameters described above is: conclusion: the proposed model was able to describe the observed data much better than a simple emax-model. incorporating two additional terms into the model, the in vitro situation could be described much better, taking the delay in onset of growth and kill into account. objectives of this study were ( ) to describe the pharmacodynamic (pd) profiles of bpr mg iv q h as a hr infusion & mg iv q h as a -hr infusion; ( ) to determine the overall probability of target attainment (pta) by weighting for the expected distributions (dis) of renal function (rfx) in the populations (pop) of interests; ( ) to determine the organism-specific pta against the pathogens encountered in phase ii trials. methods: subjects (total samples) were studied (phase i/ii subjects). samples were analysed using bignpod. to assess the impact of differing degrees of rfx impairment on pta, crcl (crcl-cockcroft & gault method) was employed as a covariate in the pop pk analysis. monte carlo simulation (mcs) ( subjects) was performed with adapt ii. overall pta was calculated for - % ft>mic. weighting for the expected dis of rfx in the pop of interests was accomplished by using the dis of crcl observed in previous registration studies of the same indications (cssti and np). dis of mics for pathogens was supplied by sponsor. results: in the pop pk analysis, the pop mean (sd) values for volume, clslope, clintercept, kcp and kpc were: . ( . ) l, . ( . ) l/hr, . ( . ), . ( . ) hr- and . ( . ) hr- , respectively. the obs-pred plot was obs = . x pred + . ; r = . after the bayesian step. in the mcs analysis of bpr mg iv q h, the pta of achieving % ft>mic & % ft>mic exceeded % for mics values £ mg/l & £ . mg/l, respectively. for bpr mg iv q h, the pta of achieving % ft>mic exceeded % for mics values £ mg/l. in the organism-specific analysis, the pta of a static effect ( % ft>mic) exceeded % for both mssa & mrsa for bpr mg iv q h. bpr mg iv q h provided a > % pta of a cidal effect ( % ft>mic) for both mssa & mrsa. for gnb, the pta of bpr mg iv q h in achieving a cidal effect ( % ft>mic) exceeded % for non-ampc-bearing gnb. for ampc-bearing gnb, the pta of achieving a cidal effect was . %. conclusions: an extensive evaluation of the pd of bpr was performed to estimate the overall activity of bpr against target pathogens. these findings need to be validated in the clinical trial arena. investigation of different levofloxacin regimens in patients with acute complicated urinary tract infections p. tenke, r. benko (budapest, szeged, hu) objective: in the present study we aimed to find out if a continuous or an intermittent levofloxacin ( · mg) treatment is more advantageous for patients with acute complicated urinary tract infection (uti) caused by urinary obstruction. we investigated if levofloxacin adsorbs to the surface of the foreign body, which was inserted with the aim of temporary resolution of ureteral obstruction. preventive effect of levofloxacin on bacterial biofilm formation and incrustation was also evaluated. methods: we enrolled and randomised patients who had acute uti caused by urinary obstruction. obstruction was resolved with double j stent (djs) insertion or percutaneous nephrostomy (pcn) and meanwhile, antibiotic treatment was started in all patients. patients (group ) were on antibiotics till the day of definitive curative operation when all foreign bodies were removed. in the other % of the patients (group ) the antibiotic therapy was stopped days after the djs or pcn insertion. short term antibiotic course -which is advisable for prevention of uti before invasive endoscopic treatmentwas administered in both groups from the day of the operation (after djs or pcn removal) and it was continued until the removal of all possible urinary foreign bodies used during the operation. in both groups of patients we recorded and evaluated early and late clinical and microbiological recovery. retrieved stents were sectioned for further laboratory examinations. adsorbed levofloxacin in the conditioning film layer and on the stent surface was detected by hplc. rasterelectron microscopy (rem) was used to examine biofilm formation and encrustation. results: we did not find any significant differences between the two groups of patients, neither in clinical (presence of fever, back pain, flank pain, leukocyte count) nor in microbiological recovery. statistical analysis showed that significantly greater amount of levofloxacin adsorbed to the conditioning film than to the stent surface in both groups of patients ( . ± . vs. . ± . in group and . ± . vs. . ± . in group ). no viable, adherent bacteria were recovered by sonication and culture in any of the patients, and no biofilms or encrustation were seen under rem either. conclusion: our data prove the hypotheses that continuous antibiotic treatment does not have any clinical or microbiological advantages in patients with indwelling ureteral stents compared to intermittent therapy. objective: the prophylaxis of bacterial infections during cardiac surgery is widely used in clinical practice. staphylococcus aureus, staphylococcus epidermidis and enterococcus spp are the pathogens most frequently involved in infective complications of cardio-pulmonary bypass (cpb) surgery. it is generally agreed that the success of prophylaxis is dependent on the ability to reach and maintain free antibiotic concentration in tissues higher than the mics for the most common pathogens. so we estimated the tissue concentrations of linezolid into sternal bone of patients undergoing cpb surgery. methods: six patients undergoing routine cpb surgery were given mg linezolid as a min iv infusion along with conventional prophylaxis of . g of cefuroxime immediately before surgery. two hours after the end of infusion blood samples and sternal bone tissues were collected. the local medical research ethics committee approved this study and all patients gave written informed consent. samples were assayed for the presence of linezolid by a high-performance liquid chromatography (hplc) method. results: following a mg infusion of linezolid, mean serum concentration for the six patients were . mg/l (range . - . mg/l) hours after the end of infusion. the concentration of linezolid into sternal bone was . mg/l (range . - . mg/ l) hours after the end of infusion. the penetration of linezolid into sternal bone was . %. conclusion: the penetration of linezolid into bone was . % of the simultaneous blood levels. in all bone samples the concentration of linezolid exceeded the mic for susceptible pathogens (< mg/l). although these data have been obtained from healthy, well-perfused bone the values suggest that linezolid may be a useful agent in the management of multidrug-resistant gram-positive bone infections. the antibacterial effect of daptomycin, teicoplanin and vancomycin against s. aureus studied in an in vitro pharmacokinetic model of infection a. noel, k. bowker, a. macgowan (bristol, uk) objectives: daptomycin (dap) is the first cyclic lipopeptide antibiotic approved for parenteral use in gram-positive infection. as yet, no comparative pharmacodynamic studies have been performed using dap and the two most common iv therapies teicoplanin (tei) and vancomycin (van). we used a pharmacokinetic (pk) model to study the antibacterial effect (abe) of these agents against two typical mrsa strains (ukemrsa & ) and a hetero vancomycin intermediate mrsa (hvisa). methods: an in vitro dilutional model was used to simulate the free drug concentration over h associated with doses of -dap mg/kg hrly (cmax . mg/l, t / h); tei mg hrly (cmax . mg/l, t / h); van g hrly (cmax . mg/l, t / h). an inoculum of cfu/ml was used and the experiments performed in triplicate. abe was assessed by area-under-the-bacterial-kill-curve - h (aubkc ) and logcfu/ml.h objectives: linezolid, the first oxazolidinone, is active against methicillin resistant staphylococcus aureus and has been effective in a variety of acute infections. however long-term administration, although desirable in bone infections caused by resistant gram-positive organisms, is hampered by the occurrence of anaemia and thrombocytopenia. administration of vitamin b has been reported to prevent myelosuppression. methods: patients attending the infectious disease clinic with bone infections caused by resistant gram-positive bacteria and treated with linezolid ( mg b.d. orally), received vitamin b ( mg o.d. orally) for the period of administration of linezolid. full blood counts were followed-up weekly. linezolid treatment was discontinued if haemoglobin declined below mg/dl or platelets below /ll. data from sixteen patients with osteomyelitis and with prosthetic joint infections were evaluated. comparisons were performed with matched historical controls receiving linezolid without b by kaplan-meyer curves with the log-rank test. results: the median follow-up of patients receiving b was . weeks and of controls weeks. in the b group % of the patients discontinued while in the control group % of the patients discontinued treatment because of side effects (p ns). % of patients receiving b discontinued due to thrombocytopenia and % due to anaemia. respective percentages in the control group were % and % (p ns for all comparisons). mean time to the occurrence of thrombocytopenia was weeks in the patients who received b and . weeks in the control patients. respective times to occurrence of anaemia were . and . weeks. all cases of myelosuppression were reversible. conclusions: administration of b failed to prevent or delay both thrombocytopenia and anaemia in patients receiving linezolid. other methods should be investigated to facilitate longer administration of linezolid in this group of patients. therapeutic drug monitoring of colistin -a -year review from a uk clinical antibiotic assay service k. bowker, a. noel, s. tomaselli, a. macgowan (bristol, uk) objectives: over the last years there has been increased use of colistin (col). however, little clinical data is available on the therapeutic levels of colistin. monitoring of col is useful in terms of therapeutic levels and avoidance of toxicity for patients with cystic fibrosis and complicated gram-negative infections. previous in vitro data has shown that col is bactericidal at col concentration is ‡ mg/l. here we assessed col data collected from our antibiotic assay service from the last seven years in order to establish if such levels were obtained. methods: col levels were determined by bioassay. data was retrospectively collected from the hospital information management system. data was assessed collectively and stratified by known cystic fibrosis patients, sex and age. objectives: ßlactams like ceftriaxone (cfx) and quinolones such as moxifloxacin (mox) are widely used to treat pneumococcal infection. we studied the antibacterial effect of (abe) after the first dose exposure to free drug serum concentrations of iv cfx g and po mox mg against s. pneumoniae strains with typical mics at low and high inocula. methods: a hollow fibre in vitro model was used to simulate free drug concentrations over h of cfx ( g h iv, cmax mg/l, auc mg/laeh, t / h) and mox ( mg, po, cmax . mg/l, auc mg/laeh, t / . h). the cfx mic was . mg/l (t>mic cfx %) and mox mic . mg/l (auc/ mic ). initial abe was measured by the slope of the log viable count - h and total abe over the dose interval ( h) by log reduction in viable count at h (d ) and the area-underthe-bacterial-kill-curve (aubkc ). inocula of cfu/ml and cfu/ml were used. results: the initial and total abe at low and high inocula were: given the pk/pd indices modelled both drugs showed a maximal effect. clearance from the model occurred at h ( inoculum) and h ( inoculum). there were no significant differences in speed or extent of abes comparing cfx and mox. conclusion: the abes of iv cfx and po mox against s. pneumoniae is similar in the first hrs of drug exposure. emergence of resistance in e. coli and ent. cloacae after exposure to ceftriaxone or ertapenem in an in vitro model of infection k. bowker, a. noel, a. macgowan (bristol, uk) objectives: emergence of resistance (eor) is an emergent factor in therapeutic choice. we studied eor to ceftriaxone (cfx) and ertapenem (ert) in e. coli (ec) and ent. cloacae (entclo), a more challenging species within inducible blactamases. methods: an in vitro dilutional model was used to simulate free drug concentrations associated with g hrly cfx (cmax mg/l, t½ h) and ert (cmax mg/l, t½ h) over h. two inocula and cfu/ml were used and eor assessed by population-analysis-profiles (pap). the area under the pap (auc-pap) was used to measure eor. ert mics were . mg/l ec and . mg/l entclo, cfx mic . mg/l ec and . mg/l entclo. experiments were performed in triplicate and mean values presented. results: observations at h were similar to those at h, hence data to h is given. at and cfu/ml, ec viable counts were reduced by fi logs, there was no eor. against entclo inoculum and , cfx resulted in an initial - log drop, then regrowth, ert produced a > log reduction. eor as measured by the mean auc-pap (n = ) with entclo is shown below: dosing with cfx resulted in eor to cfx and ert at high and low inoculum. dosing with ert resulted in no eor at inoculum, at resistance emerged to both cfx and ert. conclusions: eor depends on species (entclo > ec); duration of exposure (long > short) and agent (cfx > ert). ert appears to induce less eor both to itself and cfx than cfx does to itself and ert. however, initial use of cfx may reduce the effectiveness of ert. comparative serum activity of telithromycin, azithromycin, and amoxicillin/clavulanate against aerobic and anaerobic respiratory pathogens objectives: the purpose of this investigation was to study the clinical potential of telithromycin, a new ketolide antibiotic, for the treatment of mixed aerobic/anaerobic respiratory infections. in this study, we compared the pharmacodynamics (duration of inhibition/killing) of telithromycin (tel) to azithromycin (azi) and amoxicillin/clavulanate (a/c) against aerobic and anaerobic pathogens associated with mixed respiratory infections. methods: following written informed consent, ten healthy adult subjects (ages, - yrs) received single doses of tel ( mg), azi ( mg), and a/c ( mg) one week apart following a -h fast. venous blood samples were obtained at , , , and h after the dose and stored at ) °c. inhibitory and bactericidal titre s were determined by microdilution (s. pneumoniae & h. influenzae) and agar dilution (peptostrep. magnus, peptostrep. micros, prev. bivia, & prev. melaninogenica) procedures following clinical & laboratory standards institute methodology. bactericidal titres in serum endpoint was determined as the highest dilution of serum yielding . % killing. the median titres at each time point were calculated and the duration of activity was used for comparison of these agents. conclusions: in this ex-vivo study, we found that tel can provide prolonged ( % of the dosing interval) inhibitory activity in serum against macrolide-resistant strains of s. pneumoniae, bl pos. and neg. strains of h. influenzae, and common respiratory anaerobic pathogens. these findings suggest that tel could have clinical utility in the treatment of community-acquired mixed aerobic-anaerobic respiratory tract infections, including sinusitis, bronchitis, and pneumonia. objectives: increasing resistance in isolates of e. coli ( %) and p. aeruginosa ( %) to fluoroquinolones (fq) is a concern since these antibiotics are commonly used in the treatment of complicated urinary tract infections (utis). currently, no interpretive standards exist for ''susceptible'' isolates in urine for the newer fq. the purpose of this investigation was to evaluate the activity of high-dose levofloxacin against fqresistant urinary pathogens. methods: in this study, we determined the serum and urine levels of high dose ( mg) levofloxacin (l) as well as its bactericidal activity in urine (uba) against l-resistant isolates of e. coli (mics = to lg/ml) and p. aeruginosa (mics = to lg/ml). following written informed consent, blood and urine samples were collected from healthy adult (ages, - y/o) fasting subjects ( m and f) prior to and at . , , , , and hours after a single mg dose of l. serum and urine concentrations were measured by a validated hplc assay ( . - . % cv). the testing methodology for uba was similar to the microdilution assay used for serum bactericidal testing (clsi) with the exception that antibiotic-free urine was used to dilute these samples. the median titre ( : - : ) at each time point for the subjects was used to determine uba. results: the mean serum pharmacokinetic parameters were similar to previously published values: cmax = . lg/ml, auc = mgaeh/l, and t / = . h. mean urine concentrations ranged from lg/ml ( h) to lg/ml ( h). uba (titres > : ) was maintained for at least hours in all subjects for e. coli isolates with mics = , , and lg/ ml. for the e. coli strain with a mic = lg/ml, subjects exhibited uba at h but only subjects exhibited uba at h. similar results were observed against the p. aeruginosa isolates. conclusions: the results from this ex-vivo pharmacodynamic study in healthy volunteers found that mg of l provides prolonged (at least half the dosing interval) uba against l-resistant strains of e. coli and p. aeruginosa up to lg/ml. this suggests that a separate urinary susceptibility breakpoint is indicated for urine isolates treated with mg doses of l. objectives: exposure of methicillin-resistant s. aureus (mrsa) to acid ph restores its susceptibility to beta-lactams (sabath and al., aac, ) . in macrophages, s. aureus is mainly confined within phagolysosomes where the ph is acidic. we showed that meropenem (mem) displays similar intracellular activity against mrsa atcc and mssa atcc in macrophages. in the present study, we have investigated the intraphagocytic activity of mem and cloxacillin (clx) against mrsa clinical isolates (including one visa strain), in comparison with the reference mrsa atcc and mssa atcc strains. methods: mic's were determined in mhb (plus nacl %) by micro-dilution method. meca expression was examined at neutral and acidic ph by a semi-quantitative rt-pcr ( s rrna as housekeeping gene). intracellular activity was assessed in human thp- macrophages exposed to extracellular concentrations equivalent to human cmax (total drug; mem: mg/l; clx: mg/l) by examining the decrease in cellassociated cfu after h from the original, post-phagocytosis inoculum (controls [no antibiotic]; approx. log cfu increase). results: the table shows the mics (in broth) at neutral and acid ph and the intracellular activity for the strains studied. in atcc , meca expression was similar for bacteria maintained in broth at ph . conclusions: the intracellular environment markedly enhances the activity of beta-lactams against mrsa, probably through exposure to acid ph, although the latter does not affect meca expression. comparative activity of dalbavancin against european gram-positive isolates i. morrissey, c. dencer, j.w.t. dallow, j. childers, a. brook, j. cowan (london, uk) objectives: dalbavancin (dal) is a new semisynthetic lipoglycopeptide with a half-life of . days, enabling onceweekly dosing. this study compared the activity of dal with other agents against gram-positive isolates from europe. methods: isolates from belgium, the czech republic, denmark, finland, france, germany, hungary, italy, the netherlands, poland, spain, sweden and the uk were included. the clsi broth microdilution method was used to determine mic using dried microtitre plates. the following antimicrobial agents were evaluated: dal, vancomycin (van), teicoplanin (tei), daptomycin (dap), linezolid (lzd), dalfopristin/quinupristin (syn), erythromycin (ery), levofloxacin (lev) and tetracycline (tet). results: selected data are shown in the objectives: dalbavancin (dal) is a next generation lipoglycopeptide antibiotic in development for the treatment of complicated skin and skin structure infections (csssi). a population pharmacokinetic (pk) analysis was performed to estimate patient parameters and to determine significant covariates. incorporating the pk model, pharmacodynamic (pd) parameters were simulated to support the effectiveness of a weekly dosage. methods: the pk analysis included dal concentrations from patients across clinical trials. most patients received mg on day and mg on day . possible covariates examined included demography and concomitant medications, including medications that are considered inhibitors, inducers, and substrates of cytochrome p enzymes. the pk-pd analysis employed monte carlo simulations of time-dependent and concentration-dependent parameters. distributions of mics were obtained directly from clinical studies, and were also simulated to explore the effect of higher mics. results: dal pk fit a -compartment model with interpatient variability (ipv) on all parameters. the typical value and ipv (cv%) of clearance (cl) was . l/h ( . %), influenced by body surface area (bsa) and creatinine clearance (clcr). volume of distribution (v ) was . l ( . %) and influenced by bsa. the inter-compartmental clearance and peripheral volume were . l/h and . l, respectively. free drug concentrations were simulated using a dal protein binding of %. for a weekly -dose regimen, free dal remained above mg/l for the majority (> %) of patients for more than days. using previously described area under the curve (auc)/mic targets for staphylococcus aureus, a proposed mic of at least . mg/l was associated with a greater than % probability of target attainment. conclusions: dal pk were predictable, demonstrating low ipv. bsa and clcr were the only sources of variability, but described less than % of the ipv. pd simulations support the use of dalbavancin in a weekly regimen. objectives: methicillin-resistant staphylococcus joint infection due to peri-operative contamination is a complication after arthroplasty. the objective of this study was to assess the distribution of radioactivity in bone and related structures using quantitative autoradiography after administration of [ c]dalbavancin in rabbits. methods: new zealand white male rabbits were given a single intravenous (iv) bolus dose of mg/kg [ c]-dalbavancin (n = ) or control vehicle (n = ). plasma, cerebrospinal fluid (csf), bone, bone marrow, and nucleus pulposus were collected at , , , , and h post-dose by necropsy, homogenized, combusted, and analysed for total drug-derived radioactivity using liquid scintillation counting (lsc). in addition, the left hindlimb from rabbit/time point was flash-frozen and cryosectioned for quantitative autoradioluminography. results: [ c]-dalbavancin-derived radioactivity was rapidly and widely distributed into bone, bone marrow and, to a lesser extent, in csf and nucleus pulposus. autoradioluminography data indicated that concentration of radioactivity was highest in bone marrow, whole blood, articulate cartilage, ligament, epiphyseal plate, periostium, and meniscus. at h postdose, [ c]-dalbavancin-derived radioactivity was measurable in all tissues, and remained at relatively high concentrations in bone marrow ( . lg equiv/g), epiphyseal plate ( . lg equiv/g), periostium ( . lg equiv/g), and articular cartilage ( . lg equiv/g). in homogenized bone using lsc, mean concentration after hours was . lg equiv/g. conclusion: [ c]-dalbavancin-derived radioactivity rapidly penetrated knee joint tissues and persisted at relatively high concentrations for at least h after a single iv dose in rabbits. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound ( %) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution ( . l/kg) means that it may be removed by cvvh. cvvh is widely used in the management of critically ill patients. the objective of this study was to determine cvvh telavancin transmembrane clearance (cl) with commonly used hemofilters (an , polysulfone) at conventional ultrafiltrate flow rates. methods: tlv cl was assessed in our in vitro cvvh model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run using an (m , gambro) and polysulfone (f nr, fresenius) hemofilters. ultrafiltrate (uf) flows were , , and l/hr with sufficient blood flows [(qb) - ml/min] to maintain uf rates. blood samples were collected from the pre-filter line and uf samples from the post-filter uf port. concentrations of tlv in plasma and uf samples were assayed using validated lc-ms/ms methods. tlv cl was determined using the following formula:cl = (uf flow rate) [tlv]uf/[tlv]arterial. cl differences between the filter types were compared using a two-tailed, unpaired t-test. conclusion: tlv is substantially cleared by cvvh and cl increases significantly with increasing uf rate. cl did not differ by hemofilter type. cvvh cl at higher uf flows exceeds the total cl reported in patients with normal renal function. tlv likely will require dose adjustments in patients receiving cvvh. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound ( %) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution ( . l/kg) means that it may be removed by cvvhd. cvvhd is used in the management of critically ill patients. the objective of this study was to determine cvvhd tlv transmembrane clearance (cl) with commonly used hemodialyzers at conventional cvvhd dialysate flow rates. methods: tlv cl was assessed in our in vitro cvvhd model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run times using an (m , gambro) and polysulfone (f nr, fresenius) hemodialyzers. dialysate flows were , , and l/hr with sufficient blood flows [(qb) - ml/min] to maintain appropriate transmembrane pressures. blood samples were collected from the pre-filter port (a) and post-filter port (v), and spent dialysate samples (d) from the post-filter d port. plasma tlv concentrations (arterial and venous) and dialysate samples were assayed using validated lc-ms/ms methods and tlv cl was determined using the following formula: cl = (d flow rate) [tlv]d/(([tlv]arterial+[tlv]venous)/ ). dialytic cl between filter types was compared using a two-tailed, unpaired t-test. conclusion: tlv is effectively cleared by cvvhd. the higher permeability polysulfone dialyzer was associated with significantly increased cl vs. the an dialyzer as dialysate flow increased. the degree of tlv cl seen with cvvhd suggests that dose adjustments will be necessary in patients receiving cvvhd. objective: it is still a subject of controversy that only free, unbound drug is responsible for antibacterial activity of antibiotics. to provide further proof, that only free drug contributes to antimicrobial efficacy a comparative, doseranging time-kill curve study was performed. to exclude influence factors resulting from different mechanisms of action this was done within the antibiotic class of carbapenems, using compounds with different serum protein binding. methods: constant concentration time-kill curves were performed in % serum for the slightly serum protein bound mer-openem (~ %) and imipenem ( %) as well as for the highly serum protein bound ertapenem ( %) and faro-penem ( - %), ranging from · mic to · mic. the change in number of bacteria (cfu/ml) versus time was linked to their effect. escherichia coli atcc , klebsiella pneumoniae bay , staphylococcus aureus bay and streptococcus pneumoniae atcc were used as the test organisms. samples were taken at , . , , , and hours. the data were modelled simultaneously using the software scientist Ò for windows tm and a modified sigmoid emax model characterized by growth rate constant (k ), maximum kill rate (kmax), and concentration at half maximum effect (ec ). results: for all four bacterial strains investigated, there were dramatic increases ( - %) in ec for the highly se-rum protein bound carbapenems (ertapenem, faropenem) in the presence of serum proteins (fig. ) . for both substances no significant differences in k and kmax were determined. in contrast, imipenem and meropenem showed only minor differences in ec in the presence and the absence of % serum. conclusion: only free, unbound drug is responsible for the antimicrobial activity. analysis of these time-kill curves clearly showed that the antibacterial efficacy was significantly decreased in the presence of % serum for the highly bound ertapenem and faropenem while being unaltered for the slightly bound meropenem and imipenem. objective: numerous in vitro experiments have shown that protein binding (pb) is an important factor for antimicrobial activity, especially for highly bound antibiotics. however, the experimental conditions that simulate the in vivo situation best are still subject of controversy. therefore, an in vitro microdialysis experiment was performed that evaluates various influence factors on the pb of the highly bound betalactams ceftriaxone (pb - %). methods: a comparative, dose-ranging in vitro microdialysis study was conducted to determine free, unbound ceftriaxone concentrations in lactated ringer's solution and todd hewitt broth (thb) both with and without bovine serum albumin (bsa; sigma, st. louis) g/l and human plasma at °c. furthermore, in vitro constant concentration time-kill curves were performed, using escherichia coli atcc , streptococcus pneumoniae atcc and streptococcus pneumoniae atcc as the test organisms. the data was analysed using an appropriate pk/pd model, characterized by growth rate constant (k ), maximum kill rate (kmax), and concentration at half maximum effect (ec ) and correlated to free ceftriaxone thb concentrations determined by hplc-uv. results: there were only minor differences in both unbound drug concentrations and anti-infective activity when bsa g/ l was added to either lactated ringer's (pb % and . ± . %, with and without bsa respectively) or thb (pb % and . ± . %, with and without bsa respectively). no significant changes in k , kmax and ec were observed. however, using human plasma, unbound concentrations (pb %) %) were altered dramati-cally. conclusion: only free, unbound drug is responsible for the antimicrobial activity. however, one cannot rely on that binding to commercially purchased bsa is consistent with reported protein binding values. unbound concentrations should be measured under the respective experimental conditions to be able to correctly interpret the experimental results. in vitro postantibiotic effect of faropenem on penicillin-resistant streptococcus pneumoniae and beta-lactamase-producing haemophilus influenzae c.l. young, i.a. critchley, u.a. ochsner, n. janjic (louisville, us) objectives: faropenem (far) is an oral penem with potent activity against respiratory pathogens such as penicillin (pn)resistant streptococcus pneumoniae (sp) and beta-lactamase (bla)-producing haemophilus infleunzae (hi). the postantibiotic effect (pae) is a pharmacodynamic (pd) parameter that monitors suppression of bacterial growth following short exposure and removal of the drug. paes are clinically important for agents such as far with short half-lives ( h) . the aim of the study was to determine the pae of far on resistant phenotypes of sp and hi. methods: nine clinical isolates of sp, pn-s, pn-i, and pn-r and six clinical isolates of hi, bla-negative and blapositive were tested in pae studies. paes were determined in cation-adjusted mueller-hinton broth with - % lysed horse blood for sp and haemophilus test medium for hi. exponential cultures ( cfu/ml) were exposed to far at , and x mic. far was removed by serial washing ( , -fold dilution) prior to transfer to fresh media. control cultures were treated in the same way. bacteria were incubated with shaking and viable cfus determined at , , , , and h. counts of log cfu were plotted against time and pae defined as the difference is the time required for count in test culture and control (untreated culture) to increase log above the count observed immediately after removal. results: significant paes of > . h were observed for all strains of sp at and x mic. however, the pae was more prolonged on the pn-r strains with mean paes of . h at and x mic. among hi, little or no pae was observed on bla-negative strains but a significant pae was observed on the bla-positive isolates (mean paes of . h and . h at and x mic respectively). conclusions: far demonstrates a prolonged pae on key resistant phenotypes of sp (pn-r) and hi (bla-positive) compared with susceptible strains. the observation of pae in bla-positive hi is unique in the class of beta-lactams. far exhibits in vitro pd properties that may contribute to its clinical efficacy against pn-r sp and bla-positive hi. telavancin is more efficacious than vancomycin in a murine model of bacteraemic peritonitis induced by methicillin-resistant staphylococcus aureus s. hegde, n. reyes, b. benton, r. skinner (south san francisco, us) objective: telavancin (tlv) is a novel lipoglycopeptide that operates through multiple mechanisms to produce potent and rapid bactericidal activity against clinically relevant grampositive bacteria including methicillin-resistant staphylococcus aureus (mrsa). the present studies evaluated the in vivo efficacy of tlv vs vancomycin (van) in a model of mrsa induced peritonitis in neutropenic mice. methods: female nsa immunocompromised mice were inoculated intraperitoneally with atcc mrsa and treated, beginning at h post-infection, with subcutaneous doses (q h) of vehicle (veh) or test compound. mouse pharmacokinetic data were generated and used to choose doses of tlv ( mg/kg) and van ( mg/kg) in order to equate clinical exposures (auc's (free drug) of and lg.hr/ml, respectively). in survival studies, deaths were recorded for days post-infection and survival curves were compared using log-rank test. in bacterial titer determination studies, designated groups of control and drug-treated surviving animals were humanely euthanized at various times post-treatment and their blood and spleen were harvested to determine bacterial titers. results: mics of tlv and van were . and . lg/ml, respectively. mortality was % in animals treated with veh or van. mortality was % in tlv-treated animals (p < . vs veh and van). the pre-treatment bacterial titres were . log cfu/ml and . log cfu/g in the blood and spleen, respectively. analysis of the time kill curves for both blood and spleen revealed that tlv exhibited significantly greater killing activity than van (p < . , two-way anova). at hrs after the first dose, the titers in the blood were reduced to a greater extent by tlv () . log cfu/ml) compared to van () . log cfu/ml). at hrs after the second dose, the splenic titers were reduced to a greater extent by tlv () . log cfu/g) when compared to van () . log cfu/g). conclusions: the data described here demonstrate that tlv's in vivo bactericidal activity is superior to that of van against mrsa and results in successful infection resolution and, consequently, improved survival in the murine peritonitis model. proper use of carbapenems for blood-derived clinical isolates of pseudomonas aeruginosa y. kobayashi (tokyo, jp) methods: regimens of carbapenems were given to healthy adult subjects. changes in their blood concentrations of carbapenems were compared by using pharmacokinetic parameters (two-compartment model analysis) of meropenem (mepm), imipenem (ipm), and panipenem (papm) and by applying the lognormal distribution to the probability distribution of distribution volume and plasma half-life with monte carlo simulation (mcs). based on the data on distributions of the minimal inhibitory concentrations (mic) of various carbapenems for blood-derived clinical isolates of pseudomonas aeruginosa isolated/identified at keio university hospital between october and october (mic : mepm mcg/ml, ipm mcg/ml, papm mcg/ml), the mics in the subjects were obtained with mcs. from the changes in blood concentrations and mics in the subjects, the probability of achieving t>mic was calculated for each carbapenem regimen, using the formula reported by kuti et al.: %t>mic = ln (dose/vd*mic)*(t / / . )*( /di) <<ln: natural logarithm, dose: dose (mg), vd: distribution volume (l), t / : plasma half-life (h), di: dosing interval (h)>>. based on craig's data, the maximum bactericidal effect on gram negative bacilli is attained when %t>mic is approximately %. we focused on this information and analysed our data. results: the probability of achieving t>mic % was . % for mepm mg bid, followed by . % for ipm mg bid and . % for papm mg bid. when the dose was increased from mg to mg, it was . % for mepm mg bid, followed by . % for ipm mg bid and . % for papm mg bid. when the dose remained at mg and the dosing frequency was increased to three times daily, it was . % for mepm mg tid, followed by . % for ipm mg tid and . % for papm mg tid. regarding mepm, it was . % for mg gid? and . % for mg tid showing higher probabilities. discussion: in severe sepsis caused by pseudomonas aeruginosa, remarkably higher t>mic % was achieved with carbapenems at mg tid, although the daily dose ( mg) was lower, compared to mg bid. carbapenems with a low mic distribution, i.e. a superior antibacterial activity, showed higher probability of achieving t>mic. therefore, the optimal treatment for such sepsis is mepm mg tid. mepm mg qid appeared to provide comparable therapeutic effects with those at mg tid, the usual dose in foreign countries. penetration of moxifloxacin into normal and infected subcutaneous tissue in patients with spinal cord injury measured by microdialysis background: skin breakdowns, also termed decubitus ulcers or pressure sores, are a major complication associated with spinal cord injury, resulting in infection and tissue death. moxifloxacin (mfx) is approved for the treatment of sssi. our objective was to construct a population pk model for mfx disposition in plasma, normal and infected subcutaneous tissue in spinal cord injured patients with infected decubitus ulcer. methods: patients receiving mg mfx orally daily were enrolled in this study. blood, saliva and interstitial tissue fluid samples (microdialysis in normal and infected tissue) were collected over a time period of hrs. mfx concentrations were measured by a validated hplc. concentration-time data obtained in the present study were pooled with previously published mfx data (n = ). population pk modelling was performed with nonmem. results: the concentrations of mfx achieved in plasma, saliva, normal subcutaneous tissues and infected decubitus ulcers showed parallel profiles versus time. the pk was best described by a -compartment model with a link to interstitial tissue fluid. the population pk parameters were as follows (given as estimate with percent interindividual variability in parentheses): cl . l/h ( %); central vd . l ( %); intercompartmental cl . l/h ( %); peripheral vd . l ( %); and elimination rate constant for interstitial tissue fluid . h) ( %). with a conservative mic of . mg/l, the peak/mic ratios were higher than and the auc /mic ratios were higher than for plasma, saliva and interstitial tissue fluids. conclusions: this study showed the good diffusion of mfx into subcutaneous tissue in spinal cord injured patients with decubitus ulcers. the interstitial tissue fluids reached bactericidal levels for common bacteria found in infected skin lesions. objective: investigations of pharmacodynamic parameters such as postantibiotic effect and postantibiotic subminimum inhibitory concentration effect have been employed for design of dosing schedules of antimicrobial agents. in this study we compared postantibiotic effect and postantibiotic subminimum inhibitory concentration effect of ciprofloxacin, levofloxacin, and moxifloxacin for clinical isolates of methicillin susceptible staphylococcus aureus, methicillin resistant staphylococcus aureus and pseudomonas aureginosa. methods: the following strains were tested in this study: methicilline-susceptible staphylococcus aureus (n: ), methicilline resistant -staphylococcus aureus (n: ) and pseudomonas aeruginosa (n: ). the pae was determined by viable plate count method using mueller hinton broth. tubes containing ml of broth and the antibiotic to be tested at , , and x the mic were inoculated with approximately · cfu/ml. growth controls with an inoculum but not antibiotic were included with each experiment. result: postantibiotic effects of ciprofloxacin, levofloxacin and moxifloxacin increased with increasing concentration of the drug. the longest postantibiotic effect was observed for moxifloxacin. moxifloxacin showed no postantibiotic effect one p. aureginosa at all concentration and had no post antibiotic effect to another p. aureginosa at x mic and mic. in our study the longest postantibiotic subminimum inhibitory concentration effect against mssa was determined with moxifloxacin. similarly the moxifloxacin induced the longest effect against mrsa. however, this time frame was shorter than that of mssa. conclusions: all three antibiotics, showed for longer postantibiotic subminimum inhibitory concentration effect in all submic concentrations, immeasurable within the study period i.e. hours. lack of horizontal transmission of fluoroquinolone resistance between s. mitis and s. pneumoniae objectives: fluoroquinolone (fq) resistance can arise in s. pneumoniae through acquisition of dna from s. mitis and subsequent homologous recombination. the frequency at which this occurs is unknown, and while likely a rare event, increases in fq resistance among s. mitis may increase the rate at which horizontal transmission occurs. we sought to determine the frequency at which fq resistance could be transferred from s. mitis to s. pneumoniae or from s. pneumoniae to s. mitis. methods: s. mitis (either fq^r,tetracycline[tet^s], fq^r,penicillin[pen^s], or fq^s,pen^r) and s. pneumoniae (either fq^s,tet^r, fq^s,pen^r or fq^r,pen^s) were grown in co-culture using a pharmacodynamic model in the presence of either moxifloxacin (mxf) or levofloxacin (lfx) at salivary drug concentrations. after incubation, aliquots were plated onto either tet or pen containing sba plates to select for the recipient strains. fq susceptibility was performed using microbroth dilution. the entire parc and gyra genes were amplified and sequenced to determine if horizontal transmission occurred. results: in initial experiments tet was used as the selective agent. however tet resistance was transferred and therefore pen^r was used as a selective marker. an increase in the lfx mic in was observed in s. pneumoniae and s. mitis strain. sequencing of the parc gene revealed the selection of ser phe and ser tyr mutations in s. pneumoniae and ser phe in s. mitis consistent with fq resistance. sequencing of the entire gene failed to uncover evidence of horizontal transmission. no mutations were detected in gyra. selection of st step parc clinical microbiology and infection, volume , supplement , mutations occurred only after exposure to lfx. mxf eradicated both s. mitis and s. pneumoniae and failed to either select for resistance or support horizontal transmission. conclusions: although st step parc mutations were selected in strains ( s. pneumoniae, s. mitis), we failed to find evidence of horizontal transmission between s. pneumoniae and s. mitis under our laboratory conditions. the phenomenon of horizontal transfer resulting in fq resistance has been described, however, based on our results, we must speculate that it is an extremely rare event and not likely to be a major driver of fq resistance. of interest, the parc mutations were selected only under the selective pressure of lfx. mxf completely eradicated both s. pneumoniae and s. mitis and did not select for the development of fq^r mutations. objectives: the aim of the present study was to assess the killing activity of ertapenem (ert) and metronidazole (mtr) against four selected bacteroides fragilis strains with different mic values in an in vitro pharmacokinetic/pharmacodynamic (pk/pd) model. since anaerobes are often present in mixed infections, kill kinetics were also established for mixed inocula employing the b. fragilis strains together with four selected escherichia coli strains. the killing activity was analysed for kinetic concentrations of the antimicrobial agents simulating human serum kinetics. methods: a pk/pd in vitro model was established by adding appropriate amounts of broth every half hour. at the same time intervals samples were obtained and plated. after incubation colony forming units were counted. human serum concentrations were simulated with cmax = mg/l and t / of hours for ert and cmax = . mg/l and t / of hours for mtr. mann trend test was used for statistical analysis. results: as to be expected the e. coli strains were not killed by mtr both in pure as well as in mixed cultures whereas the susceptible e. coli strains were effectively killed by ert. in pure cultures the b. fragilis strains were effectively killed by mtr and the growth of the susceptible b. fragilis strains was reduced by ert by about two to four logs. however, in some mixed cultures the killing activity of mtr against the b. fragilis strains was significantly reduced. conclusion: the in part moderate in vitro activity of ert against the b. fragilis strains and the reduced activity of metronidazole in mixed cultures against the b. fragilis strains may explain some of the difficulties in treating mixed aerobic/ anaerobic infections. penetration of ciprofloxacin into human cerebrospinal fluid and brain tissue a. tsona, s. metallidis, e. koumentaki, j. nikolaidis, p. kollaras, g. lazaraki, p. nikolaidis (thessaloniki, gr) objectives: the aim of the present study was to determine the penetration of ciprofloxacin into cerebrospinal fluid (csf) and brain tissue of humans. methods: a total of patients undergoing brain tumor excision were evaluated. the patients received a single intravenous dose of mg ciprofloxacin. samples of blood, cerebrospinal fluid and brain (brain-adjacent tumour tissue) were collected during surgery h after drug administration. ciprofloxacin concentrations in serum, cerebrospinal fluid and brain homogenate were analysed by means of a validated hplc method. results: ciprofloxacin concentrations in plasma (mcg/ml), cerebrospinal fluid (mcg/ml) and tissue homogenate (mcg/g), respectively, after h ranged . - . mcg/ml, . - . mcg/ ml and . - . mcg/g. csf-to-serum ratio ranged between . and . . tissue-to-serum ratio ranged between . and . . mean (±s.d.) csf/serum concentration ratios and brain tissue/serum concentration ratios were respectively . ± . and . ± . . conclusion: these findings suggest that valuable informations on brain tissue penetration can be obtained only from brain material. data from csf penetration cannot be extrapolated to the brain since the blood: sf barrier differs from the blood:brain barrier. concentrations of ciprofloxacin in cerebrospinal fluid were lower than those in serum, in contrast to the brain tissue concentrations that exceeded serum concentrations. the achieved concentrations in brain tissue were generally above the mic of common pathogens in central nervous system infections (h. influenze, n. meningitidis, s. pneumoniae, l. monocytogenes, escherichia coli, aerobic gram-negative bacilli, group b streptococci, mssa). cerebrospinal fluid concentrations exceed the mics of neisseria meningitidis and most gram-negative aerobic bacilli. our findings suggests that ciprofloxacin may be an acceptable alternative for the treatment of meningitis due to susceptible gram-negative aerobic organisms and for the treatment of brain abscesses. objective: to model the performance of imipenem (imi), meropenem (mem), and ertapenem (etm) against esbl producing e. coli and klebsiella spp in order to identify possible pd differences among compounds. methods: minimal inhibitory concentrations (mics) were generated for randomly selected esbl producing isolates of ec (n = ) and kl (n = ) collected during from brazilian hospitals as part of the mystic program. mic testing for imi, mem, etm, ceftazidime (ctz), and cefotaxime (ctx) were done by e-test methodology. esbls were confirmed via ctz/clavulanate and ctx/clavulanate e-test. pd exposure, measured as percent time above the mic for free drug (ft>mic), was modelled via a subject monte carlo simulation for the following -minute infusions:imi gram every hours, mem gram every hours, and etm gram every hours, using pharmacokinetics from healthy volunteers. the bactericidal cumulative fraction of response (cfr) was calculated for each regimen against the populations of ec, kl, and against all esbl isolates together. bactericidal cfr was defined as % ft>mic for all agents. results are reported as cfr ( % confidence interval). results: isolates were % susceptible (s) to imi and mem (mic range . - . and . - mg/l, respectively), and % s to etm (mic range . - mg/l conclusions: these findings support other data that although etm is likely to be an effective empiric agent against most esbl producing ec and kl, its ability to achieve high bactericidal pd exposure will be dependent on the presence of less susceptible organisms in the population. imi and mem should remain first line for esbl infections. objectives: this study analyses eradication and resistance selection in streptococcus pneumoniae with moxifloxacin, levofloxacin and azithromycin, using a parental serotype infecting strain (a) and subsequent resistant step-mutants (isolates b, c and d) selected in vivo in a patient with pneumonia. methods: moxifloxacin, levofloxacin and azithromycin mics were , and . lg/ml for the parental strain, , , and lg/ ml for isolate b, and , and > lg/ml for isolates c and d, respectively. a pharmacokinetic computerized device was used to simulate serum and epithelial lining fluid (elf) concentrations. initial inocula was approx. cfu/ml. population analysis profiles were performed using plates with increasing antimicrobial concentrations on a mic basis. results: in serum, moxifloxacin eradicated the parental isolate (isolate a), with an auc - h/mic value of . . serum auc - h/mic values of . and . for levofloxacin and azithromycin, respectively, were not able to eradicate isolate a. in elf, moxifloxacin showed a bactericidal pattern against all isolates with a minority (approx. cfu/ml) of the survival population (isolates b, c and d) growing in plates with moxifloxacin concentrations higher than those obtained in elf. levofloxacin and azithromycin showed a bactericidal pattern only against isolate a, with the whole population of isolates b, c and d growing in plates with levofloxacin concentrations higher ( - lg/ml) than those obtained in elf, and in plates with azithromycin concentrations as high as lg/ml (for isolates c and d). in elf, moxifloxacin auc - h/mic values were . for isolate a, and . for isolates b, c and d. levofloxacin auc - h/mic values were . for isolate a, and . for isolates b, c and d. azithromycin auc - h/mic values were . for isolate a; . for isolate b; and for isolates c and d. conclusion: if prevention of resistance depends more on the eradication of possible emerging mutants in pulmonary tissues than of the parental susceptible strain, moxifloxacin concentrations in elf may provide advantages over previous quinolones and macrolides in preventing clinical failures. objectives: to explore how antimicrobial pressure influences the evolution of streptococcus pneumoniae populations sharing the same ecological niche. methods: an in vitro computerized pharmacodynamic model simulating physiological concentrations obtained over h after mg o.d levofloxacin, mg b.i.d ciprofloxacin, and mg o.d azithromycin was used to investigate its effect on a mixed culture of five s. pneumoniae serotypes (s) as an approach to ecology of population dynamics. resistance patterns were: s was susceptible to study drugs, s was low-level macrolideresistant (efflux phenotype), s was high-level macrolideresistant (erm genotype), s v was low-level quinoloneresistant, and s was high-level quinolone-resistant. initial mixed inocula (time ) included similar percentages of each serotype. results: mean colony counts in antibiotic-free plates (whole pneumococcal population) increased (from to h) from log . to . in drug-free simulations (control), from log . to . in levofloxacin simulations, from log . to . in ciprofloxacin simulations, and from log . to . in azithromycin simulations. at h of control drug-free experiments, dominant strains were s v ( . %) and s ( . %) with marginal populations of s , s and s . azithromycin selected in a much higher extent the strain with low-level resistance to macrolides (s ) than the strain with high-level resistance (s ) (accounting for . % vs. . % of total population at h). ciprofloxacin selected in a higher extent low-level (s v) than high-level (s ) quinolone resistance ( . % vs. . %). levofloxacin decreased the proportion of the predominant s v in controls to . % (an intermediateresistant strain with mic = lg/ml), and unmasked the highlevel resistant strain (mic = lg/ml) up to . %. conclusion: strain distribution in antibiotic-free environment depends on bacterial fitness in mono-and multi-strain niches. the selective pressure of antimicrobial regimens eradicate some populations and unmask minor populations, thus redistributing the whole population. selective potential only for resistance phenotypes with very low prevalence (as high-level quinolone resistance) in the community should be preferred to that selecting more prevalent resistance phenotypes. re-evaluation of the role of broad-spectrum cephalosporins against staphylococci applying contemporary in vitro results and pharmacokinetic-pharmacodynamic principals h. sader, s.m. bhavnani, p.g. ambrose, r. jones (north liberty, us) objectives: to re-evaluate the current in vitro activity and to assess the pk-pd target attainment of cefepime (cpm), ceftriaxone (cro) and ceftazidime (caz) against staphylococcus spp. methods: the potency of cpm, cro and caz against staphylococci was accessed through the sentry antimicrobial surveillance program database, worldwide. during the - period , s. aureus (sa; % oxacillin [oxa]-susceptible [s]) and , coagulase-negative staphylococci (cons; % oxa-s) were s tested against cpm, cro, caz and numerous comparators by clsi broth microdilution methods. using volunteer pk data and a linear intermittent intravenous infusion model, and an animal-derived pk-pd target of % time above mic, expected probabilities of target attainment (pta) for cephems were evaluated using monte carlo simulation. pta were determined for the following dosing regimens: cpm gm q and q hours, caz gm q hours and cro gm q hours, each representing the most common dosing patterns applied clinically. cephem susceptibility (%s) was calculated based on the current clsi ( ) breakpoints (bkps) and also on bkps derived from a pta > %. results: against oxa-s sa, mic / values were (in mg/l): / for cpm, / for cro and / for caz, respectively; and against oxa-s cons mic / values were (in mg/l) . / for cpm, / for cro, and / for caz, respectively. the calculated %s of these cephems are summarized in the table: twenty year-old clsi bkps would rank the tested agents cpm ‡ cro > caz and by pk-pd pta cpm ‡ caz > cro. cpm has a potency advantage over caz ( -to -fold) and superiority at the usual dosing over cro ( . - . %) for oxa-s staphylococci. caz pk overcomes by-weight activity disadvantages, while a low proportion (< %) of active freedrug penalizes cro in the pta calculations. pta remained at > % to a bkp of mg/l for cpm ( gm q ) and caz and to a bkp of mg/l for cro. conclusions: regardless of applied bkp (clsi or pk-pd), cpm has the widest and more potent anti-staphylococcal activity among commonly used ''third-or fourth-generation'' cephems. when used at doses ‡ gm/day, cpm assures maximal coverage of oxa-s staphylococci whether using existing (clsi) or modified (pk/pd) bkps. cro should be used with caution. methods: the mic for all strains were determined by serial two-fold macrodilutions. an in vitro kinetic model was used to investigate the antibacterial efficacy of constant drug concentrations during hours. the selection of the doses of azithromycin tested in each bacterial strains was based on their mic values. bacterial counts were determined on appropriate agar plates using an adapted drop-plate method. twelve different pk/pd models were fitted and compared to the time-kill data by using non-linear regression. results: a simple pk-pd model was not sufficient to describe the pharmacodynamic effects for the four bacterial strains. appropriate models that gave good curve fits included a saturation term for the number of bacteria (nmax), delay terms ( -e-zt) for the initial bacterial growth phase and/or the onset of anti-infective activity as well as a hill factor (h) to capture the steepness of the concentration-response relationship. azithromycin had high potency against s. pneumoniae strains and m. catarrhalis while the potency of azithromycin against h. influenzae was poor. conclusions: the developed pk/pd models are suitable for describing the pharmacodynamics of azithromycin. applications of these pk-pd models will eventually provide a tool for rational antibiotic dosing decisions. objectives: optimal antimicrobial dosage regimens aim to achieve successful clinical outcomes without drug toxicity or emergence of bacterial resistance. for concentration dependent antibiotics, such as the fluoroquinolones, in humans a cmax:mic ratio of > is considered more important for efficacy and reduced selection of resistance than prolonged antibiotic concentrations just above the mic. fluoroquinolone resistance in zoonotic bacteria is a matter of public health concern, and fluoroquinolone treatment of poultry can rapidly select for bacteria with reduced fluoroquinolone susceptibility. in this study we compared basic pharmacokinetic parameters for the recommended dose of baytril (enrofloxacin) % oral solution in poultry to . x this dose for birds dosed by continuous water (standard) compared to pulsed water treatments and dosing by gavage.methods. for the pulsed versus continuous water treatments, groups of chickens received baytril % oral solution at (recommended) or ppm continuously in the water or at (recommended) or mg/kg pulsed in the water. for each group, three birds were killed at , , , , , and hours after start of antibiotic treatment and caecal contents, liver, lung and sera were taken and the concentration of fluoroquinolone determined by fluorescence hplc. for gavage treatment, dosing was at and mg/kg by crop intubation and four birds were killed in each group at , and hours after gavage; caecal contents, liver and sera were taken and analysed as above. basic pharmacokinetic parameters were determined using pk solutions software. results: the mean fluoroquinolone cmax in caecal contents (and sera) for gavage, pulsed water and continuous water treatments respectively was . ( . ), . ( . ) and . ( . ) mg/ml after the recommended dose and . ( . ), . ( . ) and . ( . ) mg/ml after . x the recommended dose. cmax of antibiotic in liver and lung was increased by the modified regimens in similar proportions to above. both pulsed water and gavage treatment not only resulted in higher cmax values, but also a faster rate of fluoroquinolone clearance than continuous water treatment ( figure ). dosing by gavage is not practical for thousands of chickens. however, pulsed dosing at . x the recommended dose can increase cmax values about fourfold and so could improve efficacy and reduce selection of resistance, compared to the current recommended treatment regime. objectives: nephrotoxicity is the major concern arising with the use of intravenous colistimethate sodium. methods: a prospective cohort study was performed at ''henry dunant'' hospital, a -bed tertiary care center in athens, greece. patients who received intravenous colistin for at least days for the treatment of multidrug resistant gram-negative bacterial infections were included in the study. the development of nephrotoxicity through evaluations of serum creatinine, blood urea, serum electrolytes, urinalysis, and creatinine and sodium in -hour urine collection during intravenous colistin therapy was the primary end point of the study. results: twenty-six patients were included in the study, of whom received colistimethate sodium (cms) for at least days and were evaluated further. the mean (± sd)/median daily dose, cumulative dose, and duration of treatment of intravenous cms was . (± . )/ million iu, . (± . )/ million iu, and . (± . )/ days (range - days), respectively. three of the evaluable patients ( . %) developed nephrotoxicity during the intravenous treatment with cms. the cumulative dose of the administered cms was statistically correlated with the difference between the end and start of cms treatment values of serum creatinine (r = . , p = . by spearman's test). a statistically but not clinically significant decrease of the mean baseline serum sodium concentration was observed between start and end of treatment [mean . (± . ) to . (± . ) mmol/l, p = . ]. no other toxic events were noted during the intravenous administration of colistimethate sodium. conclusion: although this is an evaluation of a small number of patients, our prospective study shows that nephrotoxicity was not commonly observed in this group of patients who received intravenous colistimethate sodium. however, caution should be taken to avoid the prolonged administration of the antibiotic. objectives: the objective of the present work is quantitative structure-activity relationship (qsar) analysis of antimicrobial activity of the -thiazolidone derivatives and consequent computational design of new antimicrobials. methods: for the achievement of the formulated objectives the qsar investigation has been carried out using computational chemistry approach based on simplex representation of molecular structure (sirms). on the framework of sirms it is possible to develop the molecular design of the new effective antimicrobials. results: systematic researches of relationship between antimicrobial activity (staphylococcus aureus -methicillin-sensitive (mssa) strain, pseudomonas aeruginosa -r and s strain, klebsiella pneumoniae, candida albicans s and Ñ itrobacter freundii) and a structure of about one hundred fifty compounds ( -thiazolidone derivatives and analogs). the elucidation of structure-activity relations allows predicting biological properties of such compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of their biological effect. completely adequate statistical partial least squares models (r = . - . , q = . - . ) have been obtained for all of the studied cultures. on the base of the first ones the molecular fragments both promoting and interfering the given antimicrobial activity have been determined. they give a possibility to realize the computer high throughput screening and molecular design of active compounds. the results of prognosis are verifying by the experimental investigations. also the influence of heterocycle system evolution on antimicrobial activity has been revealed. conclusion: qsar analysis of antimicrobial activity of -thiazolidone derivatives allows us to discover that the presence of naphthalene-substituted fragment (independently on its location in molecule) has distinctly negative influence on antimicrobial action. the requirements to molecular design have been formulated. for example, high active compounds must include -indolyl fragment. computational design of the new antimicrobials based on the substituted crown ethers activity of the row of substituted crown ethers and consequent molecular design of new antimicrobials. methods: the well-known hierarchic system of qsar models based on simplex representation of molecular structure has been used for the solution of the formulated problem, within the framework of which one it is possible to develop the molecular design of the new effective antimicrobial agents. results: we tried to conduct systematic researches of relationship between antimicrobial activity (planococcus citreus, streptococcus lactis, micrococcus lysodeiktious, staphylococcus aureus, streptococcus faecalis, bacillus subtillum) about two hundred fifty crowns ethers including aromatic, cyclic and heterocyclic etc. fragments and a structure of these molecules, in particular -macro cycle size, it dentacy, lipophily, nature of the substituents, and other factors. the elucidation of similar relations allows predicting biological properties of crown compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of biological effect of such kind of compounds. completely adequate qsar models (r = . - . , q = . - . ) have been obtained using partial least squares method for all of the studied cultures. on the base of the first ones the molecular fragments with positive or negative influence on the explored properties have been determined. they give a possibility to realize the virtual screening and molecular design of compounds with the high level of target activity. the results of prognosis are verifying by the experimental investigations. conclusion: qsar analysis of antimicrobial activity of crown ethers allows us to suppose the presence of two different mechanisms of their antimicrobial action. it is discovered that the presence of diphenyloxide and tert-butyl fragments promotes; diphenyl-sulphide and diamino-biphenyl -prevents the antimicrobial action. it is shown that the hexadenthal crown ethers containing aromatic fragments with a tert-butyl group are the most perspective antimicrobials. objectives: methionyl trna synthetase (mrs) catalyses the covalent attachment of methionine to its cognate trna. rep is a synthetic inhibitor of mrs with potent antibacterial activity against staphylococcus aureus including clinically-relevant resistant strains (mic equals . to . lg/ml). we determined the biochemical potency and mechanism of action of rep and related compounds with respect to s. aureus mrs enzymatic activity. we also evaluated the enzyme kinetic properties of mutated forms of s. aureus mrs. methods: the mets gene from s. aureus was expressed in e. coli and mrs was purified to near homogeneity by ammonium sulfate fractionation and anion exchange chromatography. aminoacylation of trnamet was measured using scintillation proximity assays (spa). the kinetics of the atp:ppi exchange were determined using thin layer chromatography (tlc). mutants of s. aureus mrs were selected by serial passage and spontaneous resistance in the presence of rep . results: rep exhibited strong inhibition of s. aureus mrs in the aminoacylation reaction, having an ic limited by the enzyme concentration. in order to estimate the true inhibition constant (ki), we utilized an atp:ppi exchange assay. rep showed potent inhibition of s. aureus mrs, with a ki of pm. related inhibitors were analysed, and a correlation was observed between the ki for mrs and the mic for s. aureus. rep was found to be competitive with methionine binding, but uncompetitive with atp binding (i.e., increasing the atp concentration resulted in tighter binding of rep ). the majority of analogs exhibited comparable mechanism of action; altered mechanism of action was observed with a subset of analogs. mutated s. aureus mrs variants (derived from strains with elevated mics) showed substantially weaker binding by rep . all of the mutated enzymes exhibited impaired trna aminoacylation activity, with defects ranging from reduced turnover rates to weaker affinities for one or more substrates. conclusions: rep is a potent inhibitor of s. aureus mrs. enzymatic potency of this class of inhibitors correlates with microbiological potency. mutations that confer resistance to rep result in functionally impaired mrs, encompassing a wide variety of enzymatic phenotypes. we report here the antibacterial and antifungal activity of newly synthesized and physico-chemically characterised thioureides of -( -chlorophenoxy)-benzoic acid. the new compounds were prepared in three stage. firstly, the -( -chlorophenoxymethyl)-benzoic acid was prepared by treating the phtalide with p-chlorophenol potassium salt in xylene. the second stage was the synthesis of -( -chlorophenoxymethyl)benzoyl chloride by treating the corresponding acid with thionyl chloride using anhydrous , -dichloroethane as solvent, followed in the third stage, by the treatment of the above-mentioned chloride with ammonium thiocyanate. the -( -chlorophenoxymethyl)-benzoyl isothiocyanate resulted after refluxing the reaction mixture in dry acetone. the new compounds were prepared by refluxing the isothiocyanate with primary aromatic amines in dry acetone. the obtained compounds have been characterized by their physical properties and their chemical structures were confirmed using the spectral analysis. the aim of this study was also to evaluate the in vitro antimicrobial activity of the new compounds. the in vitro antimicrobial testing was performed by binary microdilution method, in multi-well plates, in order to establish the minimal inhibitory concentration (mic), against gram-positive (listeria (l.) monocytogenes, staphylococcus (s.) aureus, bacillus (b.) subtilis), gram-negative (psedomonas (p.) aeruginosa, escherichia (e.) coli, salmonella (s.) enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, depending on the nature of the substituents and their position on the benzene ring, both concerning the microbial spectrum and the mic value. the mics values widely ranged between mcg/ml and mcg/ml. the most active proved to be n-[ -( -chlorophenoxymethyl)-benzoyl]-n'-( , -dichloro-phenyl)-thiourea and n-[ -( -chlorophenoxymethyl)-benzoyl]-n'-( -bromo-phenyl)-thiourea, showing a large spectrum of antimicrobial activity against enterobacterial strains (e. coli and s. enteritidis), l. monocytogenes, s. aureus and candida sp. all the tested compounds were highly active against s. aureus (mic = mcg/ml). four of the tested compounds exhibited antifungal activity (mic = - mcg/ml), and p. aeruginosa as well as b. subtilis were resistant to all tested compounds. in vitro antimicrobial activities of novel dianthraquinones produced by a marine streptomyces sp. against clinical staphylococcus aureus and enterococcus faecium isolates k.l. laplante, k. lor, a. socha, d.c. rowley (providence, north kingston, us) objectives: the escalation of antibiotic resistance among grampositive pathogens presents increasing treatment challenges and requires the development of new therapeutic agents. recently we discovered a new class of dianthraquinone antibiotics produced by a marine streptomycete. the inhibitory and bactericidal activity of four dianthraquinone secondary metabolites and four semi-synthetic derivatives were measured against clinical strains of vancomycin resistant e. faecium (vre), methicillin susceptible and methicillin resistant s. aureus (mssa and mrsa, respectively). two compounds, daq a and daq , were tested against an expanded panel of pathogens. methods: thirty-two clinical strains of vre (n = ), mssa (n = ) and mrsa (n = ) were obtained from patients at the veterans affairs medical center in providence, ri. mic's were performed using methodologies described by clsi. control isolates were atcc and atcc . the bactericidal activity of each antimicrobial agent was evaluated with time-kill experiments using randomly selected mssa (n = ), mrsa (n = ), and vre (n = ) isolates tested at times the respective mic. conclusions: the potent activities and unusual structures of the dianthraquinones tested here suggest that these may provide a new molecular scaffold for the development of novel antimicrobial agents. more biological testing is warranted to more fully explore the clinical potential of these antibiotics. efficacy of the novel antimicrobial peptide plectasin to staphylococci objective: the purpose of the investigation was to investigate the in vitro efficacy and kill kinetics of plectasin against staphylococcus aureus. plectasin is a newly discovered defensintype antimicrobial peptide found in the fungus pseudoplectania nigrella which showed activity against several gram-positive bacteria including drug resistant strains (mygind ph. et al. plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. nature ; : - ). methods: all experiments were determined according to clsi/ nccls guidelines. bactericidal activity was characterized by time kill experiments at and times the mic. staphylococcus aureus (s. aureus) atcc were used as the test organism and vancomycin was used for comparison. the kill kinetics and post antibiotic effect (pae) were evaluated by cfu determination. inoculum sizes ranging from e to e cells were used to test the inoculum effect. e cells were employed for determination of mutant prevention concentration (mpc) and the frequency of spontaneous resistance. results: plectasin is bactericidal as evidenced by kill kinetics showing a . log reduction in cfu/ml after hour of incubation and a reduction of . log cfu/ml after hours. this is superior compared to the activity of vancomycin. no inoculum effect was observed in the employed range of cells. the observed pae had a duration of hours and minutes. no spontaneously resistance mutation was observed among e cells of staphylococci and the mpc were determined to be times mic. conclusions: plectasin is a novel antimicrobial peptide that shows potent antimicrobial activity against gram-positive bacteria including drug-resistant organisms. the potent, excellent bactericidal activity in vitro, lack of cross-resistance to clinical used antibiotics, low spontaneously resistance mutation frequency and good pae properties, suggest that plectasin may have potential as a therapeutic agent against staphylococci. in vitro antimicrobial activity of the novel polymeric guanidine akacid plus Ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: cationic antimicrobials are widely used for disinfection within clinical settings. in the present study the bactericidal and fungicidal activity of akacid plus Ò , a novel polymeric compound of the cationic family of disinfectants, was evaluated against quality control strains of staphylococcus aureus, enterococcus hirae, escherichia coli, pseudomonas aeruginosa, candida albicans and aspergillus niger in comparison to chlorhexidine digluconate. methods: the in vitro activity of akacid plus Ò and chlorhexidine was determined by quantitative suspensions tests according to the european committee for standardization at concentrations of . - . % against bacterial strains and c. albicans and at concentrations of . - % against a. niger after exposure for , and min in the presence and absence of . % bovine albumin and dilution in distilled and hard water. results: in the basic quantitative suspension test akacid plus Ò destroyed all bacterial pathogens at a concentration of ‡ . % in £ min contact time. chlorhexidine was also highly active against s. aureus, e. coli and p. aeruginosa, but failed to eliminate e. hirae within min. under high organic burden, the bactericidal activity of both disinfectants was slightly reduced. akacid plus Ò showed fungicidal activity against c. albicans within - min and eliminated a. niger at a concentration of ‡ % in min contact time. chlorhexidine was fungicidal against c. albicans, but did not achieve biocidal activity against a. niger. conclusion: the novel polymeric guanidine akacid plus Ò when compared to chlorhexidine digluconate showed similar bactericidal activity against s. aureus, e. coli and p. aeruginosa and superior biocidal activity against e. hirae and a. niger. investigation of emergence of bacterial resistance to the novel antibacterial photodynamic agent xf- are novel, light activated antibacterial agents ( ) active against gram-positive bacteria, which have greater potency than antibiotics. the emergence of resistance to xf- has been investigated. methods: . mg/l of xf- was added to cells/ml of mrsa. after minutes incubation in the dark the unbound xf- was removed and the culture illuminated with . j/cm of light at nm and cfu analysis undertaken to determine the number of viable cells remaining. surviving clones of the treatment were cultured and subjected to further treatment. cycles were undertaken to determine whether the number of surviving cells increased, suggesting resistance build up to xf- . results: the survival of methicillin-resistant staphylococcus aureus (mrsa) (atcc baa- ) is expressed as log n /n, where n and n are the cfu of untreated and treated suspensions, respectively. the results demonstrate that no detectable resistance build up to the activity of xf- was seen after successive treatments. a low propensity for emergence of resistance is a valuable attribute for new anti-bacterial agents. xf- might be effectively employed in the clinical setting for prophylactic use to decolonise skin and nares and therapeutic use to treat infected wounds/ulcers. objectives: the xf drugs are novel, light activated antibacterial agents ( ) active against gram-positive bacteria which have superior potency to antibiotics but possess a low propensity to induce resistant bacterial strain emergence. a novel ex-vivo porcine skin model has been developed to test the antibacterial activity of xf- on the surface of skin. methods: x cells of methicillin-resistant staphylococcus aureus (mrsa) were inoculated onto a . cm area of ex-vivo porcine skin samples, immobilised in agar. after drying, solutions of xf- were applied and after minutes, the samples were illuminated for minutes with blue light ( nm) with various total light doses using a lumacare tm lc- m lamp. cfu analysis were undertaken to determine the number of viable cells remaining after treatment. controls of drug alone and light alone were included. results: using . mg/l of xf- , cfu analysis demonstrated that at a total light dose of j/cm , there was~ % kill of bacteria. at j/cm , there was . % kill of bacteria, and . % at j/cm and j/cm . at a total light dose of j/ cm , it was found that there was a < % kill by xf- at concentrations of . , . and . mg/l. at a concentration of . mg/l, there was a > . % kill. this kill did not significantly increase at . and . mg/l. conclusions: the results demonstrate that xf- has exceptional activity at low concentrations against mrsa on the surface of porcine skin. xf- and light are non-toxic to skin at therapeutic concentrations. work is in progress to clinically evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objectives: the rise of epidemic methicillin-resistant staphylococcus aureus (emrsa) and the emergence of mupirocin resistance means that it is essential to develop new therapies that cannot be readily overcome by microorganisms. the xf series of novel light activated antibacterial agents ( ) active against gram-positive bacteria addresses this issue and have superior levels of activity to antibiotics but with less likelihood of resistance emergence. the antibacterial activity of the xf drugs against emrsa has been investigated. methods: mic and mbc assays were used to investigate the antibacterial activity of xf- , a novel antimicrobial photodynamic agent against a range of staphylococcus aureus strains. a concentration range of - . mg/l was investigated. minutes of nm light activation ( j/cm ) was applied. light alone had no effect. results: conclusions: the results demonstrate that xf- has exceptionally low mic and mbc values against all of the s. aureus strains tested. the results also demonstrate that xf- is equally effective against mrsa and methicillin-sensitive staphylococcus aureus (mssa) indicating its mode of action is independent of antibiotic resistance. xf- may therefore be useful in prevention and treatment of emrsa. xf- is non-toxic to skin at prophylactic/therapeutic concentrations and has potential for the treatment of skin sepsis and the eradication of nasal and skin mrsa carriage. work is in progress to evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objective: nxl is a novel antibacterial currently in preclinical development. the mechanism of action is directed against topoisomerase, and the spectrum of activity is exclusively against gram positive organisms. the goal of the study was to characterise the activity and time/kill kinetics against common aerobic cocci in comparison to currently marketed molecules: linezolid (lin), vancomycin (van), quinupristin/dalfopristin (q/d) and moxifloxacin (mox). methods: (i) in vitro susceptibility tests: the strains used were from the culture collection of novexel and were of clinical origin. mics were determined by an agar dilution technique. mueller hinton agar medium was used, supplemented with % horse blood for group a streptococci (gas), group b streptococci and s. pneumoniae. overnight cultures were diluted to obtain the final inoculum of cfu/spot. the mic was the lowest concentration which inhibited all visual growth ( or less colonies were ignored). (ii) time/kill kinetics: experiments were performed against strains of s. aureus (n = ) and s. pneumoniae (n = ) in ml volumes of appropriate growth medium with initial inoculum of around cfu/ml of logarithmically growing culture. timed samples over a hour period were enumerated using a spiral plating method. nxl was compared to linezolid and vancomycin and the concentrations tested were , and -fold the mic for both species. results: (i) the mic s of nxl versus comparators are shown in the table. (ii) time kill experiments showed that nxl was bactericidal against s. aureus, including methicillin resistant strains (> log reduction within - hours) compared to a slowly bactericidal effect for vancomycin ( hours). nxl and vancomycin were both bactericidal against s. pneumoniae within - hours. linezolid was bacteriostatic against all strains tested. conclusion: nxl exhibits bactericidal activity against common gram positive cocci, including strains which exhibit resistance to methicillin, vancomycin and fluoroquinolones. nxl warrants further investigation. objectives: the aim of this study was to identify bacterial proteins as targets of the endogenous antiseptic n-chlorotaurine (nct), which is a promising microbicidal agent for topical treatment of infections. in addition, a combination of nct with ammonium chloride which enhances the microbicidal activity significantly was investigated. methods: escherichia coli and staphylococcus aureus were treated with nct and nct plus ammonium chloride for different incubation times between and min -a period where killing takes place. to find out protein changes, d-page of bacterial proteins followed by mass spectrometry was performed. results: incubation in % nct revealed a change of the charge and a separation of numerous proteins into a series of spots with a different isoelectric point. moreover, in e. coli heat shock protein appeared, while ribosome releasing factor, d-ribose periplasmic binding protein, and malonyl-coa transacylase spots decreased. in s. aureus, enolase and a translation elongation factor decreased. these changes appeared more rapidly in the presence of ammonium chloride, which can be explained by formation of the more lipophilic and microbicidal monochloramine. molecular mechanisms of attack comprised mainly oxidation of thio and amino groups as confirmed with model peptides. conclusion: these results fit very well to previous preclinical and clinical findings. they indicate both surface attack and penetration of oxidation capacity into the bacteria and destruction of essential proteins by nct and nct plus ammonium chloride, respectively. objectives: ceftobiprole is a new extended-spectrum cephalosporin with activity against methicillin-susceptible and methicillin-resistant staphylococci, as well as against most enterobacteriaceae. in this study the anti-staphylococcal activity of ceftobiprole is reported from a set of isolates from a recent clinical trial. methods: consecutive clinical isolates of staphylococci from patients enrolled in a multicentre clinical trial involving complicated skin infections were examined for their susceptibility to ceftobiprole and selected anti-gram-positive agents. mics were determined using clsi methodology. results: among these isolates, staphylococcus aureus and coagulase-negative staphylococci (cons) were identified. the percentages of methicillin-resistant strains were % for s. aureus and % for cons. all strains (except one cons with a linezolid mic of mg/l) were susceptible to vancomycin and linezolid, with mics < mg/l.against methicillin-susceptible s. aureus, ceftobiprole mic and mic values were . and . mg/l, respectively, and against methicillin-resistant s. aureus, ceftobiprole mic and mic values were . and mg/l, respectively. ceftobiprole mics ranged from £ . to mg/l against methicillin-susceptible-cons (ms-cons) and methods: consecutive, non-duplicate bacterial isolates ( , strains) acquired from patients with bloodstream, respiratory, and skin and skin structure infections both nosocomial and community acquired were submitted from > medical centres in europe, the americas and the asia-pacific region. all isolates were tested using clsi/nccls broth microdilution methods against grn, the currently marketed fluoroquinolones (fq) including cipro, levofloxacin (levo), gatifloxacin (gati) and representative comparator agents. oxa-and cipro-s and -r subsets were included. a grn-s breakpoint of £ . mg/l was applied for comparative purposes only and was based upon the mic population distributions of strains that included quinoloneresistance determining region (qrdr) mutations. results: potency for grn and comparator fqs tested against sa: (see table) . key resistance patterns (%) among this sa collection included oxa ( . ), cipro ( . ), erythromycin ( . ), clindamycin ( . ), tetracycline ( . ), and trimethoprim/ sulfamethoxazole ( . %); gram-positive-targeted comparator including vancomycin, linezolid, daptomycin and quinupristin/dalfopristin all remained > % s. compared with currently marketed fqs when tested against all sa, grn was -to -fold more active (mic , £ . vs. . or . mg/ l). against both oxa-s and -r sa, grn displayed markedly enhanced potency compared with cipro and levo ( ‡ -fold), and gati ( -to -fold). among cipro-r isolates, grn also maintained ‡ -fold greater potency (mic , vs. ‡ mg/l) although overall s for all fqs was - %. compared to the fq agents tested against sa, grn was the most potent agent and maintained the broadest coverage against oxa-and cipro-r strains even when applying a very conservative epidemiologic breakpoint. when a fq is indicated for staphylococcal coverage, this des-f( ) quinolone may represent a superior alternative among fq class agents, while minimizing selection of resistance. objective: to assess the garenoxacin (grn) potency against a vast number of international respiratory tract infection (rti) pathogens, especially versus phenotypic (high mic) or genotypic (sequence change) qrdr mutants. a total of , isolates from continents were analysed ( ) ( ) ( ) ( ) ( ) ( ) ( ) table) conclusions: grn maintains clinically usable activity (mic, £ mg/l) against important community-acquired rti pathogens having r to presently marketed fluoroquinolones and against those isolates with documented qrdr mutations. continued development of this novel des-f( ) quinolone agent appears desirable. in vitro activity of garenoxacin tested against ciprofloxacin-susceptible and -resistant enterobacteriaceae and acinetobacter spp. strains collected worldwide by the sentry antimicrobial surveillance program ( ) ( ) h. sader, t. fritsche, p. strabala, r. jones (north liberty, us) objective: to evaluate the contemporary activity of garenoxacin (grn) against ciprofloxacin (cipro)-susceptible (s) and cipro-resistant (r) enterobacteriaceae (ent) and acinetobacter spp. (asp). unlike recently marketed fluoroquinolones (fq), grn, a des-f( ) quinolone lacks the c- fluorine. methods: a total of , isolates ( , ent and asp) were consecutively collected from > medical centres from bloodstream, respiratory, urinary and skin and soft tissue infections and tested by reference broth microdilution methods according to clsi/nccls methods and interpretative criteria. a grn s breakpoint of £ mg/l was applied for comparison purposes only. results: the results of the major organism groups tested: (see table) . grn showed excellent activity against this large collection of ent (mic , . mg/l) and . % of isolates were inhibited at £ mg/l. objectives: garenoxacin (grn) is a novel, broad-spectrum des-f( )-quinolone with activity against gram-negative and grampositive aerobes and anaerobes including quinolone-resistant staphylococcus aureus. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in complicated skin and skin structure infections (csssi). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn ( mg iv to po qd) or piperacillin/tazobactam ( . g iv q h) with transition to po amoxicillin/clavulanate ( mg po q h). in the second study, subjects received grn ( mg po qd) or ciprofloxacin/metronidazole ( mg q h/ mg q h). all antimicrobials were administered for to days. subjects were adults ( ‡ y) newly hospitalized or ambulatory outpatients with evidence of csssi who did not have underlying osteomyelitis. microbiologic efficacy was determined to days post-therapy. results: a total of subjects were microbiologically evaluable (grn, n = ; comparators, n = ). the disease diagnosis was similar between grn and comparators and included infected pressure sore ( % vs %), infected diabetic foot ulcer ( % vs %), major abscess ( % vs %), or postsurgical wound infection ( % vs %). the majority of common skin pathogens were eradicated by grn background: acute bacterial sinusitis (abs) is a common infection world-wide, with many patients having an associated an allergic component/history. however the role of antibacterials in these patients (pts) has not been examined. as some fluoroquinolones (fq) have an in-vitro immunomodulatory effect (ie) the clinical efficacy of gem was compared other agents in abs pts with or without allergic rhinitis (ar). methods: phase clinical trials were pooled and pts where allergic rhinitis was identified ( pts) were compared with pts not reporting ar ( pts). clinical response (success or failure) at end of therapy (eot) & at follow-up (fu, approx. - weeks after treatment) was studied. comparators (cmp) were cefuroxime (cef) and trovafloxacin (tro). results: % success based on clinical outcome at eot and fu for ar and non ar pts are shown in the table. for all treatments eot success was high for the non ar pts, but at fu this was reduced, especially with both fqs. in contrast, gem retained a high clinical success rate in pts with ar unlike cef or tro. conclusion: gem has been shown to be very efficacious in a sub group of problematic abs pts. this advantage may be due to the high antibacterial activity of gem vs key abs pathogens and/or a stimulatory ie. both being important with pts having decreased local immune defences. these data also show that not all fluoroquinolones have immuno-stimulatory properties. garenoxacin efficacy against multidrug-resistant streptococcus pneumoniae: retrospective analysis of community-acquired pneumoniae isolates obtained from nine phase ii and iii clinical studies ( ) ( ) ( ) ( ) ( ) t. black, h. waskin, r. hare (kenilworth, us) objective: garenoxacin (grn) is a novel, des-f( )-quinolone with excellent activity against s. pneumoniae, one of the most common pathogens causing community-acquired pneumoniae (cap). the incidence of infections caused by antibiotic-resistant isolates of streptococcus pneumoniae is on the increase, therefore information regarding the activity of new anti-infective drugs against populations of s. pneumoniae that are multi-drug resistant (mdr) is critical. mdr s. pneumoniae (mdrsp) includes isolates previously known as prsp (penicillinresistant s. pneumoniae), as well as strains resistant to two or more of the following antibiotics: second-generation cephalosporins, macrolides, tetracyclines, and trimethoprim/ sulfamethoxazole. methods: pretreatment sputum and blood isolates collected worldwide during grn phase / clinical cap trials ( ) ( ) ( ) ( ) ( ) were retrospectively analysed for the mdrsp phenotype. of the s. pneumoniae isolates originally identified, from subjects were subjected to secondary mdr susceptibility testing by central laboratories. confirmed mdrsp isolates were matched to individual subjects to assess clinical and microbiological outcomes for mdrsp-infections treated with grn. results: expanded susceptibility testing identified / mdrsp isolates from unique subjects. the lowest mic and mic values for mdrsp isolates tested against a panel of representative drugs were observed for grn (table ; . lg/ ml and . lg/ml, respectively). the incidence of resistance to the five classes of drugs was %, %, %, % and % for penicillin, nd generation cep., macrolides, tetracycline and tri/sulf, respectively. no isolates were resistant to grn using a proposed susceptibility breakpoint value of £ lg/ml. thirtyfive percent, %, %, % and % of isolates were resistant to , , , and drug classes, respectively. the worldwide incidence of mdrsp was % with an equivalent geographic distribution of %, % and % among north america, europe and the rest of world. overall, grn provided clinical and bacteriological success for / ( %) cap evaluable subjects with mdr infection, which was similar to clinical success for evaluable subjects with non-mdrsp cap infections / ( %). conclusions: these data demonstrate the ability of grn to successfully eradicate mdrsp associated with cap. . per cent success is shown in the table (ab, antibiotics, copd, chronic bronchitis and obstructive lung disease, hd, heart disease). results: although gemifloxacin showed lower % success than comparator against cap patients with no defined risk factor, gemifloxacin was considerably more successful than comparator against patients associated with risk factors, especially diabetic patients where comparator success was low. this advantage was often more prominent at fu than at eot. patients with other comorbidities such as renal failure or malignancy were not recruited in sufficient number for analysis. conclusions: these data support the use of gemifloxacin in the treatment of cap, especially where the patient has recognised idsa risk factors. microbiologic efficacy of garenoxacin vs. comparators against common pathogens associated with community-acquired pneumonia objectives: garenoxacin (grn) a novel, broad-spectrum des-f( )-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. grn has dual sites of inhibition (dna gyrase and topoisomerase iv) and may be less likely to promote resistance. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in community-acquired pneumonia (cap). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn ( mg po qd for d) or amoxicillin/clavulanate (a/c; mg po q h for - d). in the second study, subjects received grn ( mg po qd for - d) or levofloxacin (lev; mg po qd for - d). adults ( years of age or older) were enrolled with clinical and radiologic evidence of cap [new infiltrate(s) on chest radiograph and fever, leukocytosis, cough, chest pain, auscultatory findings, or sputum production]. the majority of subjects were fine class i/ii in both studies. bacteriologic eradication was assessed to days post therapy. results: a total of treated subjects had pretreatment pathogens (grn, n = ; comparators, n = ) . the overall eradication rate in all treated subjects was % ( / ) for grn and % ( / ) for the comparators. eradication rates for s pneumoniae were % ( / ) for garenoxacin and % ( / ) for the comparators. eradication of s pneumoniae was % and % for a/c and lev, respectively. in strains with reduced susceptibility to penicillin eradication rates were % ( / ) vs % ( / ) in favour of grn. eradication rates for h. influenzae were % ( / ) and % ( / ) for grn and comparators, respectively. lev eradicated % of h. influenzae isolates and a/c eradicated % of the strains isolated. there were very few isolates ( ) of moraxella catarrhalis in the studies. in study grn was % effective against strains of m. catarrhalis and in the other a/c was % effective against the strain isolated. grn eradicated % ( / ) of the staphylococcus aureus isolates vs % ( / ) for the comparators. conclusions: grn was highly active against pathogens commonly associated with cap including drug-resistant strains of s pneumoniae and represents an effective therapeutic option for this patient population. objectives: garenoxacin (grn) a novel, broad-spectrum des-f( )-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. there is a growing problem of resistance in strains of s pneumoniae, with multi-drug-resistant s pneumoniae (mdrsp) becoming increasingly more common. the objective of this study was to evaluate the clinical and microbiologic efficacy of grn in the treatment of communityacquired pneumonia (cap) caused by mdrsp. methods: this was a multinational, open-label, noncomparative study. subjects were adults ( ‡ and < y) with clinical (clinical signs, sputum production), radiologic (new infiltrates on chest radiograph), or microbiologic (predominance of gram-positive cocci in pairs on sputum gram-stain or a positive blood culture for s. pneumoniae) evidence of cap caused by s. pneumoniae. subjects received grn mg po qd or grn mg iv with transition to mg po qd for to days. clinical and microbiologic responses were determined at a test-of-cure visit to days posttherapy. results: a total of subjects were enrolled. of these, ( po only, iv to po) were clinically and microbiologically evaluable. clinical and microbiologic success rates were % ( / ) and % ( / ), respectively. clinical success rates were % ( / ) and % ( / ) for po and iv to po, respectively. documented s. pneumoniae bacteremia was present in % (n = ) of subjects with a clinical success rate of %. among evaluable subjects, resistance rates for s. pneumoniae were penicillin %, second-generation cephalosporin %, macrolides %, tetracyclines %, and trimethoprim/ sulfamethoxazole %. twelve evaluable subjects had pneumonia caused by mdrsp. clinical success rate was % ( / ) in subjects with mdrsp and % ( / ) in non-mdrsp subjects. clinical success of grn for strains resistant to , , , or antimicrobial drug classes, were % ( / ), % ( / ), % ( / ), and % ( / ), respectively. microbiologic success was % ( / ) and % ( / ) for mdrsp and non-mdrsp (susceptible or resistant to class) strains, respectively. grn was generally well tolerated with drug-related adverse events (ae) reported in % ( / ; po) and % ( / ; iv to po) of subjects. conclusions: grn (po or iv to po) is an effective treatment for cap caused by mdrsp and non-mdrsp. grn is well tolerated. in vitro bactericidal activity of daptomycin against staphylococcus aureus and enterococcus spp.: comparison with vancomycin, teicoplanin and linezolid h. drugeon, m. juvin (nantes, fr) objectives: the aim of this study was to evaluate the bactericidal activity (by killing kinetics) of daptomycin (dap) against staphylococcus aureus (sa) clinical isolates with different teicoplanin mics and against enterococcus faecalis (efl) and e. faecium (efm) with different mechanisms of glycopeptide resistance. dap has been compared with teicoplanin (tei), vancomycin (van) and linezolid (lin). methods: sa strains ( mssa and mrsa) with tei mic distributed from . to mg/l, enterococcus ( efl and efm) with glycopeptide phenotypes [s, r-vana, r-vanb] were studied using a killing curve method. antibiotic concentrations were used from mg/l to mg/l in two fold dilutions. surviving bacteria were counted at t , t ', t , t , t , t and t hours using agar plates with inhibitors to prevent antibiotic carry-over. antibiotics tested were daptomycin (dap), teicoplanin (tei), vancomycin (van) and linezolid (lin). results: all the sa isolates were susceptible to dap (mic = . - mg/l), to lin (mic = - mg/l), to van (mic = - mg/l) regardless of susceptibility to methicillin. dap showed the same strong concentration dependent bactericidal activity with mssa and mrsa: at t ' bactericidal activity (ba) (decrease of log cfu/ml) was observed with - mg/l of dap; at t hours, - mg/l of dap was sufficient and at t hours, ba was obtained with mg/l of dap. the other antibiotics showed a time dependent bactericidal activity but ba was observed only with long exposure ( ‡ hours) and with high concentrations. all the enterococcus isolates were susceptible to dap (mic = - mg/ l) and to lin (mic = - mg/l) regardless of the resistance to glycopeptides. ba of dap was also concentration dependent. ba was obtained with - mg/l after hours of contact and with mg/l after hours of contact for efl. ba was observed with - mg/l after hours of contact and with - mg/l after hours of contact for efm. the other antibiotics had a time dependant activity but didn't show bactericidal activity with concentrations mg/l. conclusion: the bactericidal activity of daptomycin was very strong, concentration dependent, and not influenced by the level or mechanism of glycopeptide resistance the bactericidal activity of linezolid was time dependent and observed only with the highest concentration and the bactericidal activity of vancomycin and teicoplanin was time dependent but was influenced by the mechanism of glycopeptide resistance. objectives: telavancin (tlv) is a bactericidal lipoglycopeptide with multiple mechanisms of action that is in phase clinical trials for the treatment of complicated skin and skin structure infections and hospital-acquired pneumonia with a focus on infections due to methicillin-resistant staphylococcus aureus (mrsa). this study evaluated and compared the antibacterial activity of tlv with that of other antibacterial agents against recent gram-positive clinical isolates from germany. methods: a total of aerobic gram-positive bacterial strains recently collected were included. antibiotics tested were tlv, vancomycin (van), teicoplanin, penicillin, oxacillin, ampicillin, cefuroxime, ceftriaxone, daptomycin (dap), linezolid (lzd), quinupristin-dalfopristin, clindamycin, ciprofloxacin, levofloxacin, gentamicin, streptomycin, erythromycin, telithromycin, co-trimoxazole and tetracycline. mics were determined by the broth microdilution procedure according to the guidelines of the clsi. results: tlv exhibited potent activity against all grampositive bacteria including resistant isolates such as mrsa, van-resistant enterococci, pneumococci (including multiple resistant strains with various antibiotic resistance phenotypes) and other streptococcal species. tlv showed excellent in vitro activity against the species irrespective of the antibiotic phenotype tested. for methicillin-susceptible s. aureus (mssa, n = ) and mrsa (n = ) mic of tlv for both phenotypes were . mg/l. for coagulase-negative staphylococci (n = , incl. msse, mrse, mssh, mrsh and others) mic s were . or mg/l. mic s of tlv for enterococcus faecalis (n = ) and e. faecium (n = ) were and mg/l, respectively. for van-resistant strains of e. faecalis (n = ) or e. faecium (n = ) mics for tlv ranged from . to mg/l. against streptococcus pneumoniae (n = ) tlv mics ranged from £ . to . mg/l. all streptococcus pyogenes, streptococcus agalactiae and all viridans group streptococci (n = ) had mics of £ . mg/l. conclusion: based on mic , tlv was more potent than van, dap or lzd against staphylococci, streptococci and e. faecalis. it was superior to dap and lzd against e. faecium and at least as active as dap or lzd against most van-resistant enterococci. tlv appears to be a promising new antimicrobial agent for the treatment of infections caused by gram-positive organisms including multiply resistant isolates. the extent of protein binding (pb) of dap is still under investigation and data available so far indicate pb of either % or %. therefore we tested two fscs: . (corresponding to % pb) and . (corresponding to % pb). the activity of dap was determined in mueller-hinton broth supplemented with mg/l calcium. viability counts were performed at . , . , , , , and h. one methicillin-susceptible staphylococcus aureus (mssa), two methicillin-resistant s. aureus (mrsa), one vancomycin-susceptible (van-s) and one van-resistant (van-r) enterococcus faecalis, one van-s and one van-r enterococcus faecium were tested. bactericidal activity was defined as > . % killing during incubation. results: dap was bactericidal at concentrations of . mg/l and . mg/l in all seven strains. the concentration of . mg/l was bactericidal against the two mrsa and against the van-s e. faecium. in the other four strains the maximum reduction of initial inoculum ranged from . to . log cfu/ml. in six strains a bactericidal effect at . mg/l and . mg/l of dap, respectively, occurred between minutes and h and after h in the van-s e. faecalis. van at . mg/l or . mg/l was bactericidal in only two strains after h ( mssa, mrsa). against the other five strains, van was bacteriostatic with maximum reduction of initial inoculum between . and . log cfu/ml at mg/l after h, respectively. both tpl and lzd were consistently bacteriostatic against the test strains. conclusion: dap at psc of . mg/l as well as at fsc of . mg/l showed a pronounced bactericidal effect within h in / strains. van was bactericidal in only / strains after h. compared to van bacterial killing by dap was very rapid. tpl and lzd were bacteriostatic only. the effect of human serum on the bactericidal activity of daptomycin and comparators against staphylococcus aureus and enterococcus spp. background: daptomycin is a new cyclic lipopeptide antibiotic that shows rapid bactericidal activity and has high protein binding when assessed by standard methodology. this study investigated the bactericidal activity of daptomycin and the effect of protein binding by the addition of % human serum (hs). methods: exponentially-growing methicillin-susceptible andresistant s. aureus (mssa, mrsa) and vancomycin-susceptible enterococcus faecium (vse) and -resistant enterococcus faecium (vre) (ca. cfu/ml) were exposed to daptomycin (dap), vancomycin (van), teicoplanin (tei), piperacillin-tazobactam (ptz) or linezolid (lzd) at peak (p) and trough (t) serum concentrations in mueller hinton broth supplemented with ca + to mg/l with or without hs. viable count was determined at . , . , , & h. plots were made of log reduction in viable count over time and the area-under-thecurve measured to calculate bactericidal indices (bis) from these plots (j antimicrob chemother , : - ). results: daptomycin reduced viable count of mssa & mrsa by approx. logs or more within . h and vse or vre within h at p. other agents either did not achieve this or required h to do so (not shown). bi data are shown below (>represents kill beyond the limit of detection). hs had little effect on dap kill, except against the vre at t. nevertheless, dap at t against vre was more bactericidal than any other antibacterial except dap at p. conclusions: dap was the most bactericidal agent tested as measured either by bi or rate of kill. dap at p reduced mssa and mrsa to below detection within min. the effect of hs was minimal which suggests that protein binding is either weak or highly reversible. these data support the use of dap in the treatment of infections caused by these organisms. daptomycin activity against multi-resistant staphylococcus haemolyticus bloodstream isolates from severe infections objectives: daptomycin, a new cyclic lipopeptide with activity against multidrug-resistant gram-positive pathogens including mrsa, is approved for use in cssst infections (us-fda) and is being reviewed by emea for approval in eu member countries. the rapid bactericidal activity of daptomycin, due to its unique mechanism of action, makes it an attractive antibiotic for serious gram-positive infections. the study was performed: (i) to evaluate the activity of daptomycin and other drugs against multi-resistant clinically relevant staphylococcus haemolyticus (mrsh), isolated from bloodstream infections in various hospitals in italy (ii) to determine epidemiologic and genetic correlation among strains, and (iii) to characterize the sccmec dna of these strains. methods: the mrsh strains were tested against a panel of antimicrobial agents, by broth microdilution method performed according to clsi (clinical laboratory standards institute) guidelines, including supplementation of mg/l calcium for daptomycin. moreover, phenotypic tests and antibiotic susceptibility profiling were carried out and the results compared with molecular typing analysis by using smai-pfge fingerprints and pcr to characterize the mec-complex. results: all isolates were resistant to erythromycin, gentamicin, ciprofloxacin, strains showed reduced susceptibility to vancomycin (mics mg/l), strains were resistant to cotrimoxazole, strains to clindamycin, strains to chloramphenicol and strains to tetracycline. almost all isolates were inhibited by £ mg/l of daptomycin, and only four strains exhibited a mic value of mg/l. pfge analyses showed the existence of at least two multi-resistant s. haemolyticus clones widespread in different hospitals. methicillin-resistance was correlated to the presence of the meca and preliminary results regarding the genetic element carrying the gene, showed an organization of the mec-complex of class a and class c. conclusions: our results suggest that daptomycin has excellent activity against multiresistant mr s. haemolyticus isolates, which represent a serious threat in catheter-related bloodstream infections. furthermore, the emergence of s. haemolyticus exhibiting reduced susceptibility to vancomycin is of particular concern, probably due to the common use of vancomycin as initial therapy for such infections. moreover, the use of additional molecular techniques to fingerprint isolates makes this study of clinically important cons more accurate. objectives: ceftobiprole is a new cephalosporin with a broad spectrum of action including methicillin-resistant staphylococci (mrs) as well as many other gram-positive and gram-negative pathogenic bacteria. this study investigates the structural basis for the good activity against mrs. methods: the primary beta-lactam resistance determinant of mrs, penicillin-binding protein pbp ' (or a) has been cloned and expressed as a soluble form in which the amino-terminal residues forming a membrane-anchor have been deleted. the soluble form has been crystallized and the structure of the complex formed after soaking crystals in a solution containing ceftobiprole has been determined at . angstrom resolution. additional data on the structure of the ceftobiprole-pbp ' complex formed in solution has been obtained using spectroscopic methods such as uv-circular dichroism. results: ceftobiprole reacts rapidly with pbp ' to form a stable acyl-enzyme complex. the ceftobiprole moiety is positioned deep within the active site of the acyl-enzyme complex formed with pbp ', where it forms several hydrogen bonds and hydrophobic interactions. in particular, the -aminothiadiazolylhyroxyiminoacetyl side chain of ceftobiprole sits more deeply within the side-chain binding pocket of pbp ' than does the -acylamino side chain of nitrocefin in the previously determined complex structure. the additional interactions probably add to the enhanced stability of the acyl-enzyme complex formed with ceftobiprole, compared to complexes formed with other betalactams that are inactive against mrs. significant structural rearrangements between apo-enzyme and acyl-enzyme are evident in the crystal structure and in solution. conclusion: ceftobiprole readily forms a stable inhibitory acylenzyme complex with the pbp ', the beta-lactam resistance determinant of mrs. this, together with potent inhibition of the normal complement of beta-lactam sensitive penicillin-binding proteins, accounts for its excellent activity against staphylococci and probably accounts for the low rates of resistance development observed in experimental conditions. incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in a french hospital (november -april c. morate, a. charron, c. bebear, j. maugein (bordeaux, fr) staphylococcus aureus are a major cause of nosocomial infections around the world. glycopeptides remain the drug of choice for severe infections caused by mrsa. however, after the emergence of vancomycin resistance in enterococcus and in the coagulase negative staphylococcus, strains of staphylococcus aureus with reduced susceptibility to glycopeptides (gisa) have been reported in different countries like japan, france, spain, the uk and the united states. the aim of our study was to determine the proportion of vancomycin resistance in clinical s. aureus isolates in a french university hospital, between november and april , then we wanted to define if there was an epidemic clone and study the clinical impact of these gisa strains. the protocol of detection was, first, a screening test on bhi agar containing mg/l of teicoplanin, then, the vancomycin and teicoplanin mics were determined by the method of etest with an inoculum of . mcf on the selected strains. finally, the isolates with mic of the teicoplanin ‡ mg/ l and mic of the vancomycin ‡ mg/l or mic of the teicoplanin ‡ mg/l and mic of the vancomycin £ mg/l were studied on population analysis. after that, pulsed-field gel electrophoresis (pfge) was performed on the different isolates and the pulsotypes were compared. from november to april , s. aureus isolates were collected from patients and screened for glycopeptide resistance on an initial agar screening test containing mg/l of teicoplanin. the teicoplanin mic was > mg/l for isolates ( . %) from patients and these strains were selected for the determination of the mics by ''macromethod'' etest. by this technique, strains were selected and studied by population analysis. all the profiles were compared to the reference strain mu profile. this procedure detected isolates (from patients) with heterogeneous reduced susceptibility to glycopeptides (hgisa). so the incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in our hospital was found to be . %. four strains were resistant to methicillin and were also resistant to gentamicin. the diversity of the strains was confirmed by pfge: there was not an epidemic clone in the hospital. the clinical history showed that patients had received a prior treatment with vancomycin, and that patients had a failure in treatment: of them had cystic fibrosis. objectives: enterococcus faecalis was the most prevalent organism ( . %) involved in enterococcal infections at tehran hospitals followed by e. faecium ( . %). due to widespread expansion of aminoglycoside modifiying enzymes (agmes) genes, the rate of resistance to high level concentration of aminoglycosides has increased in these years. the rate of high level gentamicin resistant isolates of enterococci (hlgr) is high in iran ( %). the aim of this study was to determine the genes encoding resistance to aminoglycosides among enterococci in iran. methods: disks containing lg gentamicin were used to detect hlgr isolates. primers specific for aac ( ') aph ( ") and aph ( ') iiia genes were used in pcr to possibly detect acetyltransferases and phosphotransferas, the common agmes among isolates of enetrococci. theses isolates were resistance to different concentration of gentamicin. results: a bp region of the aac ( ')-aph ( ") gene was amplified by pcr in % hlgr isolates as well as in % of low level getamicin resistant isolates (llgr). moreover the gene aph ( ') iiia was detected in . % and % of isolates of hlgr and llgr respectively. differences between isolates of e. faecalis and e. faecium were found in term of prevalence of aph ( ') iiia gene. conclusion: the bifunctional enzyme aac ( ')-aph ( ") is the main cause of resistance to high concentration of aminoglycosides in our collection of enterococci. this enzyme confers resistance to all clinically useful aminoglycosides with the exception of streptomycin. in the absence of aac ( ')-aph ( "), gentamicin could be used in combination therapy. prevalence and genetic analysis of methicillinresistant staphylococcus aureus expressing highlevel and low-level mupirocin resistance m. kural, t. us, y. akgun (eskisehir, tr) objectives: to investigate the genetic location of mupa gene which encoded mupirocin resistance and characterize mupirocin-resistant methicillin resistant staphylococcus aureus (mrsa) isolated from patients in a turkish university hospital by polymerase chain reaction (pcr) and plasmid analysis. methods: methicillin and mupirocin resistance were detected by disk diffusion (oxoid, uk). the etest (ab biodisk, sweden) was performed to determine mupirocin minimum inhibitory concentrations (mics). the presence of mupa and meca were detected by pcr using specific primers. plasmid analysis were used to study the genetic location of mupa gene. results: a total of ( . %) mrsa strains were identified by disk diffusion in s. aureus. of the clinical isolates ( . %) were from wound, ( . %) from blood, ( . %) from catheter, ( . %) from lower respirator tract (bronchoalveolar lavage, pleural fluid and transtracheal aspirates), ( . %) from sputum, ( . %) from urine and ( . %) from other (serebrospinal fluid, parasynthesis fluid, peritoneal fluid, and bone marrow) clinical samples. among the mrsa isolates, mupirocin resistance was detected in ( . %) strains with disk diffusion and etest. of the mupirosin-resistant isolates ( . %) expressed high-level (muh) and ( %) expressed lowlevel (mul) mupirocin resistance. all isolates were vancomycin, teicoplannin susceptible and chloramphenicol resistant with disk diffusion. isolates with high-level and low level mupirocin resistance due to the mupa gene were also detected with pcr. plasmids were detected in all of the isolates. however only the muh isolates contained a kb plasmid that encoded highlevel resistance. all of the isolates contained a . kb plasmid and resistant to chloramphenicol. conclusion: our results indicated that the mrsa clones detected in the hospital had acquired a high-level mupirocin resistant plasmid. the past observations and recent studies suggested that the numbers of such strians have increased following extensive topical use of mupirocin. the usage of mupirocin in our hospital has not yet been systematically implemented. it is frequently prescribed for the treatment of staphylococcal skin infections and less to eliminate nasal carriage of mrsa. in our hospital we should be aware of the possible emergence and increase of mupirocin highly resistant mrsa strains in the future so that we should be considered when using mupirocin to control the spread of mrsa in hospital. emergence and spread of acquired fusidic acid resistance in staphylococcus aureus objectives: a major route to fusidic acid resistance (fusr) in s. aureus involves acquisition of fusb, a resistance determinant first clinical microbiology and infection, volume , supplement , identified on plasmid pub . here we show that (i) the two currently-circulating major clones of fusr s. aureus identified to date have acquired fusb from pub (or from the same ancestral source as pub ), and (ii) that the pub -encoded fusb is only one of at least three lineages of this protein that appear to have evolved since recruitment of the original, ancestral fusb to the staphylococci. methods: plasmid purification, dna sequencing, pcr amplification, and cloning in s. aureus rn using shuttlevector pcu , were all performed using established methods. antibiotic susceptibility testing was performed by agar dilution. results: the epidemic european fusidic acid-resistant impetigo clone (eefic) and community-acquired mrsa strain st have been shown to carry chromosomal and plasmid-encoded fusb, respectively. dna sequencing of fusb and its surrounding regions in these backgrounds revealed that they are identical to sequences on pub . however, acquired fusr does not always result from acquisition of the prototypical fusb gene. a gene encoding a fusb homologue was recently identified during sequencing of s. aureus strain mssa , and we identified an additional homologue encoded in the genome of s. saprophyticus strain atcc . the products of these genes exhibit~ % homology to fusb and to each other. cloning of pcr amplicons corresponding to these genes and their upstream expression signals into s. aureus established that they both confer resistance to fus. since these functional homologues are more closely related to each other than to those from other gram-positive organisms, it is highly likely that they evolved from an ancestral fusb after its recruitment to the staphylococci. conclusions: the three members of the staphylococcal family of fusb proteins appear to have evolved from the same ancestral protein, which, based on the low level of sequence homology between fusb genes at the nucleotide level, clearly occurred well before the introduction of fus into the clinic. of the three, the fusb protein encoded by pub is by far the most successful, and this gene/plasmid represents the source of (or shares a source with) the major fusr strain lineages. telithromycin activity is reduced by efflux in streptococcus pneumoniae c. benvenuti, r. koncan, g. bahar, a. mazzariol, g. cornaglia (verona, it; ankara, tr) objectives: telithromycin shows an excellent activity against m-type erythromycin-resistant streptococcus pneumoniae, thus is commonly regarded as being capable of overcoming the efflux resistance mechanism. nevertheless, telithromycin mic values in those strains appear to be distinctly higher than in the erythromycin-susceptible ones. the possibility of telithromycin acting as an actual efflux substrate, as it was already demonstrated in streptococcus pyogenes, seemed worth investigating. methods: telithromycin mic distribution was analysed in a collection of italian s. pneumoniae strains originating from multi-centre studies ( ) ( ) ( ) ( ) . the effect of an efflux mechanism was investigated using [ h]-telithromycin. results: telithromycin mic ranges were £ . - . mg/l (mic . mg/l and mic . mg/l) in erythromycinsusceptible strains (lacking both mef and erm genes) and . - mg/l (mic . mg/l and mic . mg/l) in strains endowed with the m phenotype. a distinct telithromycin efflux was detected in the strains expressing the mef gene, but not in those expressing the erm(b) gene, nor in the susceptible strains lacking mef or erm genes. efflux reversibility by addition of an inhibiting compound (sodium arsenate) was demonstrated. an msr-like sequence was also found in all strains effluxing telithromycin, but not in the others. conclusions: this is the first time that telithromycin has been shown to be effluxed by s. pyogenes isolates. that the efflux is related to the presence of both the mef and the msr-like genes is clearly demonstrated, but -owing to the increasingly evident complexity of s. pneumoniae efflux systems -other genes might also contribute to the efflux. an unusual phenotype of enterococcus faecalis in greece expressing low-level resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin m. maniati, f. kontos, p. liakos, e. petinaki, i. spiliopoulou, a. maniatis (larissa, patras, gr) objectives: to investigate the resistance mechanism of a new described phenotype among enterococcus faecalis expressing lowlevel resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin (q-d). methods: in greece, during , three enterococcus faecalis isolates, expressing this unusual phenotype, were recovered from urine samples. the isolates were studied by pcr for the lsa-gene and by pfge. nucleotide sequencing analysis of lsa and bp of the upstream region was performed. the isolates were also tested by rt-pcr for the expression of the lsa-gene. results: the isolates belonged to three distinct clones and carried the lsa-gene. no stop codons were found in any strain, while some point mutations in the lsa-gene were detected. comparing the lsa mrna production of these unusual strains with that obtained from fully q-d resistant ones no quantitative differences were found. conclusions: the findings of the present study clearly show that the resistance mechanism of quinupristin-dalfopristin is not only correlated with the presence and the expression of the lsagene. some mutations detected in the lsa gene probably are responsible for the production of an lsa protein with decreased activity, resulting to the q-d susceptibility. the presence of erm tr gene is responsible for the macrolide-resistance of streptococcus agalactiae objectives: to investigate the mechanism of resistance to macrolides in strains of streptococcus agalactiae in the area of thessalia, greece during the period - . methods: the subject of this study were strains of s. agalactiae which were collected from clinical specimens ( % vaginal swabs) from pregnant and non pregnant women. the strains were identified by gram stain, the lancefield b antigen, and by api strep system (biomerieux, france). susceptibility to macrolides, lincosamides and streptogrammines b was studied by the disk diffusion method. the mics were also measured by the use of e-test. the differentiation between m and mlsb inducible type was tested by the double disk synergy test (ddst). the detection of the genes mef a, erm tr, and erm b was performed by polymerase chain reaction (pcr). the clonality of the resistant strains was studied by pulse-field gel electrophoresis. results: of the strains, were resistant to erythromycin, lincosamid and streptogrammines b. none was found to be resistant to erythromycin only (m-phenopype). % of the strains were mlsb constitutive phenotype, while % were mlsb inducible. all strains were found to carry the erm tr gene. only one strain was found to carry both erm tr and erm b genes. pfge analysis revealed the emergence of multiple resistant clones. conclusions: the resistance of s. agalactiae to mlsb antibiotics is related with the presence of erm tr gene in central greece. emergence of novel clindamycin resistance phenotype among invasive streptococcus pyogenes isolates in sweden a. jasir, b. luca, c. schalen (lund, se) objectives: in some recent throat group a streptococci (gas) isolates from our diagnostic laboratory total resistance to clindamycin but susceptibility to erythromycin and other -as well as -membered macrolides was found. the isolates were susceptible to -membered macrolides and streptogramin b. these atypical strains thus did not agree with previously known mls resistance phenotypes. the main objective was to characterize theses resistance phenotype and genotypes. method and results: the isolates were examined for resistance genes by pcr. out of strains one harboured an erma gene. the gene was sequenced and showed a mutation in regulatory part and was localized on a transposons. all other strains were negative for any erm genes and were also tested for s rrna mutations with negative outcome. strains were t-and emm typed and showed to belong to different types. conclusions: gas account for common human infections such as acute pharyngotonsillitis and impetigo, which untreated may be followed by the nonsuppurative complications rheumatic fever and acute poststreptococcal glomerulonephritis gas may also give rise to invasive, often life-threatening acute disease, such as scarlatina, erysipelas, endometritis, necrotising fasciitis and sepsis, often accompanied by toxic shock. without known exceptions, gas are fully susceptible to betalactams, which are first-choice drugs for treatment. in cases of allergy or intolerance to penicillins, macrolides are most used, and possibly as a consequence, a significant resistance development to these agents has evolved in many parts of the world. though the role of clindamycin for treatment of streptococcal disease is more limited this drug was shown to be particularly effective in eradicating streptococci after penicillin treatment failure of pharyngotonsillitis. clindamycin, often as a supplement to betalactams, also may have a life-saving effect in the treatment of fulminant streptococcal infections. due to its important role in the treatment of invasive streptococcal disease, resistance development to clindamycin in gas is considered highly undesirable. the alarming finding of a possibly new phenotype of selective clindamycin resistance in gas will motivate a thorough analysis of the phenotype as well as identification of its resistance determinants. a. al-lahham, m. van der linden, r.r. reinert (aachen, de) objectives: telithromycin is a novel ketolide antibiotic with significant in-vitro activity against streptococcus pneumoniae. the aim of this study is to characterize the resistance mechanisms of clinical isolates of s. pneumoniae with reduced susceptibility to telithromycin (> mg/l) and to perform the time-kill kinetics with telithromycin. methods: determination of mics was performed by the microbroth dilution method according to the clsi and the serotyping by the neufeld quellung reaction. multilocus sequence typing, sequencing of the s rrna, sequencing of genes encoding ribosomal proteins (l and l ), and ermb were performed according to standard methods. four isolates were selected for time-kill, two of which with a telithromycin mic mg/l and two strains with a telithromycin mic of mg/l. results: in two nation-wide studies and one european surveillance study (n = ) performed at the national reference center for streptococci (nrcs) in germany, reduced susceptibility to telithromycin (> mg/l) was detected in isolates ( . %). mic /mic (mg/l) of the strains to other antibiotics were as follows: telithromycin / , penicillin g / , cefuroxime / , erythromycin a > /> , clindamycin > /> , tetracycline / , and gatifloxacin . / . . two major serotypes were observed, serotype ( . %) and serotype a ( . %). all isolates possess the cmlsb phenotype (ermb positive). the isolates showed a wide range of combinations of resistance determinants including multiple alterations in the s rrna (a g, c t, a g, a t, and c t), a s n alteration in the ribosomal protein l (n = ), and a n s alteration in the erm(b) gene (n = ). the predominant clone was serotype sequence type ( of isolates), which was seen in france (n = ) and germany (n = ). telithromycin-resistance has also spread to the spain f- clone (st ; n = ) and its serotype a variant. in vitro time-kill showed a minimal kill from - hours and then regrowth. bactericidal activity was achieved only with times the mic in all strains. conclusions: although the incidence of telithromycin resistance remains rare world-wide, the spread of telithromycin resistance to multi-drug resistance clones with world-wide distribution is worrisome. gbs obtained from non-pregnant women. the erythromycin resistant-gbs were identified, phenotypically analysed, screened by pcr for mre(a) gene and for erythromycin resistance genes: erm(b), erm(tr), mef(a) and mef(e), and serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of gbs, ( . %) were erythromycin-resistant: ( . %) erythromycin-resistant gbs were isolated from vaginal swabs of pregnant women and ( . %) from non-pregnant women. the frequency of serotypes in erythromycin-resistant gbs tested, the distribu-tion of their resistance genes and the distribution of serotypes among the different genotypes are illustrated in the table. nt, nontypeable, the mre(a) gene was found in all the gbs strains tested. mics of erythromycin in erythromycin-resistant gbs were: mic and mic , > mg/l; range, to > mg/l for gbs harbouring erm(b) and erm(b)+erm(tr) and mic and mic , mg/l and > mg/l, respectively; range, . to > mg/l for gbs harbouring erm(tr). conclusion: erm(b) was the erythromycin-resistant gene most prevalent among the gbs isolates and these isolates showed the highest mics of erythromycin. the commonest serotypes among erythromycin-resistant gbs isolated were iii, ii and i, and showed genotypic variability harbouring either of the two most prevalent genes, erm(b) or/and erm(tr). methods: we studied the rates of resistance to tetracycline and minocycline among erythromycin-resistant gbs strains isolated at the university hospital lozano blesa of zaragoza, spain. isolates were subsequently phenotypically analysed by means of the disk diffusion method and screened by pcr for erythromycin and tetracycline resistance genes [erm(b), erm(tr), mef(a/e), tet(m) and tet(o)]. the susceptibility to erythromycin, josamycin, tetracycline and minocycline was tested by the agar dilution method according to the nccls. the strains were serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of isolates of macrolide-resistant gbs collected from may to april in our hospital ( . % of the total sgb isolated), ( . %) were tetracyclineresistant. the distribution of tet(m) and tet(o) among the erythromycin-resistant gbs harbouring erm(b) was ( . % and . %, repectively) and harbouring erm(tr) was ( . % and . %). the distribution of tetracycline resistance genes and serotypes among the different genotypes in gbs are illustrated in the table.*nt: non typable isolates carrying tet(m) or tet(m)+tet(o) presented the following mics: tetracycline (mic , mic , range - mg/l); minocycline (mic , mic , range - mg/l). isolates carrying tet(o) presented the following mics: tetracycline (mic , mic , range - mg/l); minocycline (mic , mic , range - mg/l). conclusion: the majority ( . %) of tetracycline-resistant gbs harboured tet(m) alone or in combination with tet(o). the most prevalent serotypes among the total of tetracycline-resistant gbs was the serotype iii ( %) and the serotype ii ( %). serotype iii was more prevalent among the gbs harbouring the tet(m) gene and serotype ii was more prevalent among the gbs harbouring the tet(o) gene. objectives: to know the prevalence of resistance to macrolides in viridans streptococci, its mechanism and the genetic elements which are involved. methods: we studied viridans streptococcus pharyngeal isolates from different patients. mics for macrolides were determined by the agar dilution method. the presence of mef and erma, ermb and ermtr genes, and the presence of mega (macrolide efflux genetic assembly) or tn in resistant, mef + isolates was determined by pcr with specific primers. the similarity to mef genes first described in pneumococci (mefe) and streptococcus pyogenes (mefa) was determined by sequencing. results: viridans streptococci isolates were resistant to macrolides ( . %). out of the resistant isolates harboured mef genes ( . %), one harboured ermb ( . %), and isolates harboured both mef and ermb genes ( . %). no isolates harboured erma or ermtr genes. we studied genetics elements which harbour mef genes in other streptococci, in mef (+) isolates. we found mega insertion element in of isolates ( . %), all of them harbouring mefe. the only isolate in which we found mefa, did not harbour mega, but tn . conclusions: m phenotype is frequent in viridans streptococci, and all of them harbour mef genes. most mlsb phenotype viridans streptococci do not harbour erm genes alone; most of them combine erm and mef genes. most isolates contained the mef sequence corresponding to mefe, and the genetic element (mega) usually described in pneumococci as harbouring this gene. one isolate contained the sequence corresponding to mefa, and the genetic element usually described in s. pyogenes (tn ). the increasing presence of macrolide-resistant pneumococci harbouring mega element might be related with its wide presence in viridans streptococci. the acquisition of mega by pneumococci from viridans streptococci through transformation is being studied. objectives: the principal mechanism of macrolide resistance in streptococcus pneumoniae in italy is target site modification mediated by erm(b). the erm(a) gene is common in streptococcus pyogenes but rare in s. pneumoniae, even if recent studies have demonstrated an increased detection of this resistance determinant. recently, a clinical s. pneumoniae isolate carrying erm(a) has been obtained from a patient with meningitis in italy. the aim of this study is the molecular characterization of this isolate. methods: antimicrobial susceptibility tests were determined by etest. the presence of erythromycin resistance determinants was detected by pcr assays. genotyping was performed by pfge and mlst. the flanking regions of erm(a) were analysed by sequencing a fragment amplified by inverse pcr. transformation and conjugation experiments were carried out. transformants were analysed by pfge and hybridization with an erm(a) probe. results: the isolate belonging to serotype (st) a, was resistant to erythromycin, inducibly resistant to clindamycin and susceptible to penicillin and tetracycline. by pcr, the only macrolide resistance determinant detected was erm(a). pfge analysis revealed the genetic correlation of this strain with other st a s. pneumoniae italian isolates. mlst confirmed this data since the isolate belonged to st , which is a single-allele variant of st , the most common st among italian st a isolates. a bp dna fragment, obtained by inverse pcr and containing the erm(a) gene, was sequenced. this fragment contains an orf upstream erm(a), the gene erm(a) identical to that described in s. pyogenes and orfs, downstream erm(a), one homologous to a hypothetical kinase and the other two to transposases of other gram-positive species. in transformation experiments the gene erm(a) was transferred to an erythromycin susceptible recipient. hybridization analysis of one transformant revealed that the size of the transferred dna fragment was approximately kb. no transconjugant was obtained in mating experiments. conclusions: this is the first italian report of an s. pneumoniae isolate carrying erm(a). erm(a) appears to be contained in a genetic element that includes two transposases, although the gene is not transferable by conjugation. objective: treatment with the first oxazolidinone antibiotic, linezolid, of infections caused by staphylococci has proved effective in most cases. in the present study we present the first three cases of linezolid resistant staphylococci in our hospital. methods: we examined three coagulase negative staphylococcus strains isolated from blood cultures (bactec, becton dickinson). identification and susceptibility testing were performed by the vitek ii automated system (biomerieux) and the results were confirmed by the api system (biomerieux) and e-test (ab biodisk, solna, sweden), according to nccls guidelines. results: three linezolid resistant staphylococci were isolated from blood cultures. identification showed that all three isolates were staphylococcus cohnii subsp. urealitycum and the mic values were lg/ml (n = ) and lg/ml (n = ) which are much higher than the value of lg/ml that characterizes sensitive strains. the isolates derived from three patients in different wards of our hospital. the first two isolates were recovered from two icu patients in april and august and the last staphylococcus cohnii was isolated from a patient in the neurosurgery ward, who is still hospitalized. all patients received prolonged treatment with linezolid. conclusions: although six linezolid resistant clinical isolates of s. aureus were previously reported in the literature, these three isolates are the first coagulase negative staphylococcus isolates resistant to linezolid. it is imperative to screen for resistance to linezolid all staphylococci and take the necessary precausions in order to prevent the spread of a linezolid resistant strain in other wards of our hospital. correlation between mic and number of mutated s rrna genes in oxazolidinone-resistant staphylococcus aureus objectives: to determine the number of mutated s rdna alleles present in clinical and laboratory-generated linezolidresistant staphylococcus aureus isolates. methods: linezolid-resistant isolates were tested, of them clinical isolates (mics - mg/l) and mutants selected in-vitro (mics - mg/l). the mutants were raised by repeated passage on increasing linezolid concentrations and their parentage was verified by pfge. mics were determined by agar dilution. genomic dna was digested with ecori and hybridized with a bp probe corresponding to domain v of the genes encoding s rrna, to determine gene copy number. pyrosequencing was used to quantify the proportions of wildtype and mutated alleles present; assays were designed to detect the presence of mutations conferring oxazolidinone resistance. pyrosequencing and hybridization data were combined to determine the number of mutated alleles present. results: resistance selected in-vitro proved less stable than that in the clinical isolates. pyrosequencing showed that all clinical isolates had the g t mutation, of the in-vitro selected mutants had g t, had t c, had t a, had a g and had g a. the s rdna copy number in the oxazolidinone-resistant clinical isolates varied from - , and from - in the laboratory-generated mutants; / laboratoryselected mutants had changes in copy number, compared with their parent strains, and had changes in fragment size, but not number. the number of mutated copies in lin-resistant isolates ranged from - in laboratory-selected mutants and from - in clinical isolates. an increasing number of mutated genes correlated with increasing linezolid mic. conclusions: in combination, pyrosequencing and hybridization successfully determined the number of mutated s rdna alleles. exposure to linezolid selected changes in s rrna gene copy number as well as sequence in % of in-vitro selected mutants. there was a positive correlation between both the number and proportion of mutated s rdna copies and mic, previously unproven for staphylococci. objectives: linezolid (lzd) is an important antibiotic for the treatment of enterococcal infections, especially when the corresponding strain possesses multiresistance including resistance to vancomycin (van). we report the emergence lzd resistance in clonally related van-susceptible and vanresistant enterococcus faecium isolates originated from an icu patient only days after initiation of linezolid therapy. patient and methods: van-resistant e. faecium was repeatedly isolated from intraabdominal cultures of a -year-old female icu-patient with infected necrotizing pancreatitis after pancreaticoduodenectomy (whipplés procedure). antibiotic susceptibility testing of the bacteria was performed by e-tests; vana gene were detected by pcr. the possible lzd resistance mechanism (mutation in the s rdna of one or more of the six s rrna alleles of e. faecium) was examined by a pcr-based method. molecular typing of the strains was performed by smai macrorestiction analysis. results: van-resistant but lzd-sensitive e. faecium (vrlse) were initially detected in intraabdominal cultures, however, already twelve days after initation of lzd therapy, van-and lzd-resistant e. faecium (vrlre) strains were detected. resistance to lzd was confirmed: mics ranged from to mg/l. all e. faecium isolates showed identical or closely related pfge patterns. throughout the icu period, van-and lzd-susceptible e. faecium (vslse) strains were repeatedly detected in the same specimens from which the vrlse and vrlre were isolated. additionally, van-susceptible e. faecium isolates with resistance to lzd (vslre) were detected. mutations in the s rdna of three out of six alleles led to lzd resistance in the e. faecium isolates examined. two weeks after termination of the lzd therapy, no lzd-resistant strain could be detected in follow-up swabs. conclusions: resistance to lzd in e. faecium can occur already shortly after the initiation of lzd therapy. assessment of antibiotic susceptibilities of all isolates at the start of therapy and regularly during the therapy is advisable, especially during therapy of severe infections. the epidemiological and clinical repercussions of resistance to lzd among enterococci cannot be predicted at this time. attention to proper dosing and prompt removal of infected devices, when feasible, could limit occurrence and spread of lzd-resistant e. faecium. objectives: to investigate the mechanisms of resistance to tetracycline in shigella spp. methods: one hundred and eleven tetracycline-resistant shigella spp strains ( s. sonnei, s. flexneri), were isolated as a cause of enteritis in our geographical area and the remaining recovered from patients with traveller's diarrhoea. antimicrobial susceptibility to tetracycline was determined by the kirby-bauer method. presence of teta, tetb and tetg genes was established by pcr. sequencing of amplified products were used to corroborate the reliability of the pcr results. maentel haenszel test was used to establish the statistical significance. results: the statistical analysis showed that the teta gene was more frequent in s. sonnei (p < . ), while tetb was more usual in s. flexneri (p < . ). although without statistical significance (p: . ), presence of non-determined mechanisms of tetracycline-resistance seems to be more frequent among s. sonnei. conclusions: species-specific differences in the distribuition of the teta and tetb genes has been shown. moreover . % of the analysed strains did not show any of the analysed determinants of tetracycline-resistance. the concomitant presence of more than one of the analysed genes is a rare event. distribution and genetic determinants of tetracycline resistance in laribacter hongkongensis isolates from humans and fish objectives: to study the distribution of tetracycline resistance and to clone and characterize a tetracycline resistance determinant in laribacter hongkongensis, a recently discovered bacterial genus and species associated with communityacquired gastroenteritis. methods: twenty-four l. hongkongensis strains isolated from patients with community-acquired gastroenteritis and l. hongkongensis strains isolated from freshwater fish in hong kong were used in this study. genetic determinants for tetracycline resistance were looked for by screening a genomic dna library of l. hongkongensis. the prevalence of teta gene in other strains of l. hongkongensis was studied by pcr using laboratory-designed primers. the presence of the tetracycline resistance determinants in plasmid was examined by southern blot analysis. results: among human and fish isolates tested, human and fish isolates were tetracycline-resistant. a -bp gene cluster, which consists of putative transposases, a tetr and a teta gene, was cloned by inserting restriction fragments of genomic dna from a resistant strain, hlhk , into pbk-cmv. the -bp teta and -bp tetr genes shared significant nucleotide sequence homology with known teta and tetr genes. while the flanking regions and ' end of the teta were identical to the corresponding regions of a tetc island in chlamydia suis, the teta was almost identical to that of transposon tn and plasmids found in many gram-negative bacteria, suggesting that illegitimate recombination may have occurred to produce the present tetracycline resistant determinant. southern hybridization suggested that the teta gene of hlhk was plasmid-encoded. the tetracycline resistance in l. hongkongensis was associated with teta. pcr amplification of the teta gene in the isolates of l. hongkongensis, including hlhk , showed the presence of teta in all the four tetracycline resistant isolates but none of the tetracycline susceptible ones. in contrast to strain hlhk , the teta of two strains were identical to that of tn , while that of the other strain was more closely related to other gram-negative bacteria plasmids. conclusion: our results indicate that horizontal transfer of genes, especially through tn and related plasmids, between l. hongkongensis and other gram-negative bacteria is probably a frequent event and is an important mechanism for acquisition and dissemination of tetracycline resistance in l. hongkongensis. succesful treatment of infective endocarditis with linezolid t. hryniewiecki, u. lopaciuk, j. stepinska (warsaw, pl) objectives: there is an increasing proportion of resistant strains causing infective endocarditis in recent years. it has changed the approach to choice of antibiotic therapy. linezolid (zyvoxid Ò ) is a new bacteriostatic antibiotic with a wide spectrum of activity against gram-positive organisms and with good efficacy in experimental animal models of endocarditis. unfortunately clinical experience with linezolid in the treatment of endocarditis is limited. the aim of the study was to observe efficacy of linezolid in the treatment of infective endocarditis. methods: the study group consisted of patients hospitalised in institute of cardiology in warsaw ( warsaw ( - due to clinically resistant infective endocarditis. the diagnosis of endocarditis was established according to the duke criteria by clinical examination, echocardiography, laboratory investigations and positive blood cultures with vancomycin mic estimation (in pts). all patients were treated surgically (valve replacement, artificial material removal) in conjunction with different conventional antibiotics and afterwards with mg of linezolid every hours intravenously. results: infective endocarditis was diagnosed as caused by mrcns in pts, mssa in pt, enterococcus faecalis in pt and staphylococcus epidermidis mr in pt. vancomycin mic vary from to mg/l. in pts culture-negative endocarditis was diagnosed. all patients were treated with linezolid intravenously to weeks (average , ). clinical response and eradication of bacteremia were achieved in all patients. leukopenia nad thrombocytopenia as an adverse reaction occurred in patient. conclusions . linezolid is effective in patients with grampositive endocarditis. . linezolid could be also effective in some patients with culture-negative endocarditis. . linezolid may provide an alternative in the treatment of infective endocarditis due to multi-resistant bacteria, in patients with resistant course or with adverse reaction to conventional antibiotics. objectives: to evaluate the safety and efficacy of lzd in a chinese population. methods: this randomized, double-blind, multi-centre study was conducted in china. after obtaining written informed consent, patients from to years of age with pneumonia (pneu) or skin and soft tissue infection (ssti) known or suspected to be caused by a gram-positive pathogen were randomized : to receive either lzd, mg, or vancomycin (van), g, each given iv q h. patients were to be treated for to days, and outcomes were assessed at end-of-treatment (eot) and follow-up (f-u) visits. results: one hundred forty-two patients were enrolled and received study medication, with pneu and with ssti. clinical assessments (effective = ''cured'' plus ''marked improvement'') for patients in the fully evaluable population are summarized in the table. the most frequently isolated pathogen was staphylococcus aureus: all isolates were susceptible to both study drugs. the eradication rates for all pathogens in evaluable patients at the f-u evaluation were / ( . %) in lzd-treated patients and / ( . %) in van-treated patients (p = . ). all patients receiving study drug were evaluated for safety. drug-related adverse events (aes) were reported in ( . %) lzd-treated and ( . %) van-treated patients. the most commonly reported drug-related aes in lzd-treated patients were mild abnormalities in liver function tests and leucopenia ( . % each); rash ( . %) was the most commonly reported ae in van-treated patients. seven ( . %) lzd-treated and ( . %) van-treated patients discontinued study drug because of an ae. conclusions: linezolid is an effective drug for the treatment of infections caused by gram-positive pathogens and is welltolerated. eradication in one patient, by rifamycinlinezolid, of a methicillin-resistant staphylococcus aureus producing panton-valentine leukocidin, responsible for relapses over months, and decolonisation of her family by mupirocin objective: we report the case of the mother who experienced relapses over the period. methods: pvl-mrsa were isolated on routine and mrsa agars (biorad). antibiotypes were studied by disk diffusion method. genetics and pulsotypes were studied by the french centre national de référence des staphylocoques in lyon (cnr). results: mrs kym had her th child on october , in the hospital of orléans. she was healthy and presented no risk factor for delivery. three weeks later she was addressed for surgical treatment of an abscess on buttocks. cultures yielded the special antibiotype: methicillin-r, kanamycin-r and tobramycin-s, of the pvl-mrsa currently spreading across europe and maghreb. the cnr found the luks-pv and lukf-pv-genes.through march , she relapsed times and was treated by pyostacin for a total of weeks. two of her children were addressed for abscesses ( buttocks, thumb) yielding the same bacteria. in march , mrs kym was addressed to the infectious diseases ward because of nasal furonculosis. samples yielded a pvl-mrsa with mls-b phenotype. treatment by rifamycin-linezolid d was initiated. the whole family was screened. the father and the boy, , who had the infected thumb months earlier, were carriers. the girl, , who had an abscess on buttocks months earlier was not. in april the whole family accepted an attempt for decolonisation by % nasal mupirocin or /d for d. cure and decolonisation were confirmed by nares and cutaneous folds samples in may and june. they missed an additional appointment in the beginning of term, but a phone call to the social worker confirmed none of them relapsed. the cnr studied strains ( from mrs kym , , from children abscesses in and , from boy's and father's nares ), and confirmed that they were all identical along the period and across the family. conclusion: short treatment with linezolid-rifamycin in the relapsing case associated with familial decolonisation by nasal mupirocin was an effective strategy to stop a time-prolonged familial outbreak of pvl-mrsa infection. multiple brain abscesses and purulent meningitis by listeria monocytogenes in an otherwise healthy man. favourable linezolid response, hampered by a suspected early drug myelotoxicity introduction: l. monocytogenes cns infection in immunocompetent adults remains rare. meningitis is the most common cns manifestation, with brain abscesses being < % of overall episodes. anecdotal episodes of cns l. monocytogenes infection were reported from immunocompetent patients where both diagnosis and treatment may be hampered by low clinical suspicion and a frequent non-specific presentation. in a -yearlong survey conducted in dallas (us), only cases of nonneonatal l. monocytogenes meningitis were found (estimated incidence rate: . %). case report: a -year-old male with a negligible history and no obvious exposure to l. monocytogenes was hospitalized owing to dizziness. a brain ct scan showed a small, late ischemic lesion. a few days later hyperpyrexia, headache, vomiting and altered mentation occurred. the csf study detected an elevated albumin content ( mg/dl), low glucose ( mg/dl) and a wbc count of cells/ll ( % neutrophils) so that ceftriaxone-chloramphenicol were immediately started. clinical-neurological conditions deteriorated while l. monocytogenes was cultured from the csf so that treatment was changed towards high-dose ampicillin-gentamicin. the persistence of severe clinical-neurological conditions and altered csf assay prompted the introduction of rifampicin-cotrimoxazole after days, but days later other focal neurological deficits appeared and a mri showed small, hyperintense focal lesions involving the medulla oblongata, interpreted as multiple abscesses. the introduction of linezolidmeropenem, despite anemia (requiring rbc transfusions after - days) led to a progressive clinical-csf improvement. our patient recovered completely and a control mri carried out month after discharge confimed the complete disappearance of the multiple brain listeria abscesses. discussion: the l. monocytogenes meningitis and multiple subtentorial abscesses (including rare localizations at cerebellum, bulb, and pons varolii), had an evolving cumbersome presentation. despite the in vitro activity of a broad spectrum of agents, multiple therapeutic changes became necessary, until the last linezolid-meropenem combination, which was proved very effective, although it was affected by relapsing anemia probably attributable to linezolid. linezolid, due to its elevated csf-brain penetration, and its activity against a broad spectrum of cns pathogens (including the intracellular l. monocytogenes), is expected to become a key antimicrobial compound, waiting for rct. discrepancy between favourable in vitro microbiological data and a severe clinical course of a staphylococcal knee and soft tissue infection responsive to oxazolidinone linezolid only after failure of all other therapeutic attempts introduction: to offer therapeutic alternatives for the emerging, multiresistant, serious gram-positive infections, novel molecules (quinupristin/dalfopristin, linezolid, daptomycin) were introduced and are made available when multiresistant gram-positive cocci are documented as no more susceptible to all available drugs including glycopeptides. however, inezolid encompasses unique tissue penetration and diffusion features (regarding soft tissues, lungs, joints and central nervous system) which make this last drug extremely promising in all circumstances where the penetration rate into infectious foci becomes critical. clinical experience: a very intriguing case report of a severe, staphylococcal knee arthtiris associated to an extensive local cellulitis/fasciitis and haematogenous dissemination occurring after a surgical curettage was characterized by a complete lack of response to a prolonged vancomycin/teicoplanin plus rifampicin therapy based on the apparently favourable in vitro sensitivity assays of methicllin-resistant staphylococci, but rapidly responded to i.v. (followed by oral) linezolid administration. the complete lack of clinical activity of a -week glycopeptiderifampicin administration cannot be explained by the in vitro measured mic values of isolated pathogens which showed complete sensitivity of staphylococcus aureus against vancomycina/teicoplanin and rifampicin and susceptibility of a concurrent hematogenous s. epidermidis strain to glycopeptidesrifampicin. since an abscess formation and an underlying osteomyelitis were carefully excluded by adequate instrumental examinations, from a theoretical point of view the active glycopeptide-rifampicin molecules should have been provided appropriate cure. on the other hand, from a strictly clinical issue, only a -week administration of i.v. linezolid followed by one more week of oral linezolid allowed to obtain a complete clinical-bacteriological cure and a complete function recovery without any sequelae after a . -year follow-up. conclusions: when the management of severe, multiresistant gram-positive infections is of concern, the in vitro activity of single drugs and therapeutic classes should be carefully evaluated in relation with the expected penetration and diffusion rates of these drugs into the relevant organs and tissues involved by the ongoing infectious localizations. otherwise, apparently unexplained failures may occur also when in vitro studies point out a complete activity of the tested compounds. epidemiology of resistance to antibiotics -ii p contemporary prevalence of bro betalactamases in m. catarrhalis: report from the sentry antimicrobial surveillance program (usa; l. deshpande, h. sader, r. jones (north liberty, us) objectives: to evaluate the prevalence of bro- and bro- among b-lactamase (bl)-producing m. catarrhalis (mcat) in the usa. although the bl-mediated penicillin (pen) resistance (r) in mcat has been stable at %, the bro- and - occurrence has not been determined in usa isolates since . bro- rates have been reported at < ( s), . ( - ), . ( - ) and . % ( - ) . methods: community-acquired mcat isolates (sentry program were tested by clsi broth microdilution methods including: , worldwide and , in north america (na). bro- and - was detected by pcr methods (levy and walker; ), compared to epidemiologic tests, and mic values. b-lactamase-positive (bl+) mcat samples per year from usa ( sites) and canada (ca; sites) were tested for the odd-numbered years. results: the bro- rate was , , , and % for , , and , respectively; rates in ca ( isolates) > usa ( ). several agents remained active: amoxicillin/clavulanate (mic , £ . mg/l), ceftriaxone (ctri; . ), cefuroxime ( ), erythromycin (£ . - . ), levofloxacin (£ . - . ), tetracyclines ( ) and trimethoprim/sulfamethoxazole (tmp/ smx; £ . / . ). pen mic distribution was tri-modal (£ . , - , > mg/l) and ctri bi-modal ( . , . ), yet bro- and - mic/zone distributions overlap (best discriminated by methicillin (mean zone, . vs. . mm) and pen ( . vs. . ) disks). possible bro- epidemic clusters could not be excluded due to a very common ribotype in centres (ca, sites; usa, ). conclusions: this bro- and - enzymes na prevalence update in mcat isolates ( ) ( ) ( ) ( ) ( ) ( ) ( ) shows stability at - % and - %, respectively. phenotypic tests (zones or mics) cannot easily distinguish between these b-lactamase types, necessitating the use of molecular applications. objective: although resistance to penicillin in beta haemolytic streptococci has not been reported yet, increasing resistance rates for alternative drugs, such as erythromycin, clindamycin or tetracycline is an emerging concern which brings the necessity to carefully monitor penicillin susceptibility. materials and methods: in order to detect any changes in penicillin mics, we performed antimicrobial susceptibility testing for all isolated beta haemolytic streptococci in our hospital between january and november . identification to serogroup level was done using a commercial latex agglutination kit (avipath strep, omega diagnostics ltd., scotland, united kingdom). results: a total of isolates were identified, distribution of groups for serogroup a, b, c and g were . %, . %, . % and . %, correspondingly. penicillin susceptibility was determined using etest (ab biodisk solna, sweden) strips according to manufacturers' instructions. when results are evaluated in year periods, mic increased from . to . mg/ml for group a, from . to . mg/ml for group b, from . to . mg/ml for group c and from . to . mg/ml for group g (table) . conclusions: even though highest mic values were to be found in group b ( . mg/ml), our results indicate the steady increase in penicillin mic for all serogroups. three group a and six group b isolates with penicillin mic of . mg/ml, reaching susceptibility breakpoint concentration according to clsi, and also highly elevated mic concentrations for group b streptococci may be messengers of possible forthcoming resistant strains. objective: to study trends in macrolide resistance rates among s. pneumoniae isolated from children aged to months attending day-care centres in france following implementation of prudent antibiotic use campaigns (alpes maritimes , france and pneumococcal conjugate vaccine (pcv) ( ) . method: nasopharyngeal aspirates were obtained from a random -stage cluster sample of children attending day-care centres in the nord (n) and alpes maritimes (am) areas during consecutive surveys between january and march march , march and . susceptibility to erythromycin and clindamycin and resistance phenotype were analysed by disk diffusion method. serotypes were determined using the quelling reaction. pneumococcal immunization status and antibiotic prescriptions over the previous months were recorded. results: sp was isolated from / , / and / children in , and , respectively (p < ) ). resistance to macrolides declined overall from . % to . % of strains between and (p < ) ). among erythromycin-resistant (e-r) isolates, percentage of erm-b phenotype increased from . % to . % (p = . ). while the proportion of penicillin non-susceptible strains declined from . % to . % of sp isolates (p < ) ), erythromycin resistance remained stable among these strains at . %. overall proportion of treated children fell from . % to . % (p < ) ) between and ; in am this reduction was observed in ( . %; p < ) ), while in n it occurred in ( %; p < ) ) and the percentage of macrolides among prescriptions fell from . % to . % (v for trend: p = . ). serotype distribution showed most e-r isolates were b, , f and f. a % reduction in serotype f was observed in am in and in n in . immunisation with pcv concerned at least . % of children in . conclusion: macrolide resistance has followed a parallel decline with penicillin resistance as a result of antibioticprescription reducing campaigns and pneumococcal immunization against the most prevalent macrolide-resistant serotypes. objective: to evaluate the prevalence of resistance of invasive strains of s. pneumoniae to erythromycin after decline in macrolide consumption. methods: the number of packages of antibiotics was obtained from the institute of public health of slovenia. for the period - the data on outpatient antibiotic consumption were collected using the atc/ddd classification (who version ) and the results were expressed in ddd/ inhabitants per day (did). all invasive strains of s. pneumoniae isolated from sterile body fluids in all slovenian hospitals were included in the study. susceptibility testing was performed using nccls approved disk diffusion test. results: during - the total use of antibacterials in slovenia decreased for . % from . did to . did. the consumption of macrolides which constituted . - . % of total use of antibacterials decreased for . % ( . to . did). short-acting (erythromycin, miocamycin), intermediate-acting (midecamycin, roxithromycin, clarithromycin), and long-acting (azithromycin) decreased for . %, . % and % respectively. in all years the use of intermediate-acting macrolides was the most prescribed subclass of macrolides corresponding for . - . did, followed by long-acting ( . - . did) and short-acting ( . - . did). the resistance of s. pneumoniae strains to erythromycin increased from . % ( / ) to . % ( / ); in children from . % ( / ) to . % ( / ) and in adults from . % ( / ) to . % ( / ) respectively. rates of the isolates resistant to erythromycin and at least of the following agents: penicillin, tetracycline, tmp/smx, chloramphenicol increased from . % ( / ) to . % ( / ); in children from % ( / ) to . % ( / ) and in adults from . % ( / ) to . % ( / ) respectively. conclusion: despite a reduction of macrolide consumption in outpatients the resistance of invasive strains of s. pneumoniae was increasing during the observation period especially in children. multiple drug resistance explains best the changes in s. pneumoniae resistance in a ten-year surveillance study in belgium objective: belgium is located between countries with very high and very low antibiotic resistance rates. modeling how resistance changes over time and place in belgium provides insights into correlates of s. pneumoniae resistance at the population level. methods: surveillance data consists of , s. pneumoniae invasive isolates from - , identified by postal code as well as clinical and demographic information. antimicrobial consumption (ims health services) is expressed in defined daily doses (ddd) per inhabitants per day. changes in resistance by month and postal code were evaluated using mixed effects models for repeated measures, using mathematical models of transmission for the curve shape, and taking into account seasonality. resistance to penicillin, erythromycin, tetracycline, and ofloxacin was considered in the analysis. results: resistance to penicillins, macrolides and tetracyclines peaked in the year , and their levels in were . %, . % and . % respectively. the shape of the curves is similar for most of the antibiotics studied, with a steep rise from to and a plateau thereafter. resistance to two or more antibiotic classes corresponded to % of all resistant isolates and in a multivariate model explains most of the variability through time and place of the antibiotics studied. resistance to only one antibiotic (any) decreased from . % in to . % in , while resistance to two or more increased . times ( % ci . - . , p < . ) from . % in to . % of all isolates years later. more than nine out of ten isolates that were macrolide or tetracycline resistant were also multiply resistant (mr). mr increases . % for each ddd of overall cumulative antimicrobial consumption, and out of all antibiotic classes, macrolides and broad-spectrum penicillins are most associated with resistance. conclusion: resistance to two or more antibiotics is the most important factor in understanding the changes over time for all studied antibiotic classes in belgium. the cumulative impact of antimicrobial exposure of separate antibiotic classes at the population level facilitates the survival and transmission of any isolate that is resistant to two or more antibiotic classes. methods: the isolates were identified by biochemical tests and specific serotyping. antimicrobial susceptibility to ampicilllin (amp), amoxicillin plus clavulanic acid (auc), cloramphenicol (cm), gentamicin (gm), cotrimoxazole (sxt), nalidíxic acid (nal) and tetracycline (tc) were established by the method of kirby bauer. the presence of beta-lactamases encoding genes (tem, carb, oxa -like) as well as the teta, tetb, tetc, tetg, cmla and flor genes, and integrons type was established by pcr, while the presence of plasmid-mediated dhfr was determined by pcr-rflp and the cat activity by a colorimetric assay. results: seven different resistance patterns were identified: i. susceptible ( strains); ii. amp, sxt, gm, a/c ( ); iii. amp, tc, cm ( ); iv. amp, tc, sxt, cm ( ); v. amp, a/c ( ); vi. amp, sxt, a/c ( ); vii ( ). -sxt. no isolate resistant to nalidixic acid was detected. resistance to beta-lactam agents was due to the presence of beta-lactamases type tem-like (pattern v), carb- (iii) and tem-like plus oxa- (ii, v, vi). meanwhile resistance to cloranphenicol and tetracycline was associated to cat activity (iii, iv) and flor (iii), and tetb (iv) and tetg (iii) respectively. no mechanism of cotrimoxazole resistance was detected in the isolates of the patterns ii, vi and vii, while dfra was detected in the isolates of the group iv. resistance to gm was associated to the presence of the gene aadb, detected in the analysis of integrons type . type integrons were detected in isolates belonging toi the pattern ii ( bp -aadb; bp -oxa , aada ), iii ( bp -carb , -aada ), iv ( bp -dfra , aada ), v and vi ( bp -oxa , aada ). conclusions: a great diversity of resistance mechanisms has been detected. those mechanisms might spread among microorganisms resulting in a serious health problem due to the limited number of antibiotic treatments available in the area. small outbreaks of veb- esbl producing acinetobacter baumannii in belgian nursing homes and hospitals through cross-border transfer of patients from northern france methods: from / to / , all belgian acute hospitals were invited to report cases of nosocomial infections/colonisations due to mdr ab isolates presenting a resistance profile similar to the french epidemic strain (resistance to all agents except carbapenems and colistin) and to send such isolates to the reference laboratory for phenotypic confirmation and for genotypic characterization (pcr of veb- and class integron, pfge typing). guidelines for detection of the epidemic strain, screening for carriage in patients transferred from hospitals or nursing homes (nh) close to the french border as well as infection control measures were sent to all hospitals. results: overall ab strains from hospitals were sent to the reference laboratory. only, of these fulfilled the phenotypic resistance patterns and were definitely confirmed as veb- ab and had a pfge pattern identical to the french epidemic clone. two mini-outbreak clusters (each involving cases) were documented in hospitals from two cities (tournai and chimay) closed to the french border. two patients died from their infection. in the first outbreak, all patients were residents who lived in the same nh. two of them were french citizens who had been hospitalised in different acute care hospitals in the north of france within the last year. in the second outbreak, the index case had also been previously hospitalised in a french hospital. secondary transmission to two other hospitalised patients occurred in this outbreak. conclusion: despite the large extension of the veb- ab outbreak in france no similar problem occurred in belgium. however, this national alert allowed to detect two small outbreaks in belgian institutions located close to the french border. in both outbreaks the epidemic strain was imported from france through patient circuits. this study illustrates that transfers between acute care hospitals and nh may explain cross-border spread of multi-resistant epidemic strains. types of extended-spectrum beta-lactamases in salmonella spp. and decreased susceptibility to fluoroquinolones objectives: the aim of this study was to determine the rate of esbl production in clinical isolates of salmonella spp. and to detect decreased susceptibility to fluoroquinolones in esbl positive isolates in turkey. methods: a total of salmonella spp. isolated from clinical samples from thirteen centres between and were included in the study. in vitro susceptibility to ampicillin, amoxicillin/clavulanic acid, cefotaxime, gentamicin, chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and ciprofloxacin were determined using the agar dilution method on mueller-hinton agar following the clinical and laboratory standards institute (clsi) guidelines. decreased susceptibility to ciprofloxacin was defined as an mic of . - mg/l. salmonella isolates were screened for esbl production by double disk synergy method using amoxicillin/clavulanic acid, cefotaxime and ceftazidime disks. types of esbl enzymes were analysed by pcr for tem, ctx-m, shv and per- genes. results: in salmonella spp. the highest level of resistance was observed against ampicillin ( . %) followed by chloramphenicol ( . %), tetracycline ( . %) amoxicillin/ clavulanic acid ( . %), trimethoprim/sulfamethoxazole ( . %), gentamicin ( . %), and cefotaxime ( . %). ciprofloxacin resistance was observed in one isolate ( . %). among salmonella isolates, ( . %) were shown to produce esbl by double disk synergy testing. these isolates were salmonella typhimurium (n = ), serogroup c (n = ) and salmonella enteritidis (n = ). three isolates were from fecal samples two were from urine and one was from blood. one of the esbl producing isolates were susceptible to cefotaxime in vitro. two isolates showed decreased susceptibility to ciprofloxacin. all the esbl producers were resistant to ampicillin, amoxicillin/ clavulanic acid, chloramphenicol and harbored ctx-m type enzymes. in three isolates a tem-type enzyme was also present. conclusion: albeit being rare, esbl production is an important resistance factor among salmonella spp. in order to prevent treatment failures, decreased susceptibility to fluoroquinolones should be investigated routinely in invasive isolates as well as esbl production. incidence of faecal carriage of esbl-producing enterobacteriaceae in hospital and community patients during two non-outbreak periods of time the identities of the esbl-producing isolates recovered during were: e. coli (n = ), k. pneumoniae (n = ), p. vulgaris (n = ) and e. cloacae (n = ), and isolates recovered during were: e. coli (n = ), k. pneumoniae (n = ), k. oxytoca (n = ), p. vulgaris (n = ), p. mirabilis (n = ), e. cloacae (n = ) and e. aerogenes (n = ). conclusions: a dramatic, significant increase in the frequency of faecal carriage of esbl-producing isolates was demonstrated in among hospitalized ( . %) and ambulatory patients ( . %).the results revealed that the prevalence of faecal carriage among ambulatory patients and hospitalized patients was not significantly different in both periods of time. outpatients came from the community carrying enterobacteria harbouring esbl in the intestinal tract, suggesting that the community could be a reservoir for these microorganisms and enzymes. methods: a total of k. pneumoniae, k. oxytoca, e. coli, c. freundii, s. marcescens, and e. cloacae from university hospitals, isolated from blood, wound, urine, sputum and other clinically significant specimens were proven to produce esbls. antimicrobial susceptibility was determined according to clsi, ; conjugation on a solid medium was performed; isoelectric focusing was followed by bioassay; pcr with beta-lactamase group-specific oligonucleotides was applied, followed by nucleotide sequencing; rapd with eric- a and eric primers was performed. results: mic of ceftazidime varied from to > mg/l, mic of cefotaxime - - mg/l; the addition of sulbactam : reduced mic > -fold. transconjugants exhibited resistance both to extended-spectrum cephalosporins and aminoglycosides in of strains. according to their pi, two clusters of betalactamase producers could be described: first one -esbls focussed at pi . , and the second -pi at . . results from pcr confirmed the presence of two groups esbls: tem and shv. sequencing of representative strains showed the presence of shv- in two participating hospitals and of shv- in only one strain e. cloacae, while tem- like enzyme was found in centres and had a clonal dissemination. objectives: during treatment with selective decontamination of the digestive tract (sdd), four strains of multidrug-resistant (mdr) gram-negative bacteria (three escherichia coli strains and one klebsiella pneumoniae) were isolated at the intensive care unit (icu) in the academic medical center (amc) in amsterdam. these isolates were extended spectrum betalactamase (esbl) positive. we investigated whether this was due to interspecies transfer of resistance genes. methods: the strains were typed by amplified fragment length polymorphism analysis. the plasmids from these strains were characterized by restriction fragment length polymorphism. resistance genes of the mdr-strains were characterized by pcr and sequence analysis. results: aflp analysis confirmed that the three mdr e. coli isolates represented three different strains. the mdr-strains were shown to harbour the same plasmid with identical extended-spectrum â-lactamase (esbl) genes; ctx-m- and shv- . conclusions: identification of the emergence of such mdr gram-negative bacteria and recognition of resistance plasmid transfer during sdd treatment is crucial for optimal application of this regimen in icus. the use of the third generation cephalosporins in sdd may associate with emergence and increase in the prevalence of esbls. therefore, for optimal screening of resistance to cephalosporins in icus, the screening for esbls should be included. objective: carbapenems are the drugs of choice for the treatment of serious infections caused by esbl-producing enterobacteriaceae and the emergence of carbapenem resistance is rarely documented. we investigated pairs of carbapenem-susceptible and resistant k. pneumoniae isolates from three patients, collected before and after therapy with carbapenems. methods: pre-and post-therapy pairs of esbl-producing k. pneumoniae isolates were from three patients with urinary catheter-associated infections who were treated with ertapenem (erp, cases) or meropenem (mem, one case) in a district general hospital with a low incidence of esbl-producing organisms ( . / bed days), and meropenem use of ddd/year. isolates were compared by pfge of xbai-digested genomic dna. mics were determined and interpreted by british society for antimicrobial chemotherapy methodology. blactx-m alleles were sought by multiplex pcr. outer membrane proteins (omps) were extracted, and analysed by sds-page. results: the three patients relapsed following erp or mem therapy, and the post-therapy isolates from repeat urine samples were resistant (table), with mics erp>mem>ipm. all six isolates from the three patients belonged to the same pfge strain, but transmission of the resistant variants is unlikely as the patients were geographically and temporally unrelated and separate selection of resistance in individual patients seems more likely. all isolates had a group ctx-m esbl; the resistant isolate in each pair had lost a major omp, consistent with a porin, compared with its susceptible 'parent'. all three patients were successfully treated with amikacin. the emergence of carbapenem resistance in ctx-m-producing k. pneumoniae following therapy severely limits treatment options. whilst unusual in general, such selection has occurred repeatedly with this strain. wide geographic spread of diverse acquired ampc beta-lactamases in escherichia coli and klebsiella spp. in the uk and ireland objective: to determine the distribution of genes encoding acquired ampc beta-lactamases in cephalosporin-resistant isolates of e. coli and klebsiella spp. submitted to the uk national reference laboratory. methods: mics were determined by agar dilution and interpreted according to breakpoints of the british society for antimicrobial chemotherapy. isolates of e. coli or klebsiella spp. resistant to cefotaxime and ceftazidime, irrespective of addition of clavulanic acid, were inferred to have possible ampcmediated resistance. genes encoding six phylogenetic groups of acquired ampc enzymes were sought with a multiplex pcr assay (perez-perez & hanson. j clin microbiol ; : - ) . selected isolates were compared by pfge, and selected blaampc amplicons were sequenced. results: e. coli isolates and klebsiella spp. from separate patients yielded pcr amplicons indicating the presence of genes encoding acquired ampc enzymes. forty of these e. coli isolates (from hospitals) produced cit-type enzymes, (from irish hospitals) produced acc types, and a dha type. the klebsiella spp. produced acc ( isolates from irish hospitals), fox ( isolates from welsh hospitals) or dha ( irish isolate) enzymes. genes encoding ebc-/ent-and moxtype enzymes were not detected. twelve e. coli isolates from one hospital all produced a cit-type enzyme; these isolates belonged to an epidemic uk strain, designated strain a; isolates also contained blactx-m- linked to an upstream copy of is , as is characteristic of strain a; isolates lacked blactx-m- . sequencing of a representative blaampc amplicon indicated production of a novel cmy- variant in these isolates. conclusions: diverse acquired ampc enzymes are present in e. coli and klebsiella spp. in the uk and ireland, with cit-types the most common, and acc types linked to ireland. the broad resistance profiles of ampc enzymes compromises patient management. hence, the acquisition of a cmy- -like enzyme by epidemic e. coli strain a suggests that acquired ampc enzymes are poised to become an important public health issue in the uk. objective: to characterize the spectrum of activity and potency of dor (formerly s- ) and comparator agents against contemporary wild-type bacterial isolates from medical centres in europe and the middle east in . dor is a novel parenteral -b-methyl carbapenem in late stage clinical development whose molecular structure confers stability to b-lactamases and resistance (r) to renal dehydropeptidases. methods: the collection included non-duplicate, consecutive clinical isolates from patients in medical centres in europe ( ), turkey ( ) and israel ( ) that were submitted to the dor surveillance program ( ) for identification confirmation and susceptibility (s) testing. mic values for > antimicrobials were determined using nccls broth microdilution methods ( ) . a tentative dor susceptible (s) breakpoint of £ mg/l (£ . mg/l for s. pneumoniae) was used for comparative purposes; clsi ( ) criteria were used for other tested agents. results: antimicrobial activities of dor and other carbapenems vs. selected isolates. dor consistently displayed activity against staphylococci and streptococci (mic , . and . mg/l) most similar to that of imipenem, and against e. coli and klebsiella spp. (mic , . and . mg/l, respectively, including . and . % of strains that met esbl screening criteria), most similar to that of meropenem. enterobacter spp. isolates, including . % that were ceftazidime-r (indicative of ampc production), were also highly s to dor and other carbapenems ( . to . % r). dor also provided slightly enhanced coverage against p. aeruginosa ( . % s) and acinetobacter spp. ( . % s) compared to other carbapenems. carbapenem r among these latter strains is, however, a particularly worrisome development. conclusions: dor is a new carbapenem with a competitive profile that incorporates both potent gram-negative and grampositive activity, with enhanced activity against the commonly occurring non-fermentative gram-negative bacilli. carbapenems are assuming a greater therapeutic role in many nations as multi-drug resistance (including emergence of ambler class a, c and d b-lactamases) spreads, necessitating their accelerated development. phenotypic and genetic characterisations of enterococcal isolates in tehran sewage, with emphasis on detection of vana and vanb genes objectives: enterococci are members of the normal gut flora of animals and humans and are thus released into the environment directly or via sewage outlets, where they can survive for long time periods. during the last decade the concern has been focused on enterococci that are resistant to the glycopeptide antibiotic vancomycin [vancomycin-resistant enterococci (vre)]. the aim of the study was to detect and to analyse the biochemical diversity of the entrococci strains in tehran sewage and to determine the genetic characterization of vre. methods: a total of isolates of enterococci were selected on me agar medium. all of the isolates were identified at the species level by the common biochemical tests. drug susceptibility test of isolates was done by disk diffusion method with antibiotics vancomycin, erythromycin, gentamicin, tetracycline, chloramphenicol and ciprofloxacin. the mic was also done by macrobroth dilution assay. analysis of the plasmid profiles and the pcr tests for vana and vanb genes were done. methods: we studied vre isolates collected in the north and center of portugal ( portugal ( - from: (i) clinical isolates from hospitals in different cities, (ii) faecal samples from healthy volunteers, (iii) river water samples, (iv) samples collected downstream of hospital sewage water, (v) samples from urban sewage water, (vi) swine faeces (vii) poultry food samples for human consumption. identification and characterization of vancomycin resistant genes vana, vanb, vanc and vanc were determined by a multiplex pcr. the backbone structure of tn was characterized by a pcr overlapping assay ( overlapping fragments), and further sequencing. conclusion: beta-lactamase production among hi strains has declined significantly since among children attending daycare centres as antibiotic prescriptions fell among this population. results: the mic distribution of am showed % of strains (n = ) with a mic > mg/l and % with a mic of > mg/l, indicating that resistance to am is still relatively rare and does increase as compared to nethmap . the lognormal distribution of both am and amc ( strain r) extended to mg/l but showed tailing to mg/l. this may indicate hidden less susceptible strains but could equally well be explained by testing circumstances, since the left part of the mic distribution showed comparable tailing. all strains were susceptible to moxifloxacin, levofloxacin and cefotaxim. the lognormal distribution of sxt extended to . mg/l with % of strains showing higher values. doxycyclin resistance was less than %. most of the strains were resistant to clarithromycin and azithromycin with a mic > . mg/l for both. conclusions: resistance of hi to common antimicrobials in the netherlands is still low and does not increase. objectives: s. pneumoniae (sp) and h. influenzae (hi) are the two most common pathogens associated with community-acquired pneumonia. changes in the prevalence of resistance or multidrug resistance (mdr) among these pathogens have important therapeutic ramifications. the global surveillance initiative is a longitudinal study that benchmarks antibacterial resistance among respiratory pathogens. methods: during , sp and hi were isolated from patient specimens collected at hospital laboratories in france (fr), germany (ger), italy (it), spain (spa), and the united kingdom (uk). isolates were centrally tested by broth microdilution against lev, penicillin (pen; sp only), azithromycin (azi), erythromycin (ery), clindamycin (cli), ceftriaxone (ctx), cefuroxime (cfx), and trimethoprimsulfamethoxazole (tmp-smx) (nccls, ) . data were analysed according to pen resistance, mdr, and b-lactamase status. mdr was defined concurrent resistance to ‡ of the following agents: ctx, cfx, ery, lev, pen, and tmp-smx. results: for sp, pen r was . % in ger, . % in the uk, . % in it, . % in spa, and . % in fr. azi r was . % in the uk, . % in ger, . % in spa, . % in it, and . % in fr. overall, lev r was rare (£ %) and mic s = mg/l in all countries. . % of isolates were susceptible to all of the drugs tested, the most common phenotype encountered. the prevalence (%) of mdr among sp ranged from . in uk to . in fr. resistance to pen, ery, cfx, and tmp-smx was the most prevalent mdr phenotype found in europe. overall . % of mdr sp were susceptible to lev. for hi, b-lactamase rates varied by country from . % in it to . % in fr. based on mic lev and ctx were the most active agents tested against hi, regardless of b-lactamase status. conclusions: lev showed potent activity against sp and hi. for sp, lev activity was independent of resistance to pen or mdr phenotype. lev maintained consistent activity against sp based on mic , regardless of country studied. antimicrobial surveillance data from studies such as the global offer guidance to physicians for empiric prescribing. sxt was obtained ( sxt was obtained ( - . conclusions: our results suggest that beta-lactamase production does not constitute a threat in hi therapy since values were almost constant. although with an unregulated fluctuation on arnblp percentages, it seems that this mechanism is gaining importance in relation to beta-lactamase production. thus, we conclude the need to be aware of arnblp, as these strains are difficult to detect using the nccls ( ) breakpoints. further molecular studies of the resistance genes responsible of this resistance mechanism are needed. resistance of beta-lactamase producer strains, to other antibiotics decreased during the period of study, due to the diminished use of these antibiotics. this study shows the importance of monitoring antibiotic resistance in hi in order to detect emerging mechanisms. antimicrobial susceptibility of respiratory haemophilus influenzae strains in northern greece k. koraki, p. karapavlidou, d. sofianou (thessaloniki, gr) objectives: to investigate the antimicrobial susceptibility of haemophilus influenzae, one of the most frequent bacterial pathogens of respiratory tract infections. treatment of these infections is most often empirical and considerable geographical resistance variation has been reported. methods: eighty h. influenzae strains were collected from respiratory tract specimens (sputum, bronchoalveolar lavages, endotracheal secretions) in a -year period ( ) ( ) ( ) ( ) ( ) . identification was made by colonial morphology, gram staining characteristics, x-and v-factor requirements and api nh (biomerieux, france). antibiotics were selected to reflect representative current treatment options and susceptibility was determined by kirby-bauer disc diffusion method on haemophilus test medium according to nccls guidelines. results: out of the h. influenzae strains were isolated from children and from adults. % of isolates came from children admitted to the intensive care units and . % from cystic fibrosis patients. a seasonal trend was reported for infections since . % of isolates were collected during springtime and % during autumn months. overall ampicillin resistance was . % and resistant strains were isolated exclusively from children. ampicillin resistance was doubled among cystic fibrosis patients ( . %). all isolates were susceptible to amoxicillin/clavulanate, chloramphenicol, ciprofloxacin and imipenem. the rank order of cephalosporin activity was cefotaxime and ceftriaxone ( %) followed by cefuroxime and cefaclor ( . % and . % respectively). trimethoprim/ sulfomethoxazole was active against . % of isolates while erythromycin was the least potent antimicrobial agent with % of isolates being susceptible to it. no multiresistant phenotypes were detected. conclusion: our results demonstrated that ampicillin resistance among h. influenzae in our area is still relatively low and overall antibacterial susceptibility rates are high. knowledge of antimicrobial resistance among these pathogens is imperative for physicians to choose the most appropriate therapeutic agent. results: nosocomial gram-negative uropathogens were studied. most common uropathogens were p. aeruginosa ( . %), e. coli ( . %), k. pneumoniae ( . %), followed by a. baumannii ( . %), enterobacter spp. ( . %), s. marcescens ( . %), proteus spp. ( . %) and other gram-negative rods ( . %). resistance rates (i+r, %) among p. aeruginosa were: gentamicin - %, levofloxacin - %, ciprofloxacin - %, cefoperazone - %, cefoperazone/sulbactam - %, cefepime - %, piperacillin - %, amikacin - %, ceftazidime - %, imipenem - %, meropenem - %, piperacillin/tazobactam - %, polymyxin b - %. resistance rates (i+r, %) among e. coli were: piperacillin - %, ticarcillin/clavulanic acid - %, amoxicillin/clavulanic acid - %, ciprofloxacin - %, gentamicin - %, moxifloxacin - %, levofloxacin - %, cefoperazone - %, ceftriaxone - %, cefepime - %, ceftazidime - %, cefoperazone/sulbactam - %, piperacillin/tazobactam - %, amikacin - %, all strains were susceptible to ertapenem, imipenem, meropenem. resistance rates (i+r, %) among k. pneumoniae were following: piperacillin - %, cefoperazone - %, ceftriaxone - %, gentamicin - %, amoxicillin/clavulanic acid - %, cefepime - %, ceftazidime - %, ciprofloxacin - %, piperacillin/ tazobactam - %, moxifloxacin - %, cefoperazone/sulbactam - %, levofloxacin - %, amikacin - %, ertapenem - %, imipenem and meropenem were active against all isolates. conclusion: p. aeruginosa, e. coli and k. pneumoniae are the main gram-negative uropathogens in russian icus patients. imipenem, meropenem, ertapenem showed prominent activity against e. coli and k. pneumoniae. cefoperazone/sulbactam, piperacillin/tazobactam, amikacin exhibited considerable activity versus e. coli, while k. pneumoniae were more resistant to them. p. aeruginosa were highly resistant to all tested antimicrobials except polymyxin b, thus leaving virtually no choices for therapy in terms of acceptable patient safety. results: overall gram-negative anaerobic bacteria from patients were studied. isolation sites were represented by intraabdominal - ( . %), soft tissue - ( . %), prostate fluid - ( . %), bone - ( . %), and dental - ( . %) infections. susceptibility of ( . %) prevotella spp., ( . %) bacteroides spp. (predominantly b. fragilis group - strains), ( . %) fusobacterium spp., ( . %) porphyromonas spp., and ( . %) veillonella spp. to ampicillin, clindamycin, metronidazole, imipenem, ertapenem, amoxicillin/clavulanic acid and cefoperazone/sulbactam was determined. all species were susceptible to carbapenems. in prevotella spp. there were % and % strains resistant to ampicillin and clindamycin and % of strains with intermediate resistance to metronidazole. among bacteroides spp. % of strains were resistant to ampicillin and % to clindamycin. no resistance to metronidazole was detected in bacteroides spp. objectives: the objectives of this study were to: analyse our current blood culture practice; describe the frequency of occurrence and antimicrobial susceptibility of bloodstream infections (bsi) isolates; determine the contamination rate. methods: we performed a prospective survey of all positive blood cultures received in the department of microbiology of tartu university hospital ( beds) in . blood culture system used was bactec . duplicates within one week were excluded. isolates were identified using conventional microbiology methods and susceptibility tests were those recommended by nccls. to determine extended spectrum beta-lactamase (esbl) producers an e-test with cefepime and cefepime combined with clavulanic acid was used. nosocomial infections were defined according to cdc criteria. results: during study period blood culture bottles were received, comprising blood culture sets ( . sets per patient-days). these resulted in ( . %) positive blood cultures, ( . %) were considered contaminants and contamination rate was . %. a total of bsi episodes involving patients were identified and ( %) of these were nosocomial. the incidence of nosocomial bsi (n-bsi) and community-acquired bsi (ca-bsi) was . and . per patient-days, respectively. polymicrobial bsi was detected in patients. among n-bsi dominated coagulase-negative staphylococci ( / . %), staphylococcus aureus ( / . %), klebsiella spp. ( / %), and escherichia coli ( / . %). the most frequent pathogens of ca-bsi were e. coli ( / . %), s. aureus ( / . %), haemophilus influenzae ( / . %), and streptococcus pneumoniae ( / . %). susceptibility to oxacillin of s. aureus and cons was % and . %, respectively. all s. pneumoniae isolates were susceptible to penicillin. . % of e. coli strains were susceptible to ciprofloxacin, . % to ampicillin, and % to gentamicin. susceptibility of klebsiella spp. to both ciprofloxacin and gentamicin was . %, and to ampicillin . %. . % of klebsiella spp. and none of e. coli isolates were esbl-producers. the susceptibility patterns of n-bsi and ca-bsi pathogens were similar to each other. conclusion: compared to west and north european countries our number of blood culture sets per patient-days is low. this may explain the relatively low incidence of bsi. the interventions to reduce contamination rate need to be implemented. the susceptibility among bsi isolates was high. recent outbreaks of c. difficile associated diarrhoea (cdad) reported in north america, united kingdom and the netherlands have emphasized the importance for an ongoing surveillance of cdad. the aims of the present study was to determine the epidemiology of cdad over the past years and the rate of nosocomial transmission in our acute care hospital ( -beds). materials and methods: all the cases of cdad diagnosed between january st and december st were retrospectively reviewed. a cdad case was defined as diarrhoea in hospitalised patients with a positive result for c. difficile cytotoxin or with a positive toxigenic culture. cdad was considered as severe if patient fulfilled at least of the following criteria: fever > . c abdominal pain or leukocyte count > , /mm or if the patient had an endoscopically proven pseudomembranous colitis or complications (toxic megacolon, perforation…). cdad was considered as community acquired if the diarrhoea occurred in patients within h after admission and if the patient had no history of hospitalisation in the previous months, otherwise cdad was considered as nosocomial. all the strains were serogrouped and characterized by toxinotyping and pcr-ribotyping. detection of toxin a, toxin b and binary toxin was performed by pcr. results: cases of cdad were diagnosed: clinical charts could be reviewed and strains were studied. global incidence of cdad was . per thousand discharges with higher rates in and . diarrhoea was community acquired in % of patients. for patients with nosocomial cdad, transmission of the strain from patient to patient (i.e. strain with the same serogroup and pcr-ribotype than the strain from another patient hospitalised in the same ward in the previous months) was demonstrated in . % of cases. binary toxin was positive in % of strains. binary toxin was associated to a more severe diarrhoea (p < . ) and to a higher case fatality (p < . ). a specific clone accounted for % of all the strains (serogroup h, pcr-ribotype '' '') but this clone was found both in nosocomial or community cases. three strains belonged to toxinotype iii but further investigations are needed to know whether these strains correspond to the hypervirulent strains involved in recent outbreaks. conclusion: incidence of cdad is low in our hospital and cross infection is limited. these results also suggest that strains with binary toxin might be more virulent. the development and application of a new exact typing method for clostridium difficile: multilocus variable number of tandem repeat analysis objectives: to study the epidemiology of clostridium difficile, a typing method with a higher discriminatory power, typeability and reproducibility than currently available methods is required. multi-locus variable number of tandem repeat analysis (mlva) is a new candidate technique, that has already been tested successfully on a number of bacterial and fungal species. using the whole genomic sequence, we developed mlva for c. difficile and compared the method to standardized pcr-ribotyping. additionally, mlva was tested on a collection of the new emerging hypervirulent pcr-ribotype strains. methods: short tandem repeat loci ( to bp) were identified using tandem repeat finder v . on the genome of c. difficile strain . amplification of the repeats was performed using a single pcr-protocol. pcr-fragments were analysed using multicoloured capillary electrophoresis on an abi , with a rox -marker as internal marker for each sample. the number of repeats per fragment was subsequently determined.the discriminatory power of the mlva was tested on reference strains representing serogroups and toxinotypes. the ability to subtype specific pcr-ribotypes was investigated with subtypes of pcr-ribotype (rep-pcr types - ), tcda-/tcdb+ strains of pcr-ribotype , and strains belonging to pcr-ribotype . of these type strains, were isolated from outbreaks and from endemic cases. results: a total of regions with short tandem repeats were identified. mlva discriminated all reference strains and the known reference strains of pcr-ribotype (rep-pcr - ). two mlva-types were recognized among tcda-/tcdb+ strains; the differences were present in only one of the repeat-regions. of pcr-ribotype strains, outbreak-related strains were identical to each other. interestingly, two endemic type strains differed from the other strains in of the regions. conclusion: mlva is a highly discriminatory genotyping method for c. difficile and is capable to subtype various crribotypes. mlva is also an important new tool to study the epidemiology of the emerging pcr-ribotype strains. comparative study of clostridium difficile diarrhoea in elderly patients treated with moxifloxacin versus amoxycillin for lower respiratory tract infections l. mooney, m. wilcox (leeds, uk) fourth generation fluoroquinolones such as moxifloxacin have improved anti-anaerobic activity. consequently, these new agents could induce c. difficile infection (cdi) by inhibition of 'protective' anaerobic flora. recent reports have suggested such an association. however, further studies are warranted to determine the risk of cdi in elderly in-patients treated with these agents, and notably where exposure to cd is measured/ controlled. methods: we prospectively investigated the propensity of moxifloxacin (mox) or amoxycillin/macrolide (aml/mac) to induce cdi when used to treat lower respiratory tract infections (lrtis) in elderly in-patients, using a -ward, crossover design ( months total). patients prescribed mox or aml/mac were monitored for gastrointestinal symptoms. diarrhoea was assessed as due to cd, viral or other cause. relevant clinical data were collected. concurrent epidemiological surveillance was also performed to determine environmental exposure to cd. results: patients were studied, receiving mox and had aml/mac. univariate analysis indicated that there was no significant difference between mox and aml/mac patients in gender, age ( . vs . mean years, respectively), or duration of hospitalisation (total, prior to and post diarrhoea). duration of antibiotic therapy did not differ significantly between mox and comparator patients (either total days or days before diarrhoea onset). there was a significant association between mox and overall risk of diarrhoea. however, there was no significance between mox treatment and cd, viral or other cause of diarrhoea. risk factor analysis to inform on possible confounders was performed. initial epidemiological survey results indicate that there was no change in environmental exposure levels to cd on each hospital ward. molecular typing of all clinical and environmental isolates of cd is ongoing. conclusions: although recent reports have highlighted a risk of cdi associated with fluoroquinolones (and increased age), none have specifically studied hospitalised elderly populations prospectively and controlled for exposure to cd. diarrhoea occurs relatively frequently after antibiotic therapy in the elderly. mox was associated with an increased rate of diarrhoeal symptoms, but causes other than cdi explained this association. mox treatment was not significantly associated with cdi when compared with amox/mac treatment for lrti in elderly in-patients. prevalence and association of macrolidelincosamide-streptogramin b resistance with resistance to moxifloxacin in clostridium difficile strains isolated from symptomatic adults and children hospitalised in two university hospitals in warsaw h. pituch, d. wultanska, g. nurzynska, p. obuch-woszczatynski, f. meisel-mikolajczyk, m. luczak (warsaw, pl) objectives: clostridium difficile is the main aetiological agent of nosocomial diarhoea. clindamycin, penicillins, and cephalosporins have been associated with cdad. however, several case reports of fluoroquinolone-associated c. difficile diarrhea have been published. c. difficile strains usually exhibits susceptibility to metronidazole, and vancomycin. we describe prevalence and association of macrolide-lincosamide-streptogramin b (mlsb) type resistance with resistance to moxifloxacin of c. difficile strains isolated from adults and children. methods: eighty-three c. difficile strains recovered from adults and children hospitalised in two university hospitals were investigated (hospital : adults n = , and children n = ; hospital : adults n = ). toxin types were determined by commercial test for toxin a and cytotoxicity test for toxin b. tcda, tcdb were detected by pcr. mics of erythromycin, clindamycin, moxifloxacin, vancomycin and metronidazole were determined by e-test (ab biodisk, sweden). the ermb gene was detected by pcr. results: sixty-seven ( %) c. difficile strains were toxigenic. among these, were a+b+, and were a-b+. all strains were susceptible to vancomycin and metronidazole. high level resistance to erythromycin, clindamycin and moxifloxacin was found in %, %, % of the tested strains, respectively. twenty-one c. difficile strains harboured high level resistance to erythromycin, clindamycin and moxifloxacin, simultaneously. among these, all were a-b+ and were isolated from adults, only. twenty-one of the macrolide-lincosamide-streptogramin b (mlsb)-resistant a-b+ strains carried the erythromycin resistance methylase gene (ermb). conclusion: resistance against clindamycin, erythromycin and moxifloxacin among polish a-b+ c. difficile strains was very frequent. fluoroquinolone resistance is associated with resistance to mlsb antimicrobials. we suggest that increasing use of fluoroquinolones is selective pressure for clonal dissemination of a-b+ c. difficile strains. fluoroquinolones use is a strong risk factor for cdad in our hospitals. acknowledgement: this work was supported by the ministry of scientific research and information technology, grant no. p d . national surveillance to the incidence of clostridium difficile-associated diarrhoea in the netherlands s. paltansing, r. guseinova, r. van den berg, c. visser, e. van der vorm, e.j. kuijper (leiden, amsterdam, nl) objectives: the recent outbreaks of clostridium difficileassociated diarrhoea (cdad) due to the new emerging pcrribotype , toxinotype iii strains has renewed the interest of cdad as an important nosocomial infection. to determine the incidence of cdad in the netherlands, we conducted a prospective surveillance study in hospitals in the netherlands. clinical microbiology and infection, volume , supplement , methods: from may st to july st of , participating hospitals registered all patients diagnosed with cdad. a standardized questionnaire was devised to obtain patient information. faeces samples or isolated strains were sent to the reference laboratory at the lumc for culture and the presence of genes for toxins a and b (tcda and tcdb). pcrribotyping was performed according to the method of bidet and toxinotyping as described by rupnik et al. results: routine methods to diagnose cdad in laboratories included combinations of cytotoxicity tests ( %), enzymeimmunoassays ( %) and culture of toxinogenic strains ( %). in total, patients with cdad were reported. the overall incidence (median) of cdad was for , patient admissions and varied from to . of patients with cdad, % was community acquired. the median age of patients with nosocomial acquired cdad was years. of patients with cdad, ( . %) died during the study period. at least different pcr-ribotypes could be recognized among strains. type was identified in patients from hospital. toxinotyping revealed the presence of at least different types. of strains, % were tcda+/tcdb+, % tcda-/tcdb-and % tcda-/tcdb+. conclusions: the incidence of cdad in the netherlands is lower than reported in usa and canada, but varied considerably per hospital. the new emerging type was found in patients from hospital with a high incidence of cdad ( per , admissions). outbreak of clostridium difficile pcr-ribotype toxinotype iii in harderwijk, the netherlands objectives: since , several epidemics of clostridium difficileassociated diarrhoea (cdad) caused by c. difficile pcr-ribotype toxinotype iii have occurred in usa, canada, and the uk. in april , the first outbreak encompassing patients was observed in a medium large hospital of beds in the netherlands. the isolated strain was completely resistant to erythromycin and ciprofloxacin. the patient characteristics, predisposing factors and outcome of cdad were studied. methods: a case-control study was performed in patients and at random selected controls without diarrhoea who stayed at the same department as the patients when the diagnosis of cdad was made. standardized questionnaires were designed to collect data from the patient records and all surviving patients were interviewed months after the diagnosis. faeces samples were cultured for the presence of c. difficile and isolates were typed. results: the incidence of cdad increased from per , patient admissions in to . per , admissions in . between april and september , patients with cdad due to type were identified. of patients, ( %) died of which ( %) as a direct result of cdad. eleven ( %) patients experienced one or more relapses. the average age of the cases was yrs, . % of the patients was male. in a multivariate analysis, antibiotic use (or . , p < . ), duration of hospital stay (cases days, controls days; p < . ) and tube feeding (or . , p = . ) were found to be significantly associated with cdad. in particular, the use of ciprofloxacin (or . , p < . ) and cephalosporins (or . , p < . ) were associated. no association was found between the use of protonpump inhibitors and the risk of cdad. the use of erytromycin was significantly higher in cases ( . %) than in controls ( . %) in a univariate analysis (p < . ), but this relation was not significant in a multivariate analysis. conclusion: antibiotic use (especially ciprofloxacin and cephalosporins), duration of hospital stay and tube feeding were significantly associated with cdad caused by c. difficile type , toxinotype iii in the netherlands. we could not confirm the previously described relation between use of protonpump inhibitors and risk of cdad. clostridium difficile pcr ribotype , toxinotype iii in the netherlands objectives & methods: shortly after the reports in june of clostridium difficile pcr ribotype , toxinotype iii in england, this more virulent type was also detected in the netherlands. in response, the dutch centre for infectious disease control has undertaken measures to monitor and control the outbreak. c. difficile guidelines for infection control and treatment were formulated, separately for hospitals and nursing homes. the leiden university medical centre serves as a reference centre for diagnostics and typing of c. difficile. laboratories are encouraged to send in samples for typing in case of a clear rise in the incidence in c. difficile, rapid spread, or several clinically suspect cases.organisation-based surveillance was set up: questionnaires are sent monthly to institutions with c. difficile associated diarrhoea (cdad) outbreaks to obtain information on incidence, c. difficile testing strategies, antibiotics use and control measures taken.measures taken in hospitals dealing with an outbreak of type include: treatment of cdad with vancomycin in stead of metronidazole, emphasis on frequent and thorough cleaning and disinfection, isolation of all patients with diarrhoea until tested negative for c. difficile toxin, cohort isolation of cdadcases if individual isolation capacity is exceeded and strong restriction of certain antibiotics, including fluorochinolones. results: until november st, , samples from institutions have been sent in for typing, resulting in type positives. epidemic spread of type has been detected in hospitals and one nursing home. furthermore, in retrospective studies in four hospitals isolated cases of type were detected. it became clear that in one region with three hospitals, the cdad incidence had already risen in , and , respectively . unfortunately, no samples from that period were available for typing. in the hospitals with epidemic spread of type , a wide range in the monthly incidence of cdad was observed, from to per , admissions during the outbreaks. the incidence in the pre-epidemic period varied from to (see figure) . conclusions: the outbreaks in hospitals are difficult to control: most hospitals continue to have new cases for a long period, although the incidence is decreasing in several hospitals. fortunately, once a c. difficile outbreak in a hospital is recognised, spread to other hospitals has not been observed. objectives: c. difficile is a major cause of antibiotic associated diarrhoea (aad) and colitis (c). the aim of this study was to determine the incidence of these infections in our hospital ( beds), during a period of months (march-october ) . methods: a number of liquid stools from equal adult patients (mean age y, m: , f: ) receiving broad spectrum antibiotics (especially cephalosporins) were plated in ccfa (oxoid) and anaerobic brucella agar (ba), after alcohol shock procedure. if the culture was positive, an immunochromatographic test was performed for toxin a (colorpactm toxin a, bd, usa). if the last test was negative, a rapid enzyme immunoassay was performed for toxins a+b (immunocard, meridian bioscience inc. cincinatti, ohio). results: c. difficile was isolated in / ( . %) samples. seventeen men (pathological -p, pneumological -pn, surgical -s, urologic -u, outpatients -o, , , , , respectivly) and women (p: , pn: , o: ) harbored c. difficile in their intestin. twelve out of strains ( %) produced toxin a, while the remaining ( %) produced toxin b. eleven patients had severe diarrhoea ( - days). one patient got endoscopic examination, which confirmed colitis findings. the two outpatients received oral cefuroxime in the preceding week of the positive culture. conlusions: ( ) the incidence of c. difficile infections in this study is among these reported in international bibliography ( . %). ( ) since toxigenic b c. difficile strains were demonstrated in half cases, the use of the tests detecting both toxins a and b by clinical laboratories is recommended.( ) molecular technics application (e.g. pfge and ribotyping) will offer a better knowledge of c. difficile spread in our hospital. assay of the cytotoxicity of stool samples to cells in tissue culture is commonly considered the 'gold standard' for detection of c. difficile toxin. however the method is slow and therefore its use can result in delayed patient treatment and implementation of infection control measures. we undertook a comparison of two microtitre plate-based elisa kits (techlab c. difficile tox a/b ii and meridian premier toxins a & b) and three rapid immunoassay card kits (remel xpect clostridium difficile toxin a/b, meridian immunocard toxins a & b and techlab tox a/b quik chek) with an in-house cytotoxin assay. all samples tested had been referred for routine microbiological examination. toxin tests were done on unformed samples from adult hospital in-patients and bone marrow transplant recipients and on samples where c. difficile toxin testing was requested by the referring clinician. all kits were used according to manufacturers' instructions. three hundred and thirty three specimens were tested using all five kits and cytotoxin assay. sensitivities and specificities were calculated both (a) assuming the cytotoxin assay to be the 'gold standard' (universally correct) test and (b) taking a concensus view that any sample with at least two tests positive is truly positive. data are shown in the table below. overall, the microtitre plate-based elisa kits were more sensitive than the rapid immunoassay card kits. the cytotoxin assay was negative for seven samples that were positive by at least two other tests. thus the plate-based elisa kits were also more sensitive (but less specific) than the cytotoxin assay if consensus data was used to judge true positivity. we conclude that some immunoassay kits offer an acceptable alternative to cytotoxin assays for the detection of c. difficile toxin, allowing more rapid diagnosis. location of the enterotoxin gene in strains of clostridium perfringens associated with gastroenteritis objectives: clostridium perfringens type a is a common cause of food poisoning and is also associated with non-food borne gastroenteritis including antibiotic associated, infectious and sporadic diarrhoea. the disease symptoms are due to an enterotoxin produced when the organism sporulates in the human small intestine. the c. perfringens entertoxin gene (cpe) has been shown to be located either on the chromosome or on one of two large plasmids and it is generally accepted that c. perfringens strains associated with food poisoning have a chromosomal cpe gene whilst strains isolated from non-food borne diarrhoea have a plasmid encoded cpe gene. spores from strains possessing a chromosomal cpe gene have been found to be far more heat resistant than spores from strains with a plasmid encoded cpe gene. heat resistant spores are more able to survive the cooking process and go on to cause food poisoning, thus explaining why most food poisoning strains have been found to have chromosomally located cpe genes. the purpose of this study was to determine the location of the cpe gene in a range of c. perfringens strains from the uk, including those from both food borne and non-food borne illness. method: a multiplex pcr assay described by miyamoto et al., ( ) was used to determine the location of the cpe gene in strains of c. perfringens isolates associated with food borne illness and strains associated with non-food borne illness. results: by multiplex pcr assay % of c. perfringens strains associated with food borne outbreaks in the uk were found to have a plasmid encoded cpe gene. these findings have not been described before. all strains associated with non-food borne illness had the cpe gene located on one of two plasmids, as anticipated. conclusions: a significant number of food borne outbreaks of c. perfringens food poisoning were found to be caused by strains of c. perfringens carrying a plasmid encoded cpe gene. since strains of c. perfringens with a chromosomal cpe and plasmid cpe genes have different physiological characteristics this may have a profound impact on their mode of transmission. references miyamoto, k., wen, q. and mcclane, b. a. ( ) multiplex pcr genotyping assay that distinguishes between isolates of clostridium perfringens type a carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an is -like sequence, or a plasmid cpe locus with an is sequence. journal of clinical microbiology , - . novel multiplex-pcr method for simultaneous detection of clostridium difficile toxin a and toxin b and the binary toxin (cdta/cdtb) genes applied on a danish cohort k.e.p. olsen, s. persson (copenhagen, dk) objectives: a new multiplex pcr method was developed for the detection of the clostridium difficile toxin genes: tcda, tcdb, cdta and cdtb. this method was applied on clostridium difficile strains isolated from danish hospitalised patients with diarrhoea in the period from april to october , in order to investigate the present toxin profiles and their correlation to sex and age. method: a -gene multiplex pcr method was developed for the simultaneous amplification of the four clostridium difficile toxin genes tcda, tcdb, cdta, cdtb and s rdna as an internal positive control. template dna was prepared from plate grown bacterial colonies by a simple boiling procedure, and amplicons were visualized by standard gel electrophoresis. results: three different toxin profiles were detected in the danish cohort: tcda+, tcdb+, cdta+/cdtb+; tcda+, tcdb+, cdta-/cdtb-and non-toxigenic tcda-, tcdb-, cdta-/ cdtb-. the prevalence of the binary toxin genes in this study was % of the clinical isolates.more than half of the strains ( %) were isolated from the elderly part of the population (> years), and % of these strains displayed the tcda+, tcdb+, cdta+/cdtb+ profile. of the non-toxigenic strains, % of the patients were females. one fourth of the strains isolated from children under years of age were non-toxigenic. in four patients, two different toxin profiles were obtained from independent faecal samples. conclusion: this method offers a one-step, rapid and specific identification of clostridium difficile toxin genes. this specific toxin profiling allows an evaluation of the pathogenic potential of the isolated clostridium difficile and surveillance of emerging toxin profiles. further studies of the isolated toxigenic clostridium difficile strains will include gene deletion analyses of the tcda and the tcdc (toxin regulating gene) which independently have been observed to cause enhanced pathogenicity. prevalence of clostridium difficile-associated diarrhoea in hospitalised patients with nosocomial diarrhoea in university of medical sciences hospitals, tehran, iran objectives: this study was aimed at determining the prevalence of clostridium difficile associated diarrhoea in hospitalized patients with nosocomial diarrhoea at three university hospitals in tehran from december to august . methods: during the study period, the stool samples of hospitalized patients with nosocomial diarrhoea were cultured and tested by stool cytotoxin assay, toxigenic culture and also of the samples were examined by enzyme immunoassay. results: in ( . %) of samples c. difficile grew and stool samples (prevalence: . %) were toxin positive by stool cytotoxin assay, enzyme immunoassay or toxigenic culture. there were no significant relationships between c. difficileassociated diarrhoea and sex and age of patients. the results of the present study showed that among requested samples the highest percentage of c. difficileassociated diarrhoea was observed from the transplantation department ( . %), followed by icu and paediatric section. objectives: the prevalence of toxigenic clostridium difficile (c. difficile) has been reported about - % in korea. toxin a(-)/ toxin b(+) variant c. difficile strain is also important in nosocomial c. difficile infection. however, characterization of clostridial toxin (toxin a, toxin b) had not been studied. methods: we used pcr for toxin a and toxin b genes in c. difficile isolates from patients admitted in three tertiary hospitals during january to december, . primers for toxin a genes were nk -nk , nk -nk and nk -nk and toxin b gene was nk -nk . results: toxin a and toxin b positive rates using nk -nk , nk -nk and nk -nk were concordant and ranged from . % to . % in hospitals. the proportions of non-toxigenic strains were - %. however, we could differentiate toxin a(-)/toxin b(+) variants using nk -nk primers. the proportion of toxin a(-)/toxin b(+) c. difficile variants were . %, . % and . % in hospitals respectively. objective: administration of antibiotic drugs has long been known to cause alterations in the gut ecosystem. in some patients, these alterations may create a niche that allows the overgrowth of some pathogens such as clostridium difficile, the main causative agent in nosocomial infectious diarrhoea. a predictive tool to assess the risk of development of clostridium difficile, would be of utmost clinical relevance. it remains to be determined whether specific patterns in pre-existing gut microbiota can predict the risk of onset of clostridium difficile, upon initiation of antibiotic treatment. using samples from subjects enrolled in a previously published clinical study on antibiotic-associated diarrhoea (aad), we investigated the potential relationship between their dominant faecal microbiota and the subsequent development of clostridium difficile when subjects received antibiotics. methods: temporal temperature gradient gel electrophoresis (ttge) was used to assess dominant species distribution in gut microbiota. each electrophoregram was digitised from the migration distances and a regression model [partial least square-discriminant analysis (pls)] was built to investigate the correlation between pre-treatment dominant faecal microbiota and the acquisition of clostridium difficile during antimicrobial chemotherapy. results: this pls model could explain % of the subsequent onset of clostridium difficile. this result supports the concept of ''permissive'' flora with preliminary data focusing on clostridium coccoides-phylogenetic group. conclusion: to our knowledge it is the first time that dominant faecal microbiota is found to heighten susceptibility to the subsequent onset of clostridium difficile upon initiation of antibiotic treatment. these findings insinuate that strategies reinforcing the control of dominant faecal microbiota at homeostasis would be of clinical relevance. this study has been partially financed by biocodex laboratories. objectives: selective therapy of c. difficile diarrhea (cdd) requires the reduction of pathogen counts in the colon, but spare the normal flora. to determine if par is selective for cdd, serial stool samples were collected at study entry, at day , and weekly x during the conduct of a phase a study of cdd treatment. methods: patients (n = ) were randomized to receive , or mg twice daily of par for days. no prior therapy was given to patients; receive or doses of standard therapy. as treatment controls, additional patients were treated with vancomycin mg qid for days. five well persons donated stools as normal flora controls. fresh stool samples were cultured - , , , for c. difficile vegetative and spore forms; faecal filtrates were tested for cytotoxin b by cell assay. strains were characterized by tcda/b, ermb, cdta/b pcr and by ribotyping. at study entry and day , aerobic and anaerobic faecal flora cultures, diluted - , , , , were examined for major floral shifts. since bacteroides group organisms are ubiquitously present and cultivable, this genera was selected as a indicator of the integrity of the microbial flora. results: at study entry, mean log cfu + sd vegetative counts of c. difficile (all par patients) were . + . , range - . ; at day , with the exception of one patient receiving mg, all other patients had c. difficile quantitative counts reduced < log /gm faeces. vancomycin was similarly effective. at study entry, bacteroides group counts were < , - , & . - log cfu/gm in~ / each of patients. all normal stools showed complex, multi-genera in high counts, with - bacteroides group species > log cfu/g. mean + sd of log cfu of bacteroides group counts/g feces wet weight at study entry and day for mg/day (n = ) were . + . / . + . (p = . , wilcoxon matched pairs signed-ranks test, tailed); for mg/day (n = ) were . + . / . + . (p = . ); for mg/day (n = ) were . + . / . + . (p = . ); and for vancomycin (n = ) . + . / . + . (p = . ). conclusion: patients with cdd have variably impaired normal flora. par was effective in all dosages in eradicating c. difficile. a dose-dependent reduction in bacteroides counts was not observed. vancomycin significantly reduces bacteroides counts during cdd treatment. par is effective against c. difficile in-vivo, and is relatively sparing of the normal flora. results: the three rt pcr assays were able to detect all enterovirus strains in cell culture supernatants. however the detection limit of the mgb rt pcr was to log more sensitive in out of dilutions assays of vc supernatants compared to the rab and ver rt pcr. all ver and mgb negative csf were vc negative. thirty-two csf specimens from patients suspected of viral meningitis were positive by all rt pcr ( . %), whereas only were found positive by vc ( . %). the rab rt pcr failed to detect csf confirmed positive by vc ( echo and non typable ev). among samples positive by rt pcr, sensitivity of ver, mgb and rab was respectively %, % and . %. conclusion: in our laboratory, mgb rt pcr has a good correlation with ver rt pcr whereas rab rt pcr is less sensitive especially for the detection of echovirus . the mgb rt pcr seems to be the most sensitive of the rt pcr. further studies, including more ev strains should help to precise the sensitivity of this assay. a. dalwai, s. ahmad, e. hussein, a. pacsa, w. al-nakib (kuwait, kw) objectives: enteroviruses generally share tissue tropism and present with overlapping disease spectrum, however certain enteroviruses may be over represented in certain diseases than others. coxsackievirus a though has been reported to cause several diseases such as febrile illness, herpangina, aseptic meningitis and acute flaccid paralysis, the frequency was very low. the study aimed to determine the prevalent enteroviruses causing non-specific febrile illness, aseptic meningitis, encephalitis, neonatal disease and myositis, in kuwait. it also aimed to study the association between a certain enterovirus and a particular disease and its severity. methods: diagnosis of enteroviral infection was based on detection of enteroviral rna by semi-nested rt-pcr of a portion of the 'utr of the enteroviral genome followed by southern hybridization with an enterovirus specific probe to confirm the results. the enterovirus was genotyped by sequencing of the 'utr, the vp and a portion of the vp encoding regions, and the sequence was analysed by blast analysis, clustalw alignment and phylip phylogenetic analysis package. results: enteroviruses were the only etiological agents detected in % ( ) of disease cases investigated. coxsackievirus a was identified to be the second most predominant enterovirus ( %; of cases genotyped) associated with disease, after only echovirus ( %; / ). although identified in all the diseases investigated, coxsackievirus a occurred less frequently in cns disease cases ( %; / ) than in febrile illness cases ( %; / ). in a preliminary study, it was also predominantly detected in % ( / ) of myositis cases. the 'utr of this virus showed % homology with that of coxsackievirus a prototype strain (parker strain) whereas the vp and the adjoining region showed greater homology to human enterovirus b genotype sequence. conclusions: coxsackievirus a was determined to be an emerging enterovirus associated with different diseases in kuwait. it was frequently represented in mild febrile illness and myositis cases than in cns disease suggesting that the isolate might be less neurovirulent. molecular analysis suggests that the isolate might have emerged due to recombination between coding and non-coding segments of coxsackievirus a and human enterovirus b group genomes. acknowledgement: supported by research administration project grants mi / , ym / and college of graduate studies, kuwait university. the new proposed enterovirus type is causing meningitis in spain introduction: several new proposed enteroviruses (ev) have been recently described, including the named ev [ ] . a total of isolates of this serotype were identified from to in america, africa or asia associated mainly with acute flaccid paralysis or unspecified disease. objective: to determine if this new serotype circulates in spain and what type of disease produces. methods: a total of ev isolates coming in to the spanish enterovirus reference laboratory were studied both by micro neutralization assays and by typing pcr [ ] . in the isolates in which ev was suspected by the mentioned methods complete vp gene was amplified and sequenced with specific designed primers. results: four isolates from two different regions of spain were identified as ev (more than % of homology with the published sequences). three of them corresponded to aseptic meningitis in children and were isolated from csf. discussion: the present work demonstrates that this new proposed virus circulates also by europe and is associated to aseptic meningitis. till the moment it seems that is represented in a minor proportion ( / studied), however the possibility of spreading of this viral infection should be considered, as evs may behave in that way, as previously have been demonstrated [ ] . objectives: rotavirus is the most important cause of severe gastroenteritis in infants and young children through the world and is responsible of , deaths annually, mostly in developing countries. therefore, development of rotavirus vaccine is a high priority. rotavirus strains with g types account for the majority of the diarrhoea episodes. recently, a monovalent g attenuated rotavirus vaccine was licensed in mexico. in view of a hypothetical introduction of such vaccine in europe, we investigated the variability over time of vp antigenic genes of g rotavirus strains in our area. methods: fifty strains were selected from a total of g strains obtained from children of less than years of age hospitalised with acute gastroenteritis at the ''g. di cristina'' children's hospital of palermo in the period - . the selected strains were genotyped by rt-pcr and of them were submitted to vp gene sequence analysis. results: all but one of the strains were genotyped as g p( ). the vp sequences of of them were distributed into lineages i and ii. lineage i included strains from different years in the range - . lineage ii included strains from different years in the range - . the degree of similarity among the nucleotide sequences of italian strains in each lineage were comprised between % and %. an alignment of the deduced amino acid sequences showed major lineage specific amino acid changes in the variable antigenic regions with respect to the reference wa strain. conclusions: sequence analysis indicated that in palermo there was co-circulation of g strains belonging to two different lineages. overall, the g strains showed a high degree of similarity inside each lineage and shared specific amino acid modifications. the antigenic differences between circulating strains might permit them to escape neutralization and persist in the infantile population. our results suggest that rotavirus strains belonging to the two g lineages should be both included in a rotavirus vaccine preparation. epidemic spread of recombinant noroviruses with four capsid types in hungary objectives: noroviruses (''winter vomiting diseases'') are the predominant etiological agent in hungary and common pathogen worldwide in outbreaks of gastro-enteritis in humans. noroviruses are genetically diverse group of viruses with multiple genogroups (gg) and genotypes. more recently, naturally occurring recombinant noroviruses were identified. these viruses had a distinct polymerase gene sequence (orf , designated ggiib/hilversum) and were disseminated through waterborne and food-borne transmission in europe. our aim was to characterize these emerging recombinant noroviruses causing outbreaks of gastro-enteritis in hungary. methods: stool and rna samples -from norovirus outbreaks between january and may -containing ''ggiib/ hilversum polymerase'' (ggiib-pol) were selected for analysis of the viral capsid region (orf ) by reverse transcriptionpolymerase chain reaction (rt-pcr) followed by sequence-and phylogenetic analysis. results: forty ( . %) of confirmed norovirus outbreaks were caused by the new-variant lineage with the ggiib-pol. viral capsid region was successfully characterized in ggiibpol outbreaks. four different recombinants were detected with capsids of hu/nlv/ggii- /mexico/ (n = , . %), hu/ nlv/ggii- /snow mountain/ (n = , . %), hu/nlv- /ggii/hawaii/ (n = , . %) and hu/nlv/ggii- / lordsdale/ (n = , . %). interestingly, outbreaks caused by recombinant ggiib-pol strains mostly associated with outbreaks among children ( . %) and had non-winter seasonality. conclusions: epidemic spread of emerging multiple recombinant norovirus strain ggiib-pol were detected in hungary that became the second most common norovirus variants -next to the endemic ggii- /lordsdale virus -causing epidemics of gastroenteritis in the last . years. the respiratory infections are the most common diseases in the world being the origin of a great morbidity and mortality especially in infants and elderly. ( ) human metapneumovirus (hmpv) was first described in dutch children with acute respiratory tract infections (artis) in june . ( ) very limited studies data are available from tropical and developing countries. we sought to determine the role of hmpv in upper and lower respiratory tract infections in cuban patients and correlated the presence of virus with clinical characteristics of the disease. between october to september clinical samples received from the national surveillance program of artis at the national reference laboratory of respiratory viruses, for virological study, were used to detect hmpv by rt-nested pcr, amplifying a conserved fragment of nucleotides in the polymerase gene. we found rna hmpv in . % of samples from the patients with artis. . % of individuals who tested positive for hmpv were under months of age. patients with evidence of hmpv had symptoms consistent with either upper or lower respiratory tract disease or both. . % of hmpv positive individuals were detected during august-october (table ). the results of this preliminary study shows that hmpv is present among cuban patients with arti. constitute the first report of the frequency of hmpv infection in a non-preselected group of cuban patients with ages ranged from months to years old. it should be noted that this is the first report of hmpv infection in central america and in the caribbean region, further confirming the worldwide distribution of the virus ( ) ( ) ( ) . detection of human metapneumovirus in paediatric nasopharyngeal aspirates by a taqman minor groove binder probe assay: a one-year prospective study in belgium w. verstrepen, p. bruynseels, a. de smet, a. mertens (antwerp, be) objective: human metapneumovirus (hmpv) has a relative high incidence in acute respiratory infections in children but is difficult to isolate in culture. the aim of the study was to decrease the number of undiagnosed viral respiratory infections in our hospital by means of a taqman minor groove binder (mgb) probe assay. methods: from october to september a total of nasopharyngeal aspirates from children presenting at our paediatric facility were analysed. rna extracts from specimens negative for rsv, parainfluenzavirus and influenzavirus with an (in) direct immunofluorescence assay (ifa) were subjected to a taqman mgb probe assay in parallel with a previously published taqman assay. results: of the specimens, ( %) were positive by ifa for either rsv ( ), parainfluenzavirus ( ), influenzavirus a ( ) or influenzavirus b ( ). hmpv was detected in ( . %) of the remaining specimens subjected to the newly developed pcr. of the patients with a positive hmpv assay, / ( . %) presented with respiratory symptoms. % of the positive specimens were from children less than year as compared to only % from children older than years. viral load was highest in children less than year. a prominent seasonal variation was noted since more than half of the positive specimens occurred during the months march and april. there was no significant difference in the proportion nor viral load of positive specimens from ambulatory patients, patients admitted to a general ward or patients requiring intensive care. as compared to the published taqman assay, diagnostic sensitivity and specificity were . % and . % respectively, whereas ppv and npv were . % and . %. method comparison (nccls guideline ep- a) failed to demonstrate a significant difference between both assays when the threshold cycle (ct) was between and . strongly positive specimens (ct < ) were associated with a lower ct using the published taqman assay. however, the new taqman mgb probe assay appeared to be more sensitive for weakly positive specimens (ct > ). conclusion: the number of viral respiratory infections confirmed in our hospital was substantially increased by means of the hmpv taqman mgb probe assay. the new assay is a reliable alternative to the previously published taqman assay for detection of hmpv in nasopharyngeal aspirates. nucleic acid sequence based amplification and molecular beacon detection for the real-time identification of respiratory syncytial virus in paediatric respiratory specimens r. manji, f. zhang, c. ginocchio (lake success, us) background: respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract infection in infants and young children, with bronchiolitis and pneumonia being the major clinical manifestations. the rapid diagnosis of rsv infections is of central importance for individual patient management (rational use of antibiotics and antiviral agents), hospital infection control and monitoring epidemiological disease patterns. this study included a technical validation and a retrospective clinical evaluation of a real time nasba assay for the detection of rsv a and rsv b in paediatric respiratory samples. methods: samples tested included: dilution panels of in vitro transcribed rna, local rsv isolates, isolates of common respiratory pathogens, and frozen respiratory specimens (nasopharyngeal aspirates, washes or swabs) from children (age range: d to yr) who were evaluated in the paediatric emergency department for respiratory disease. nucleic acid (na) isolation, amplification and detection were performed using the nuclisens easyq basic kit and nuclisens easyq rsv a+b reagents (biomérieux). specimen nas and a rsv specific internal rna control (ic) were co-extracted using nuclisens magnetic extraction reagents and the nuclisens minimag instrument (biomérieux) and co-amplified using a single rsv specific primer pair. included in the reaction were a rsv specific molecular beacon ( '-fam) and an ic specific molecular beacon ( '-rox). target amplification and continuous monitoring of emitted fluorescence were performed using a nuclisens easyq analyzer (biomérieux). results were compared to direct immunofluorescence (dfa) and/or viral culture using r-mix cells (diagnostic hybrids, oh). results: the limit of detection for rsv was rna copies/rxn and the % detection rate was copies/rxn. the assay was % specific for rsv with no cross reactivity to other respiratory pathogens. the nasba assay detected % more positive specimens than dfa and % more positive samples than vc. the npvs of the assays were: nasba . %, dfa . % and vc . %. the nuclisens easyq rsv assay demonstrated superior sensitivity to both dfa and viral culture for the detection of rsv a and b from respiratory specimens. the assay was easy to use, required minimal hands on time ( hr) and a faster time to results as compared to rapid culture ( hr vs. - hr). respiratory syncytial virus (rsv) is a major cause of acute lower respiratory tract infection in infants and young children. it has previously been shown that hrsv isolates can be divided into two antigens groups a and b. the g protein is the most divergent both between and within the two subgroups and appears to accumulate amino acid changes with time, suggesting evolution under selective pressure. our knowledge of the molecular epidemiology of rsv has so far been based mainly on studies done in the developed world with e temperate climate. very limited epidemiological data are available from tropical and developing countries, where rsv infections may follow a different pattern. in this report we examine the molecular epidemiology and evolutionary pattern of the g protein of both subgroups a and b rsv through consecutive epidemics in cuba. sixthly four nasopharyngeal swabs were collected from children under years of age with respiratory disease to different hospitals in cuba between and , to examine the molecular epidemiology and evolutionary pattern of the g protein of rsv. all samples collected from to were rsv subgroup a; however both subgroups co-circulate during . the cuban isolated from to showed a great homogeneity between them and were resemble to an ancient strain (long) with only five nucleotide differences, this also occur in and with two strain. furthermore was detected different size of g protein ( or for rsv a and for rsv b) due to change in stop codon used he genetic homogeneity of the cuban isolates ( ) ( ) ( ) and their resemble to an ancient strain such as long was an unusual finding in our country. in both subgroups was observed the predominance of strains with almost similar sequences. phylogenetic analysis for subgroup a strains showed that strains were cluster in different genotypes with virus isolated in different geographic regions. both subgroups co-circulated during and clustered whit south african strains that circulate during at the same time. point mutations in respiratory syncytial virus detected by lightcycler pcr and melting curve analysis u. germer, l. nielsen, k. boye, h. westh (copenhagen, dk) objective: the objective was to analyse rsv real-time pcrpositive isolates from clinical samples, which appeared to belong to three different groups according to melting temperature (tm) of the amplicons. the analysis was done according to genotypic and phenotypic difference and related to geographical distribution. materials and methods: nasopharyngeal aspirates were collected from children with respiratory distress in the city of copenhagen. viral rna extracted using the magnapure lc automated extraction system was amplified in a real-time rt-pcr previously described ( ) . five samples from each of the three groups with different tm's were selected for bidirectional dna sequencing using the rsv primers. sequences were analysed using chromas lite version . . results: a total of clinical samples were analysed. ( %) of the samples were positive and ( %) were negative for rsv. three distinctive groups with different tm's could be identified from the melting curve analysis. group (n = ) had a tm with a median of . °c, group (n = ) and (n = ) had lower tm's with a median tm of . °c and . °c respectively. sequence analysis of amplicons showed that the difference in tm was due to differences in genotype between the three groups. genotype and were closely related, differing only in two nucleic acids in position (c to t) and (a to t). both were silent mutations. only position is targeted by the probe. genotype and were both blasted to a complete genome sequence of respiratory syncytial virus subgroup a (genbank rsu ) with the highest identity score for genotype . genotype sequences were blasted to human respiratory syncytial virus mutant cp subgroup b (genbank af ). geographical analysis showed a higher prevalence of the mutant strain (genotype ) in the northern areas of the greater copenhagen area compared to central, southern and western areas (p = . ). conclusion: we found three genotypes of rsv according to the tm of the pcr product. two of the genotypes were closely related with only two point mutations and the same phenotype. genotype was mainly found in clinical isolates from the northern part of copenhagen, suggesting a local epidemic spread. objectives: biomerieux is developing a real-time nasba assay to detect influenza a and b rna in different kind of respiratory clinical samples, by using the nuclisens Ò easyq basic kit in combination with specific primers and molecular beacons. methods: nasal/throat swabs in transport medium from hospitalised children ( - yrs from edouard herriot hospital, lyon, france) were used for this evaluation. influenza rna is isolated using the nuclisens Ò minimag extraction. an internal control is added to the sample prior to nucleic acid extraction. the assay is designed to detect in a single tube, using a three-label approach, the internal control and both influenza a and influenza b rna. amplification reactions were performed in a nuclisens Ò easyq analyser allowing real-time detection. the results of the clinical samples were compared to cell culture results. results: among swabs tested, real-time nasba detected ( . %) samples for influenza a and ( . %) samples for influenza b. comparatively, by cell culture only ( . %) samples were identified as influenza a and non as influenza b. interestingly, influenza a positive sample identified by cell culture was found negative in real-time nasba. conclusions: the data showed that nuclisens Ò easyq influenza a/b assay detected % more influenza a virus than cell culture method. moreover, real-time nasba detected influenza positive samples, which were not detected by cell culture. with this assay a qualitative detection of influenza a and influenza b viruses in a single reaction can be done within hours. it provides a valuable alternative to cell culture method for the clinical management of patients with influenza infections. results: patients have developed mumps meningitis and patients were diagnosed with mumps meningoencephalitis. age limits were from to years and sex ratio m/f was / .clinical manifestations involved fever ( %), stiff neck ( %), nausea and vomiting ( %), headaches ( %), photophobia ( %) and neurological manifestations such as: equilibrium disorders and drowsiness ( %), convulsions ( . %), cerebellum syndrome ( . %). meningeal symptoms have occurred shortly after parotiditis in % of cases and before parotiditis in % of cases; the other cases have evolved without parotid swelling. other localizations of the mumps infection were: parotiditis ( %), pancreatitis ( %), submaxillitis ( %) and orchitis ( %). lumbar puncture yields csf containing between and wbc/mm . the predominating cells were usually lymphocytes, but % of the patients have polymorphonuclear leukocyte predominance at the first puncture. protein levels are normal to mildly elevated in all cases and hypoglycorrachia was founded in % of the patients. therapy for mumps meningitis was symptom-based (analgesics and antipyretics) in % of cases and glucocorticoid therapy in % of cases. conclusions: ( ) neurological involvement in mumps occurred in . % of cases; ( ) men are afflicted two times more often as women, but the age distribution is the same as for uncomplicated mumps; ( ) mumps meningitis was the only localization of the mumps infection in % of cases. mumps is acute generalized infection occurs primarily in schoolaged children and adolescents. most prominent manifestation of mumps is swelling and tenderness of salivary glands especially parotid gland. uveitis is a rare manifestation of mumps. here we present a mumps case complicating with uveitis. years old paediatric nurse was admitted to our emergency department because of headache and malaise. on physical examination bilateral parotid enlargement was noticed. opthalmology consulatation revealed anterior uveitis. local prednisolon and cyclopentholate treatment were prescribed. lumbar puncture revealed lymphocytic pleocytosis without hypoglychorachea and elevated protein levels. mumps igm was found positive. differential diagnosis made with other viral infections and sarcoidosis. her headache diminished day after the hospitalisation. uveitis responded very well to local therapy and patient got well in weeks. clinical and epidemiological aspects of a measles epidemic, bucharest, romania results: there were cases; sex ratio m/f: / . the mainly affected age group is under months ( . %) followed by months- years ( . %), - years ( . %), > years ( . %) and - years ( . %). . % cases were hospitalacquired (mostly in paediatric clinics), . % were communityacquired; in . % cases the source was unknown. the most common clinical features were fever ( %), rash ( %), conjunctival hiperemia ( . %), cough ( . %), micropoliadenopatia ( . %), diarrhoea ( . %). pulmonary complications were described in . % of cases; . % of them were bacterial pneumonia, . % were viral pneumonia. in . % of cases we diagnosed acute stomatitis, in . % bacterial conjunctivitis; in . % of cases -otitis; in . % of cases -pharingitis, and in one case ( . %) -urinary tract infection. . % of the patients were previously diagnosed and treated for pulmonary tb. all cases were confirmed serologically through detection of specific igm antibodies. patients ( . %) had severe clinical forms of measels. the evolution was good in all cases. conclusion: . this year in the south-east part of the country, evolves a measels epidemic with different features comparing to the previous one ( ) ( ) we investigated the recombinant proteins np and hn to develop new antigen with useful properties for applied in elisa test systems. methods: significant antigenic epitopes of nucleoprotein (np) and haemagglutinin (hn) measles virus strain edmonston were generated by computer analysis. using standard geneengineering techniques was evaluated two fusion peptides np and hn consist from only linear t-cell antigenic determinants. the virus-neutralization activity of hyperimmune serum on recombinant proteins was determined by plaque reduction neutralization test (prn). the level of specific igg in serum to genotypes a, d , and d of measles virus was determined by enzyme-linked immunosorbent assay (elisa). we used recombinant proteins np and hn as antigens for elisa. results: hyperimmune serum was collected from mice after immunization by np and hn recombinant proteins. the level of neutralize activity was measured in the prn assay with strain edmonston. the titre reached up to : . and : . for np and hn recombinant proteins, respectively. interestingly that, hyperimmune serum on recombinant protein np in elisa reacted both with np (titre : ) and with hn (titre : ), and in turn serum on recombinant protein hn reacted only with hn (titre : ). the estimation immunological properties of proteins with use of the panel of serum ( samples) collected from patients. the diagnosis of measles infection was confirmed in laboratory (by rt-pcr). the nucleotide sequences of rt-pcr products used for genotyping of mv. selective interaction of antibodies in elisa with recombinant proteins in relation to various genotypes is revealed. the interaction with genotypes a and d was expressed with high level of correlation whereas with genotype d any serum did not react authentically (as the control was used recombinant protein n of sars virus). conclusion: we have shown that neutralize antibodies formed hot only on superficial proteins such as hn, f and sh but also on core proteins such as np. our data demonstrate that the recombinant proteins np and hn could be a cost-effective alternative to current whole virus based elisas for surveillance for immunity to measles and could more efficient in detecting susceptibility to measles in relation to genotypes a and d . episode : a pregnant woman with thirty-eight week of gestation was hospitalized in obstetrics clinic with the complaints of fever, malaise, and severe vaginal bleeding. on admission, white blood cell count was /mm , haemoglobin was . g/l, platelet count was /mm . the level of ast was iu, alt iu, lactate dehydrogenase iu, and creatinin phosphokinase iu. the baby was delivered by cesarean section. in serum cchf igm was positive by elisa, and per oral ribavirin was administered after delivery. at the first day of delivery, the clinical and laboratory of findings of the baby were found to be normal. however, on his th day, he died because of massive bleeding. his cchf igm was found to be negative. episode : a pregnant woman with week of gestation was admitted to the hospital. her complaints were fever, malaise, headache, myalgia, nausea, vomiting, diarrhoea, and subconjuntival bleeding. in her laboratory investigation, white blood cell count was /mm , haemoglobin level was . g/l and platelet count was /mm . the level of ast was iu, alt iu, and ldh iu. in her serological analysis cchf igm and cchf virus -pcr was found to be positive. at the twenty six week of gestation in obstetric ultrasound, fetal intraabdominal fluid was visualized and amniocentesis was performed. in serological analysis of amniotic fluid cchfv-pcr was found to be negative. intraabdominal fluid had increased and scrotal edema was visualized at thirty eighth weeks of the gestation. after her vaginal delivery, baby was severely ill and was operated with the diagnosis of necrotizing enterocolitis. his laboratory findings were normal except high white blood cell count. on his fifth day, thrombocytopenia occurred and he died because of massive bleeding. his cchf igm and pcr were negative. conclusion: to our knowledge, these are the first episodes of intrauterine cchf infection. these episodes show that cchfv can transmit through placenta. obstetricians in endemic countries should consider cchf infection among the patients with massive bleeding and thrombocytopenia. objective: to detect the asymptomatic crimean congo hemorrhagic fever virus (cchfv) infections in an endemic area, and calculate the attack and the infection rate. methods: the study was performed in a cchf endemic region. the household members of the index cases were screened for cchfv igg and igm by elisa. the data related to risk exposure were obtained by a structured form. results: eleven index cases were admitted to the clinic, household members of these cases were screened. all the index patients had positive igm or pcr for cchfv. among the household members, three individuals had igg positivity (%), and only one patient had igm positivity. none of the screened individuals had symptoms. the mean age was (sd ), and % of the subjects were female. tick bite was detected a risk factor (p = . ) for cchv infection, whereas patient care and contact with body fluids of the patients were not (p > . ). eighteen patients had the history of tick bite, and became infected ( %), and five ( %) became ill. among the infected eight individuals, five became ill ( %). conclusion: although we consider that some of the patients do not notice tick bite, we can still suggest that the infection rate of the virus is rather high compared to similar diseases. tick bite is the major risk factor, in comparison to exposure to blood and body fluids of the infected cases. results: children were included in our study. distribution according sex was: . % female and . % male. median of age was years (iqr = ). during the follow-up study we recorded years when the number of cases increased. the distribution of cases among the study was: . % in , . % in , . % in , . % in , . % in and . % in . the proportion of paediatric patients also varied from; . % in , . % in , . % in , . % in , . % in and . % in . in panama city we recorded . % of the infants. we detected an increase in the number of patients in the rain season, from may till november. the mean of days between the onset of symptoms and the first blood sample was . days (ds: . ) a second sample was obtained in . % of our infants with an average time of . days (ds: . ). the frequency of classical symptoms related to dengue virus infection was: fever ( . %), severe headache ( . %), chill ( . %) rash ( . %), myalgia ( . %), retro-orbital pain ( . %), arthralgia ( . %), gastrointestinal symptoms ( . %), inflamed pharynx ( . %), cough ( . %), mild respiratory symptoms ( . %) and diarrhoea ( . %). in our infants the symptoms which were detected first were; fever, severe headache, chill, myalgia, retroorbital pain, arthralgia, mild respiratory symptoms, cough and inflamed pharynx. we did not observed differences on clinical features between girls and boys. however, we detected detected significative differences among symptoms when we compared infants who were £ years old with those who were older (p < . ). four of our patients died because of dengue hemorrhagic fever. conclusion: dengue is endemic in panama as in most tropical countries and is one of the world s major emerging infectious disease. more data about this illness are needed to elaborate sanitary programmes which contribute to control this infection. diagnosis of dengue infection by enzyme-linked immunosorbent assay and reverse transcriptionpolymerase chain reaction from oral specimens dengue. salivary elisa has been shown by various investigators to be useful for dengue diagnosis. we sought to perform a pilot evaluation of diagnostic value of elisa and pcr of oral brushes and saliva for dengue diagnosis in adults. methods: adults with acute fever and suspected of dengue infection admitted to our university hospital were enrolled. dengue diagnosis was made by standard elisa using serum or plasma. patients with negative elisa served as controls. buccal mucosal cells were collected for rt-pcr and saliva for both rt-pcr and elisa at least twice, - days apart. our elisa criteria for saliva were single igm > units or single igg > units or -fold increase in igg titre with the second titre > units for secondary dengue infection. criteria for primary dengue infection were the same as secondary infection plus igm:igg ratio of over . . results: cases and controls were enrolled. our country is endemic for dengue and thus there was no primary dengue adult case in this study. as the study was performed in hospitalized patients, most of the first samples were collected one day before or on the day of defervescence. the specificities of either methods and the sensitivity of elisa method for saliva were %. sensitivities were approximately - % for rt-pcr using buccal cells or saliva specimens. however, a combination of rt-pcr results for both types of oral specimens gave a sensitivity of %. the results are summarised in the table. conclusions: collection of oral specimens is less invasive and may be more acceptable in certain situations. a single, acute specimen is adequate for diagnosis by rt-pcr. our specimens, however, were collected late in the course of illness which affected the sensitivity of rt-pcr's. earlier specimens may give a better yield. a study in paediatric patients is needed to assess the value of these methods for primary dengue infection. objective: the aim of this study was to assess the proportion of seropositives against hantaviruses among healthy blood donors. methods: volunteer donors were recruited by the institute of transfusion medicine, representing the demographic situation in the tyrol regarding gender and residence. sera were tested for igg with a commercially available elisa. positive samples were confirmed by a commercially available dot blot which was also used for identification of the serovar. setting: the study area comprises north tyrol (austria, north of the main ridge of the alps), south tyrol (italy) and east tyrol (austria, both south of the main ridge of the alps). south tyrol belongs to the catchment area of the etsch river, which drains into the adriatic, while north-and east tyrol are part of the catchment area of the danube, which drains to the black sea. results: none of samples from the italian part of the study area yielded a positive result, wherein of donors of the austrian part turned out to be seropositive. two patients were positive for hantaan, patients were positive for puumala, one patient was positive for dobrava and one patient had antibodies against hantaan and dobrava. only one of those patients reported extensive travelling abroad. conclusions: evidence was found for the occurrence of hantaviruses in the austrian part of the region covering the catchment area of the danube, but not in the italian part of the study area covering the catchment area of the etsch river. seropositivity to hantaviruses differs by hydrogeographic areas. objectives: canine coronavirus (ccov) is an enveloped, singlestranded rna virus, belonging to group i coronaviruses within the family coronaviridae. two different ccov genotypes have been recognised, that are designated ccovs type i and type ii on the basis of their genetic relatedness to feline coronaviruses (fcovs) type i and type ii, respectively. ccov is usually responsible for mild, self-limiting infections restricted to the enteric tract. we report the molecular characterisation of a pantropic variant of ccov that caused fatal disease in pups. methods: ccov type ii strain cb/ was isolated from an outbreak of fatal disease affecting seven dogs housed in a pet shop in apulia region, italy and characterised by fever, lethargy, inappetance, vomiting, haemorrhagic diarrhoea, neurological signs, and severe lesions in the parenchymatous organs. in all tissues, ccov antigen was detected by immunoistochemistry and ccov type ii rna was identified by genotype-specific realtime rt-pcr. the ' end of the genome of strain cb/ was determined by amplification of seven partially overlapping fragments. the pcr-amplified products were subjected to direct sequencing and the obtained nucleotide (nt) sequences were assembled and analysed using the bioedit software package and the ncbi's and embl's analysis tools. genbank accession number dq was assigned to the sequenced . -kb fragment. the inferred amino acid sequences (aa) were compared to the analogous proteins available in the online databases. results: the structural proteins s, e, m, n of strain cb/ displayed a high degree of aa identity to the cognate orfs of ccov type ii, although the s protein showed the highest identity to type ii fcovs. while the nonstructural protein (nsp) a had the same length of known ccovs, the nsp b was -aa shorter than expected due to the presence of a -nt deletion at position and to a frame shift in the sequence downstream the deletion that introduced an early stop codon. conclusions: association of strain cb/ to a severe, fatal disease of dogs, together with virus isolation from organs with remarkable lesions, strongly suggests that this virus has changed the tropism, acquiring the ability to spread from the enteric tract to the internal organs. by sequence analysis of the viral genome, the only striking change was the truncated form of nsp b, but the role of the deletion in the orf b in determining the patho-biological change deserves more in-depth investigation. objectives: to perform a surveillance study for sars coronavirus (sars-cov)-like virus in non-caged wild animals from the wild of hong kong special administrative region (hksar). methods: from summer to spring , bats, rodents and monkeys from locations in hksar were captured. nasopharyngeal and anal swabs and blood samples were collected and tested for sars-cov-like virus rna by rt-pcr using conserved primers targeted to a -bp fragment of the rna-dependent rna polymerase (pol) gene. the complete genome of the sars-cov-like virus from bats (bat-sars-cov) was sequenced using rna extracted from three anal swabs of three bats as template. phylogenetic tree construction was performed using neighbor-joining method with growtree using jukes-cantor correction. prediction of signal peptides and cleavage sites was performed using signalp, transmembrane domains using tmpred and tmhmm, potential n-glycosylation sites using scanprosite and protein family analysis using pfam and interproscan. antibodies were detected using a recombinant bat-sars-cov nucleocapsid protein enzyme immunoassay and neutralization assay for human sars-cov. results: we identified a coronavirus closely related to sars-cov (bat-sars-cov) from ( %) of anal swabs of wild chinese horseshoe bats by rt-pcr. sequencing and analysis of three bat-sars-cov genomes from samples collected at different dates showed that bat-sars-cov is closely related to sars-cov from humans and civets. phylogenetic analysis showed that bat-sars-cov formed a distinct cluster with sars-cov as group b coronaviruses, distantly related to known group coronaviruses. most differences between the bat-sars-cov and sars-cov genomes were observed in the spike gene, orf and orf , which are the regions where most variations were also observed between human and civet sars-cov genomes. in addition, the presence of a -bp insertion in orf of bat-sars-cov genome, not in most human sars-cov genomes, suggests that it has a common ancestor with civet sars-cov. antibody against recombinant bat-sars-cov nucleocapsid protein was detected in % of chinese horseshoe bats using an enzyme immunoassay. neutralizing antibody to human sars-cov was also detected in those with lower viral loads. conclusion: our data support the existence of sars-cov-like virus in chinese horseshoe bats in hksar. noroviruses are genetically heterogeneous and form at least genotypes within genogroups, gi, gii, giii, giv, and gv, based on the capsid genes. human novs cause an estimated million cases of illness annually in the united states alone and > % of nonbacterial epidemic gastroenteritis worldwide. porcine calicivirus have been found to be genetically similar to human gii novs or to sapoviruses but calicivirus rna has been detected at low frequency by rt-pcr in adults or fattening pigs. the close genetic relationships between human and porcine novs raise public health concerns regarding their potential for zoonotic transmission and as a potential source of new epidemic human strains. methods: a total of faecal samples of nursing and weaning piglets with enteritis were collected during - in porcine herds in italy. an additional samples were include in the analysis, that had been collected during a rotavirus (rv) surveillance study in - , all which tested positive to rv by electron microscopy and by rt-pcr. viral rna was extracted by the guanidine thiocyanate/glass milk method to eliminate enzyme inhibitors. primer pair con -con , targeted to the vp outer capsid protein, was used for rv detection. a degenerated version of primer pair / was used for nv detection, that targets a conserved region in the rnapolymerase. results: nov rna was detected in / of the screened samples, while rvs were detected in / samples. mixed infections nov+rv were found in samples. screening of the rv positive samples allowed detection of mixed infections with novs. conclusions: in previous investigations novs were detected in of , normal slaughtered pigs in japan, in of pooled pig faecal samples of -to -month-old fattening pigs in the netherlands and in out of healthy adult and finisher pigs in the united states. interestingly, in this study a high rate of positivity to novs ( / ) was found in nursing and weaning piglets with diarrhoea, a finding that may suggest a higher frequency of infection by nov in young pigs or an association between nov infection and occurrence of enteric disease. altogether, these findings demonstrate that novs are common in porcine herds in italy and provide new insights into the ecology of novs. detection of calicivirus genome in calves using ni/e primers m. mahzounieh, t. zahraeisalehi, e. moghtadaei khorasgani (shahrekord, tehran, ir) caliciviruses may cause a wide spectrum of disease in animals and are important etiological agent of viral gastroenteritis in humans. members of the family caliciviridae are small nonenveloped viruses to nm in diameter. they possess a single stranded poly adenylated rna genome. caliciviruses have been isolated from mink, dog, cattle and non-human primates. "norwalk-like viruses" (nlvs) are the most common cause of acute non-bacterial gastroenteritis in humans. cattle may be a reservoir of nlvs although never bovine nlvs have been found in humans. in this study, we try to detect enteric caliciviruses genome from faecal samples of dairy cattle herds in shahrekord area using reverse transcriptase polymerase chain reaction (rt-pcr) assays specific for nlvs found in humans. the primers used for pcr amplification were ni and e , which amplify a -bp product for the detection of both genogroups i and ii srsv rna in fecal material. our results showed that nine specimens ( %) were positive. these findings suggest that calicivirus infection is endemic in dairy herds in shahrekord, iran and may be have an important role in calf diarrhea. objectives: reoviruses are non-enveloped, -segmented dsrna viruses. in humans and mammalians three distinct serotypes exist, whose prototypes are strains lang (t ), jones (t ) and dearing (t ). although reoviruses have been isolated both from the enteric and respiratory tract, no diseases has been clearly associated to reovirus infection in humans. the potential association with extra-hepatic biliary atresia, myocarditis, and, above all, neurological and cutaneous diseases require further investigations. reoviruses are ubiquitous and scarcely speciesspecific. reovirus identification is usually based on electronic microscopy or gel electrophoresis and reovirus incidence seems to be very low in humans and most mammalians. in this study, we investigated the presence of reoviruses in dogs by means of molecular methods. methods: one hundred ninety-two rectal samples from dogs with diarrhoea, ocular swabs, nasal swabs and oropharyngeal swabs from dogs with ocular/nasal discharge were subjected to an rt-pcr assay targeting a conserved region of viral genome segment l (primers l -rv /l -rv ). positive samples were characterised by polyacrylamide gel electrophoresis (page), serotype-specific rt-pcr assays targeting segment s and sequence analysis. to increase the sensitivity, a nested pcr using primers l -rv /l -rv was performed on samples tested rt-pcr negative. results: only faecal swabs ( . %) were found positive (rt-pcr product of bp). by using a serotype-specific rt-pcr assay and/or sequence analysis, two strains was characterised as type and the other ones as type . page of viral dsrna confirmed the genetic characterisation. unexpectedly, in secondround pcr faecal samples ( %), ocular swabs and nasal swabs yielded a bp product, while no oropharyngeal swab was positive. conclusions: these data suggest a wider distribution of reoviruses in dogs than previously thought, even if most reovirus infections were detected only by nested pcr. the ability of reoviruses to induce disease in dogs, alone or in synergism with other pathogens, is still unclear, since attempts to reproduce a specific disease in germ-free dogs have given contradictory results. due to their poor species-specificity, reoviruses may be easily transmitted from animals to humans (and vice-versa). further studies are required to understand reovirus ecology and their potential zoonotic impact. objectives: parvovirus b is a member of a family parvoviridae. on the basis of genetic distances and evolutionary relationships, human parvoviruses are divided into three genotypes: genotype i corresponding to b -related isolates, genotype ii to lali-related isolates, and genotype iii to v -related isolates. parvovirus b causes a common exanthematous disease in childhood or adult age, arthropathy, hydrops fetalis, various haematological disorders and myocarditis. up to now, we have had no data of the prevalence of b virus in slovenian population. consequently, we also lack information on the genotypes of parvovirus b that are involved in the patients who suffer from the infection. methods: to gather information of the genetic variants of b virus present in slovenia, we extracted dna from serum samples that were sent for serologic diagnostic of parvovirus b infection and were positive for specific igm in the period from january to june . nearly half of all patients were children and young adults up to years. the ns region of parvovirus b was amplified by the nested pcr (primers pb f , r and pb f , r ). all pcr products were directly sequenced. the results of our study show that dna of parvovirus b was present in all samples that tested positive for specific igm antibodies. after the first round of pcr reaction, samples were positive, and after the second reaction, all samples were positive. altogether unique genotype variants of parvovirus b were identified and all were clustered in the genotype i group of b -related isolates. most of the distinct genetic variants differed in % to % from the sequences deposited in gene bank. the majority of sequences obtained from the b virus epidemic in represents a single variant of genotype i with the gene bank acc. no. aj . we also found that different genetic variants of parvovirus b were circulating in and were % or % identical to the genotype i variant with the gene bank acc. no. z . in our study, we were not able to identify any variants of other rare genotypes (lali or v ). conclusion: parvovirus b dna was successfully amplified from all igm positive serum samples of the patients. the genotype i of parvovirus b is dominating in infections with parvovirus b in slovenia. objectives: great britain has been free of animal brucellosis since (european commission decision / ). the main source of infection for uk residents is through contact with infected material in foreign countries. the objective of this study is to type human samples received in the uk since using variable number tandem repeats (vntr) molecular typing to confirm results obtained by classical typing and relate these results to the suspected source of infection. methods: classical typing is traditionally used and is based on the phenotypic attributes of each strain and biovar. vntr typing is a recently developed molecular method, which is based on short repeats contained in the dna that can be amplified to give a banding pattern specific to each strain. results: results found using both methods are consistent. the results show geographical differences, consistent with observations of strain genotype distribution found in animal brucellosis. conclusion: patient history has been gathered where possible giving information on recent travels. along with results found by classical typing and confirmed by vntr typing we can draw a picture of the sources of infection. these results illustrate the potential of vntr typing as a tool to aid conventional approaches to epidemiological traceback that, in the presence of a suitably comprehensive database of strain genotypes, could help identify the source of an infection. is -fingerprinting of brucella isolates from humans e.stubberfield (surrey, uk) objective: brucellosis is a zoonotic disease usually associated with cattle, sheep, goats and pigs. human infection has been attributed to b. melitensis, b. abortus, b. suis, b. canis and b. maris. although the uk is officially bucellosis free there are a number of human cases due to travel and occupation that are submitted to our laboratory for diagnosis. definitive diagnosis of brucella is by bacteriological culture and microbial tests (classical typing), however these require skilled personnel and the results can be subjective. there are a number of molecular tests that have been developed to assist with diagnosis more rapidly and in some cases to strain level less subjectively. is -fingerpinting is a molecular technique that has proved useful for the identification of brucella isolates to species and in some cases stain level. is -fingerprinting relies on the variable number and location of the is mobile genetic element found in all brucella isolates. method: brucella isolates from humans have been tested. genomic dna was extracted, digested using restriction endonuclease ecor , and electrophoresed. southern blotting was performed, hybridising with a dig-labelled is probe. results: the number of brucella is copies range from to more than . brucella melitensis remains the most commonly acquired brucella species of travellers, while occupational infections have included b. abortus isolated from cattle farmers and b. suis associated with pig butchers. two marine brucella strains have been isolated originating from an occupational perspective (a laboratory worker) and a natural setting from an unknown source. unusual patterns have been observed, of which are unique. one of the new patterns has been observed only in isolates originating in east african countries. conclusion: although the diagnosis of brucella to species and strain level is not essential for the treatment of human brucellosis, it is useful for epidemiological studies. is fingerprinting is able to identify the three biovars of b. melitensis, many other techniques do not offer this capability, because of this it may be a useful test in epidemiological studies. this method remains an important diagnostic tool for brucella identification. rapid diagnosis of brucellar epididymo-orchitis by real-time pcr assay in urine samples objectives: to study the diagnostic yield of a real-time pcr assay in urine samples for the rapid diagnosis of brucellar epididymo-orchitis, in comparison with conventional microbiological techniques. methods: ten consecutive patients with brucellar epididymoorchitis were included in the study. the diagnosis of brucellosis was established according to one of the following criteria: first, isolation of brucella spp in blood or any other body fluid or tissue sample or, second, the presence of a compatible clinical picture together with the demonstration of specific antibodies at significant titers or seroconversion. epididymo-orchitis was diagnosed in patients with scrotal enlargement, swelling and pain not due to other causes.for dna amplification we used a sybr green i lightcycler-based real-time pcr assay. the assay amplifies a bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein (bcsp ). the pair of nucleotide primers b ( ' tgg ctc ggt tgc caa tat caa ') and b ( ' cgc gct tgc ctt tca ggt ctg ) were used in the amplification process. after dna amplication, we performed melting curve analysis to verify the specificity of the pcr products. in order to study the specificity of the technique, all the samples from the patients with brucellosis were paired with an equal number of samples from controls with urinary tract infection. (e. coli four cases, k. pneumoniae two cases, p mirabilis two cases, and c. freundii and p. aeruginosa one of each). results: the mean age was . years (range - ). the duration of the symptoms prior to diagnosis was . ± . days (range: - ). b. melitensis was isolated from blood cultures in nine cases ( %). wright's seroagglutination was negative or inconclusive in % of cases. brucella was isolated from urine in only one case whereas real-time pcr assay in urine was positive in nine ( %) cases and the results were available in four hours, whereas the mean time to availability of the final blood culture results was . days (range . - days). real-time pcr was negative for all the control samples from patients with urinary tract infections. conclusion: sybr green i lightcycler-based real-time pcr assay in urine samples is highly sensitive and specific, easy to perform and could provide the clinician with the results in under five hours. the technique could be a practical and useful tool for the rapid diagnosis of genitourinary complications of human brucellosis. objectives: although the united kingdom remains brucellosisfree, there are more than , new cases of human brucellosis reported each year according to the world health organisation. uk residents returning from worldwide travel may have encountered exposure through contact with infected animals and animal products such as dairy produce and meat. phenotypic characterisation or classical methods remain the definitive diagnosis though require skilled personnel and have their limitations. the increasing range of molecular techniques can aid the rapid detection and characterisation of brucella species and their biovars and may have significance in epidemiological studies. methods: a study of brucella reference and field strains of mainly human isolates from different geographic locations were analysed for diversity of their genes encoding the outer membrane proteins (omps) , a and b. pcr products of the three genes digested with seven restriction enzymes were analysed for polymorphisms. results: a re-occurring unique pattern profile seen only in human isolates was observed originating in some european countries and beyond. a growing database of strain types giving a recent overview of brucella infection of humans of many countries. conclusions: molecular typing methods may have an advantage over classical typing concerning brucella melitensis, the most common brucella infection of humans. the characterisation of human brucella isolates may be useful in epidemiological studies for a variety of purposes. objectives: a study to demonstrate the rapid detection and speciation of campylobacter jejuni and of campylobacter coli isolates directly from enrichment broth using a taqman Ò assay. single nucleotide polymorphism analysis of mapa positive strains was used for rapid identification of c. jejuni clonal complexes. methods: thermotolerant campylobacter species were initially confirmed by culture according to the modified draft iso method, where water samples were filtered through . mm pore size nylon membrane. the filters were transferred to selective enrichment in preston broth to improve their recovery and therefore detection of any campylobacter cells present. dna was extracted directly from the enrichment broth culture for real-time detection of c. jejuni and c. coli using the taqman Ò . samples, which were map a positive were, further characterise by single nucleotide polymorphism profiling for rapid recognition of c. jejuni clonal complexes. results: environmental samples, which were confirmed by culture were also map a positive by taqman Ò . snp profiling of mapa positive isolates identified clonal complexes, which are predominantly contained in isolates of human disease and chicken. conclusions: this study has demonstrated the feasibility of rapid detection and identification of c. jejuni and c. coli following short enrichment incubation using a taqman Ò assay. a rapid turnaround time of between - h per batch of samples was achieved. snp profiling offers important epidemiological grouping at strain level, enabling accurate and phylogenetically valid strain identification for c. jejuni, which may have important host associations for tracing sources of infection and consequently improve public health responses. objectives: campylobacter jejuni and c. coli are recognized as the most common causes of acute bacterial gastroenteritis in humans, c. jejuni being the predominant species in most developed countries. the hippurate hydrolysis test is widely used to differentiate c. jejuni from other campylobacter species. about % of c. jejuni isolates fail to hydrolyze hippurate under laboratory conditions. molecular methods represent an alternative to the phenotype-based methods. we tested two multiplex pcr assays for species identification of human campylobacter strains and compared the results with the hippurate hydrolysis test. methods: campylobacter strains isolated from patients were tested for hippurate hydrolysis with rosco diagnostic tablets. hippurate-negative and hippurate-positive strains were selected for two multiplex pcr assays. one pcr-method was based on distinctive ceue-genes of c. jejuni and c. coli, the other pcr-method detected genes from five major clinically relevant campylobacter species: hipo from c. jejuni, glya from c. coli, c. lari and c. upsaliensis, sapb from c. fetus subsp. fetus, and s rrna gene from campylobacter spp. as an internal validation control. results: the c. jejuni hipo gene was detected in all of the hippurate-positive strains and of the hippurate-negative strains. the c. coli glya was detected in of the hippuratenegative strains. in one hippurate-negative strain, sapb from c. fetus subsp. fetus was detected. species-specific genes were detected in of the strains with the ceue-based pcr assay. c. jejuni ceue was detected in hippurate-positive and hippurate-negative strains. c. coli ceue was detected in hippurate-negative strains. conclusion: all hippurate-positive strains were identified as c. jejuni. of the hippurate-negative strains, % were identified as c. coli, whereas % were identified as c. jejuni and one strain as c. fetus subsp. fetus. the results of the two pcr assays were concordant, although some strains could not be identified with the ceue-based pcr assay. the results suggest that molecular species identification should be performed on hippuratenegative strains after the hippurate hydrolysis test for accurate species identification. multiplex-pcr is quick and easy to perform. using the pcr assay that simultaneously detects five campylobacter species also diminishes the need for further phenotypic testing. phenotypic typing of cryptosporidium species isolated from children in kuwait: a role in unique transmission j. iqbal, p. hira (safat, kw) background: cryptosporidiosis is recognized worldwide as a significant cause of diarrhoeal diseases in both adults and children especially in children less than years of age. objective: cryptosporidium spp. isolated from young children in kuwait were characterized at the molecular level to understand the transmission of infection. the study was approved by the ethical committee, faculty of medicine, kuwait. methodology: over a period of years, faecal specimens from kuwaiti children with persistent diarrhoea found to be positive for cryptosporidium spp. by microscopy were genotyped and sub-typed with a small subunit rrna-based pcr-restriction fragment length polymorphism analysis. informed consent was taken from all individuals included in the study. results: the median age of infected children was . years, and the majority of the infections (> %) occurred during the cooler months january-april, indicating a marked seasonal variation. more than % of the children with cryptosporidiosis had only cryptosporidium infection. socio-demographic information did not reveal any particular mode of transmission of infection. genotyping of the organisms isolated showed that ninety-two ( %) of the children had c. parvum, ( %) had c. hominis, and ( %) had both c. parvum and c. hominis. altogether, subtypes of c. parvum and c. hominis were observed. objectives: the intracellular respiratory pathogen chlamydophila pneumoniae (cp) might be involved in the pathogenesis of atherosclerosis. several studies have demonstrated a serological association between cp and cardiovascular disease and dna from the bacteria has been found in various atheromatous vessels. after infection in the respiratory tract, cp is believed to be disseminated systemically within alveolar macrophages. the prevalence of cp within peripheral blood mononuclear cells (pbmc) has in some studies been shown to be higher in patients suffering from cardiovascular disease than in control patients. we investigated the presence of cp dna in aortic heart valves and pmbc in patients ( men; women; mean age years) undergoing aortic valve replacement because of aortic stenosis. also, the presence of cp mrna was investigated in the sclerotic aortic heart valves as a marker of viable bacteria. methods: dna was extracted from aortic valve biopsies and pbmc using the qiaamp dna mini kit (qiagen). mrna and dna were extracted from another piece of the same biopsy using trizol (invitrogen). real-time pcr directed against the chlamydia momp gene was used to detect cp-specific dna and mrna. patient sera were tested for cp-specific igm, igg and iga antibodies by the microimmunofluorescence technique. results: cp dna was found in aortic heart valves from % ( / ) of the patients and in pbmc from % ( / ) of the patients. in one patient cp dna was found in both pbmc and heart valve. no patient had cp-specific igm antibodies. in patients that were pcr-positive for cp dna in the aortic heart valves, % had igg ‡ : and % had iga ‡ : . in patients that were pcr-negative in the aortic heart valves, % had igg ‡ : and % had iga ‡ : . cp-specific mrna in aortic heart valves will be presented on the poster. conclusion: cp-specific dna was found in sclerotic aortic heart valves from % of patients undergoing aortic valve replacement. this confirms previous investigations supporting a role for cp in the pathogenesis of aortic valve sclerosis. the prevalence of cp in pbmc was % which is comparable to that reported in healthy blood donors and lower than that recorded in patients suffering from other cardiovascular diseases. if the bacteria are involved in the pathogenesis of aortic sclerosis they have likely been spread to the aortic valve long before the patient is in need of surgery because of the stenotic valve. introduction: diarrhoea is one of the most common causes of morbidity and mortality among young children in developing countries. diarrheagenic e. coli strains include several emerging pathogens of worldwide public health. six important categories are entero-aggrigative e. coli (eaec), entropathogenic e. coli (epec), enterotoxigenic e. coli (etec), enterohemorrahgic e. coli (ehec), entroinvasive (eiec) and shigatoxin-producing e. coli (stec). this study investigated the role of different diarrheagenic e. coli in iranian children with acute diarrhoea by molecular methods and the antibiotic susceptibility of isolated strains. methods: from april to january , one thousand eighty five children with acute diarrhoea in tehran hospitals in were enrolled in the study. the fecal samples were cultured on macconkey for conventional bacterial pathogen and sorbitol macconkey agar for non sorbitol fermenting phenotype, than they were incubated in ordm;c. the primary stool cultures were subjected to six different pcr reactions targeting stx and stx gene, heat-labile enterotoxin (lt) producing gene, heatstable enterotoxin (st) producing gene, eae gene and pcvd plasmid. the kirby -bauer disc diffusion method was used for antibiogram of isolated strains from different diarrheagenic e. coli by different antibiotics. results: two hundred seventy one diarrheagenic e. coli strains were detected. stec was the most prevalence with ( . %). the frequency of other strains was . %, . %and . % for etec, eaec and epec, respectively. out of stec isolated strains (% . ) had stx or stx gene, and strains had stx and stx gene. the eae gene was found in ( . ) stec strain. out of tested strains, ( . %) were resistance to ampicllin and cefalotin, and ( %) to streptomycin. conclusion: in this study stec was the most frequent associated with diarrhoea. the strong association between use of antibiotics and colonization with antibiotic resistant e. coli, suggest a major role for selection of resistant strains while using antibiotics. the existence of other unknown intestinal adherence factors has been suggested by the isolation of stec strains that lack the eae gene but are still associated with bloody diarrhoea or hemolytic ureamic syndrome (hus). since there is no specific treatment, there is an urgent need for effective preventive measures based on detailed understanding of the epidemiology of stec infections. identification of shiga toxin-producing escherichia coli in raw beef using dna hybridization with digoxigenin-labelled probes and multiplex pcrs m. weiner, j. osek (pulawy, pl) shiga toxin-producing escherichia coli (stec) is an important cause of bloody diarrhoea, haemorrhagic colitis, haemolytic uremic syndrome and thrombotic thrombocytopenic purpura. transmission of stec occurs through consumption of contaminated food, especially meat, dairy products and water. objectives: to develop a three-steps procedure based on two multiplex pcrs and dna hybridization with digoxigeninlabelled probes for identification of stec in raw beef. methods: beef samples inoculated with different number of e. coli o :h cells were incubated in tsb medium at °c for h. the cultures were then transferred to tsb with mitomycin c and incubated for another h. the resulted cultures were used as a source of dna template. the mpcr- was established to identify shiga toxins genes (conserved sequence). the positive culture samples were subjected to dna hybridization with dig-labelled probes as follows: the culture was diluted and inoculated onto agar plates supplemented with tergitol Ò and incubated at °c for h. then, the nylon membranes were put on agar plates, carefully removed and incubated in denaturation, neutralisation and equilibration solutions following incubation with the stx-specific dig-labelled probes, anti-dig conjugates and finally developed with enzyme substrates (bcip and nbt). dark spots visible on the membranes were compared with the respective bacterial colonies on the original agar plates. the corresponding bacterial colonies were isolated and characterized using the mpcr- test which allows amplification of stx (shiga toxin type ), stx (shiga toxin type ), rfbo (e. coli o ) and flich gene (h antigen). an internal control of amplification (e. coli s rrna gene) was also included in both mpcr tests. results: the first mpcr resulted in two amplification products: bp for stx and bp for s rrna genes. the positive meat samples were further tested with dna probes and positive colonies were then characterized with the second test (mpcr- ), generating the amplicons either of bp (stx ), bp (stx ), bp (rfbo ), bp (flich ) or bp ( s rrna). the specificity of this procedure was confirmed by testing e. coli o :h , o :h-and non-stec bacteria. the sensitivity of the method was estimated as cfu/g of meat. conclusion: the obtained results demonstrated the high specificity of the procedure developed and the possibility of using it for identification of shiga toxin-producing e. coli in raw beef. correlation between virulence pattern, phylogenetic group and extended spectrum betalactamases genes in escherichia coli strains isolated from blood cultures m. damian, c. usein, d. tatu-chitoiu, s. ciontea, d. jardan, a. palade (bucharest, ro) e. coli, heterogeneous species consisting of commensal and pathogenic strains, is causing a broad spectrum of human diseases, including extra intestinal and enteric infections.the strains isolated from invasive infections were documented to be carriers of a large number of genetic structures coding for virulence, as well as for resistance to antimicrobial agents. the aim of this study was to evaluate the virulence of strains in comparison with the presence of esbl genes and their distribution among the different phylogenetic groups. a total of e. coli strains, isolated from blood cultures, in hospitalised patients, adults and children, were screened for virulence factors-encoding genes (pap, sfa/foc, afa, hly, cnf, aer and fimh), for genes encoding resistance to extended spectrum betalactam antibiotics (bla shv and bla tem genes) and the appurtenance to one of the main four phylogenetic group based on presence or absence of markers chua, yjaa and tspe .c three strains, negative for all virulence genes, were included in the phylogenetic group a. ten strains, which were positive for five or six virulence genes, were identified as b group. no matter the phylogenetic grouping, the remaining strains possessed at least one virulence gene. no strain was pcr positive for all seven virulence genes targeted. among the strains which were positive in the double disk test, strains exhibited both bla shv and bla tem genes and strains only bla tem gene. restriction with pst i and dde i and sequencing of the amplicons were performed in order to identify the type of esbl gene expression product. taking into account the link between phylogenetic group and virulence, we obtained a good correlation for the bacteremic e. coli strains analysed, but there was no relationship with the production of esbls. isolation of shiga-toxin producing escherichia coli from meat samples, phenotypic and genotypic characterisation of isolated strains f. baghbani-arani, f. jafari, m.r. zali, s. salmanzadeh-ahrabi (tehran, ir) objective: shiga-toxin producing escherichia coli (stec) is an emerging foodborne pathogen of worldwide public health importance. this bacterium has been reported as an etiological agent of many outbreaks and sporadic cases. definition of the diversity and antimicrobial susceptibility of (stec) may be helpful in the management of sporadic cases and outbreaks. studies in different countries show that food items maybe contaminated by this pathogen. the present study was carried out to determine the frequency of contamination of meat samples by stec collected in tehran as well as defining genotypes, serotypes, antibiogram susceptibility patterns and molecular diversity of isolated bacteria. methods: from july to june , beef samples were collected from different part of tehran. a grams of each samples was enriched in ec broth and subculture on mac-conkey agar. dna was extracted from a loop full of bacteria taken from primary first streaking area of mac-conkey agar and was subjected to three different pcr reactions targeting stx , stx and eae genes. as much as colonies required were tested for finding the colony responsible for positive results in the first pcr. antibiogram susceptibility patterns of isolated strains were determined by standard disk diffusing method. the antimicrobial agents were used at this study. all isolates were serotyped by slide agglutination test using standard antisera (mast groups) subtyping of strains was done with rapd-pcr by primer. results: among samples, ( %) samples were positive and their genotypes were as follow: ( . %) stx +, stx -, eae-, ( %) stx -, stx +, eae-, ( . %) stx -, stx + and eae+. ( . %) stx +, stx +, eae-, ( . %) stx +, stx +, eae+. among these positive samples strains were isolated. according to the antibiotic susceptibility tests, all isolates were resistance to erythromycin (e) and oleandomycin (ol), and were sensitive to imipenem (i); gentamicin (g) norofloxazin (nx) enterofloxazin (ex) ciprofloxazin (cf) and ceftazidim (ca). in otyping and htyping the most frequency were o ac and h serotypes. analysis of isolates by rapd-pcr yielded different patterns. conclusion: our results show that contamination of meat samples by stec is a life-threatening health problem. combinational analysis of antibiogram susceptibility patterns and serotypes with rapd-pcr patterns can aid to survey the characteristics of stec strains. factors affecting the conjugative transfer of plasmid pip in enterococcus faecalis a.m. al-qurashi (dammam, sa) objectives: factors which are known to influence plasmid transfer were studies using the conjugative plasmid pi , which encodes erythromycin resistance, in enterococcus faecalis. methods: the donors strains streptococcus a agalactiae v (group b) is resistant to in rifampicin and fusidic acid, non hemolytic and b-lactamase-negative. it contains the broad host range plasmid pi , which confers resistance to erythromycin and chloramphenicol. the recipient is enterococcus faecalis strain jh - group d. results: transfer of pip occured on a agar, on filters and in broth cultures at relatively high densities ( - bacteria/ml). transfer frequency was largely unaffected over a wide range of temperatures ( - °c). the ph of the medium, in the range ph - had little effect on the transfer frequency. log phase cultures and donor: recipient ratios of : - : were required for optimal for plasmid transfer. conclusion: factors which modified the transfer efficiency of the conjugative plasmid pip were mating media, solid or liquid environment, mating time, mating temperature, selection temperature, growth temperature of donor and recipient of prior to mating, ph culture age, and donor/recipient cells ratio, to obtain a better understanding of this plasmid and its transfer process will help understand what role they may have in the dissemination resistance among streptococcal and enterococcal populations. enterococcus faecium blood-culture isolates collected during a five-year period h. billströ m, Å . sullivan, b. lund (stockholm, se) background: enterococcus faecalis and enterococcus faecium have during the last years become a significant nosocomial problem. this could be due to the enterococcus hardy nature combined with intrinsic and acquired antibiotic resistance. since most individuals harbour enterococci in their normal intestinal microflora there has been a discussion regarding the origin of these isolates. during the last ten years the isolation ratio between e. faecalis and e. faecium have shifted from : to : . this could be because of increasing antibiotic resistance among infectious e. faecium isolates compared to infectious e. faecalis ones. it is possible that this increase also depends upon different virulence genes such as enterococcal surface protein (esp), hyaluronidase variant gene (hylefm) and e. faecalis antigen a variant (efafm). objectives: the objectives in this study were to determine the presence and frequencies of seven different enterococcal virulence genes in infectious isolates. further objectives were to see if the number of virulence genes in these isolates vary or increase over time. methods: a total of strains isolated from bacteraemia patients during year - at the karolinska university hospital, huddinge were used. all isolates were screened for seven different virulence genes using a multiplex pcr. these seven virulence genes were aggregation substance (asa), cytolysin (cyt), collagen binding protein (ace), e. faecalis antigen a variant (efafm), enterococcal surface protein (esp), gelatinase (gel) and hyaluronidase (hyl). results: according to the results about half of all isolates were esp-positive. the prevalence of the other virulence genes asa, efafm, gel and hyl were detected, but in low frequencies (< %). conclusion: it seems like the esp gene is the most dominant virulence gene in e. faecium isolates. the occurrence of virulence traits in these isolates further indicates that the potential to cause infection is potentiated among this enterococcal population the data from this investigation supports the hypotheses that enterococci causing infection in hospitalized patients are probably of nosocomial origin rather than endogenous. objectives: the ability of l. monocytogenes to tolerate alkaline stress is of particular importance, as this pathogen is often exposed to such stress in food processing environments cleaned with alkaline detergents or in the mildly alkaline ph values which prevail within engulfing phagolysosomes. this study aims to investigate the alkaline tolerance response (altr) in listeria monocytogenes s using dna microarray technology. knowledge of the alkaline-induced stress response will be useful in understanding how this pathogen tolerates alkaline stress. methods: transcription profiling of l. monocytogenes s was carried out at , and min at high ph in order to capture an early, an intermediate and a prolonged expression response to alkaline stress using oligo arrays from the pathogen functional genomic resource centre. to verify the microarray results the regulation of some ph stress response genes were confirmed by real time quantitative polymerase chain reaction (rt-pcr). results: about genes were upregulated and genes (of open reading frames represented on the arrays) were down regulated at least . fold upon alkaline shock. many of the repressed genes encode enzymes that are involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating a metabolic adjustment of the cells to the high ph. notably, the strongest alkaline-inducible genes were involved in the membrane transport systems. conclusion: the analysis of the data revealed that cells sense and respond to alkaline stress with an extensive program of changes in gene expression. interestingly, there is a strong correlation between the altr and virulence gene expression. comparison to various microarray data already in the literature revealed similarity between the response to alkaline stress and the transcriptional response to stresses such as osmotic shock. engineering improved listerial stress tolerance "with a twist"! r. sleator, c. hill (cork, ie) objectives: to engineer listeria monocytogenes strains with a significantly improved ability to tolerate stresses encountered in the external environment and during gastrointestinal transit, thus, improving listeria's efficacy as a potential vaccine and drug delivery platform. methods and results: using a directed evolution approach, based on a random mutagenesis strategy involving the e. coli xl -red mutator strain, we generated a mutant variant of the listerial betl gene (designated betl*), encoding a secondary betaine uptake system. the mutant betl* promotes a dramatic increase in resistance to a number of biologically relevant stresses when expressed in a variety of different surrogate hosts. using a luciferase (lux) reporter system in combination with the ivis imager system (xenogen corporation, alameda, ca), we tracked betl* expression, in real time, both in vitro under various environmental stresses and in vivo in animal models of infection. in each case strains expressing betl* demonstrated a marked improvement over those expressing wild type betl, both in terms of gene expression and bacterial growth. sequence analysis of the mutated gene revealed a single nucleotide deletion in the spacer region between the - and ) promoter elements upstream of the betl coding region. this deletion presumably introduces a conformational 'twist' in the putative promoter, thereby increasing its transcriptional output. furthermore, the betl* mutation appears to counter the heretofore unreported 'twisted' cell morphology observed using scanning electron microscopy of l. monocytogenes grown at elevated osmolarities. conclusions: it is possible to selectively improve genes required for bacterial stress survival both inside and outside the host. such mutated genes systems may ultimately be used for the construction of more physiologically robust bacterial based vaccine and drug delivery platforms. a.r. samarbaf-zadeh, s. tajbakhsh, s.m. moosavian (ahwaz, ir) introduction: peptic ulceration following infection of stomach with h. pylori is a common disease. accurate and rapid detection of the bacteria can lead to implementation of appropriate treatment and recovery. this research was undertaken to evaluate the sensivity and specificity of fluorescent in-situ hybridization (fish) in the detection of h. pylori in patients who were suffering from dyspepsia. methods: for this purpose, one hundred gastric biopsy samples taken from antrum and corpus of stomach by endoscopy were tested by fish and compared with conventional culture method complemented with biochemical tests. results: fish detected h. pylori in clinical samples while conventional method detected samples. the sensivity and specificity of fish for detection of h. pylori were calculated as % and % respectively. conclusion: the findings of this study suggest that fish is a highly suitable and rapid method for diagnosis of h. pylori, especially when the samples are taken from the antrum and the corpus of the stomach this technique potentially can be applied routinely for detection of this bacterium in clinical samples. objective: numerous studies have demonstrated that h. pylori is ubiquitous; approximately % of the world's population is infected with the organism. gastroduodenal diseases associated with h. pylori infection are manifested principally in adults. however, it's usually during chilhood that the infection is acquired, and it is possibile that mucosal and humoral responses at this time may determine, at least in part, the course of the natural infection. our study will describe the prevalence of the h. pylori oral carriage in children resident in bari, south of italy, using the pcr method. methods: the evaluation was performed in children, with ages ranging from to years, from primary school district of local health unit of bari, italy (ausl ba/ ). the school and the class have been selected using the cluster sampling method. a standardized questionnaire was used to verify socio-economic standard, hygiene and history of previous gastrointestinal disorder. a standard full-mouth examination was made to detect periodontal diseases, then dental plaque and saliva collected from children were placed in pbs and transported in laboratory. h. pylori infection status was checked by pcr method. dna was extracted from oral samples by the boiling method and evaluated for the presence of h. pylori caga and urea genes using commercial kit (ab analitica, padova). results: a total of children ( females and males) partecipated to the study. the presence of gene coding for caga was found in children ( %), but gene urea was detected only in ( %). the bacteria was detected in saliva, supragingival and subgingival plaque, suggested that these sites may be considered reservoirs for h. pylori in ureasi-positive patients. there was statistically significant relationship between who didn't wash their hands frequently and the presence of urea gene (o.r. . ). conclusions: current knowledge implies that acquisition of h. pylori seems to occur predominantly in childhood and that once acquired the infection persists life-long in most infected subjects. it has been reported at a worldwide level that h. pylori infection prevalence in children varies between % and % and increases with low socio-economic and educational levels and age. the results of this study suggest that oral carriage of h. pylori may play a role in the transmission of infection and that the hand may be instrumental in transmission. the role of helicobacter pylori in otitis media with effusion t. yilmaz, m. ceylan, y. akyon, o. ozcakir, b. gursel (ankara, tr) objectives: otitis media with effusion (ome) is such a common disease of childhood and its pathogenesis still remains unsettled. pepsinogen and pepsin has been shown in the middle ear fluid of patients with ome, indicating that gastric juice could reach as far as middle ear. if gastric juice could enter the middle ear, helicobacter pylori, a common inhabitant of gastric juice and mucosa, would also be expected to be found in the middle ear of patients with ome. the objective of this study was to evaluate possible role of helicobacter pylori in pathogenesis of otitis media with effusion. methods: the study group consisted of children who are to undergo bilateral ventilation tube insertion, adenoidectomy, tonsillectomy with a diagnosis of ome, adenoid hypertrophy and chronic tonsillitis. the control group consisted of children who are to undergo adenoidectomy, tonsillectomy with a diagnosis of adenoid hypertrophy and chronic tonsillitis. for the study group, middle ear fluid was aspirated and a small biopsy was taken from the promontorium mucosa. for the control group, myringotomy was done and a small biopsy was taken from the promontorium mucosa. for both groups, mm deep tissue specimens were obtained from tonsil and adenoid. for all the specimens taken from the patients, culture and a nested-pcr were performed to show helicobacter pylori. results: middle ear fluid culture was positive for h. pylori in patients and mucosa culture was positive in patient only. in the control group middle ear mucosa cultures were always negative. when culture and pcr results were combined together; the middle ear was positive for h. pylori in patients in the study group and in patients in the control group. this difference was statistically significant. h. pylori presence in the tonsillar and adenoid tissues by culture and pcr was also significantly more frequent in the study group compared to the control group. conclusion: this study is the first to grow h. pylori in the middle ear in ome. significantly increased colonization by h. pylori of the middle ear, tonsillar and adenoid tissue in patients with ome indicates that the bacteria reaching the middle ear through gastroesophageal reflux might be involved in the pathogenesis of ome. for ome cases resistant to medical treatment it may meaningful to evaluate the patient for gastroesophageal reflux and h. pylori. distribution of the serine-aspartate repeat protein-encoding sdr genes among nasal carriage and invasive staphylococcus aureus strains objectives: this study was designed to examine the distribution of the sdr genes among nasal carriage and invasive staphylococcus aureus strains as well as methicillinsensitive s. aureus (mssa) and methicillin-resistant s aureus (mrsa). methods: the presence or absence of the sdr genes using dna from s. aureus strains was determined by a novel triplex pcr procedure. the two-tailed fisher's exact test was used to analyse the distribution of the sdr genes among s. aureus strains originating from different hosts. p values less than . were considered a statistically significant difference. results: the sdr locus was found in all investigated s. aureus strains although in strains it contained only the sdrc gene (sdrd -sdre-). the sdrc + sdrd -sdre-gene profile was exclusive to mssa strains (fisher's exact test; p = . ) and was not found in the strains collected from bone infections (p = . ). we also found a strong association between the presence of the sdrd gene and mrsa strains (p < . ). conclusion: our findings suggest that mssa strains with the newly uncovered sdrc + sdrd -sdre-gene profile have a substantially decreased potential to establish bone infection. sequencing of luks-pv and lukf-pv in methicillin-sensitive and methicillin-resistant staphylococcus aureus of diverse genetic backgrounds in a swedish county c. berglund, b. sö derquist (Ö rebro, se) objectives: community-aquired methicillin-resistant staphylococcus aureus (ca-mrsa) have been reported to carry the loci for panton-valentine leukocidin (pvl) in high frequency. the aim of this study was to describe variations within the pvl genes (luks-pv and lukf-pv) in methicillinsensitive and methicillin-resistant s. aureus of diverse genetic backgrounds. methods: twelve pvl-positive s. aureus were characterised by multilocus sequence typing (mlst) and mrsa also by staphylococcal cassette chromosome mec (sccmec) typing. ten of these were isolated between - in Ö rebro county, sweden. oligonucleotide primers were designed to yield a product size of~ bp including luks-pv and lukf-pv and flanking regions by pcr amplification. cyclic sequencing was performed with several sets of primers to overlap the sequences on both strands and was separated on abi prism Ò genetic analyzer (applied biosystems). the nucleotide sequences were analysed using abi prism Ò autoassembler tm dna sequence assembly . . software and compared using bioedit . . . results: analysis with mlst differentiated the pvl-positive ca-mrsa into six different sequence types (st , , , , and ) with either sccmec type iv, iv c, v or unknown types. six additional sts (st , , , , and new) were detected among the pvl-positive methicillin-sensitive s. aureus. sequencing luks-pv and lukf-pv revealed eight point mutations among these isolates with twelve different origins. five substitutions had occurred in luks-pv and three in lukf-pv. only one substitution was nonsynonymous (histidine fi arginine). conclusion: the pvl-genes were well conserved despite the different genetic origins of the isolates analysed. the pvl is an extracellular product and the genes are not subject to any selective forces and thereby diversify very slowly. additional nonsynonymous mutations might result in a non-functional toxin. the first case of staphylococcus pseudintermedius in humans isolated from an icd lead l. van hoovels, a. vankeerberghen, k. van vaerenbergh, a. boel, h. de beenhouwer (aalst, be) introduction: staphylococcus pseudintermedius is recently described as a new coagulase-positive species from animals (devriese et al., ) . the pathogenic significance of this novel species remains unclear and to our knowledge no human infection due to s. pseudintermedius has been reported to date. here, we present the first isolation of s. pseudintermedius in humans with important clinical significance. patient and methods: a -year old male patient was referred to our centre for an ischemic cardiomyopathy and ventricle tachycardia for which he recieved an implantable cardioverterdefibrillator (icd) in january . in august he presented with complaints of migration of the icd device. clinical examination revealed perforation of the icd pocket. infection was suspected and confirmed by the presence of pus in the pocket. the infected icd was completely removed and several samples (ventricular lead, pus and a tissue sample from the pocket) were sent for culture.bacteria obtained by routine culture were further characterised by phenotypical identification, pastorex Ò staph-plus (biorad), api staph Ò (biomérieux) and phoenix Ò (bd). for molecular analysis, pcrs were performed targeting the nuclease (nuc) and coagulase (coag) genes of s. aureus. additionally, sequencing of the s rrna gene was performed and further analysed using blast. results: staphylococci with identical phenotypical appearance were isolated from of the icd samples (lead and pus). colonies were beta-hemolytic on sheep blood agar, dnase and coagulase positive but clumping factor, mannitol and pastorex Ò negative. biochemical identification by api staph Ò and phoenix Ò gave a presumptive identification of s. aureus with a confidence value of respectively , % and %.the pcrs for the nuc and coag genes were both negative. s rrna gene sequencing resulted in the identification of s. pseudintermedius based on a % sequence similarity with a previous reported sequence by devriese et al. conclusion: this case report describes the first identification of s. pseudintermedius as a significant pathogen in human. growth characteristics and commercial identification systems misidentify the organism as s. aureus. when confronted with an inconsistent phenotypical identification pattern, clinical labs should consider the use of s rrna gene sequencing for final confirmation. characterisation of staphylococcus aureus isolates recovered from dairy sheep farms (agr group, adherence, slime, resistance to antibiotics) e. vautor, m. sabah, g. mancini, m. pepin, h. carsenti-dellamonica (sophia-antipolis, nice, fr) objectives: the purpose of this study was to investigate staphylococcus aureus natural isolates associated with dairy sheep mastitis for epidemiological key features (agr group, adherence, slime production and antibiotics resistance). methods: the s. aureus isolates (n = ) were recovered from a field study in the southeast of france in - ( from subclinical mastitis, from clinical mastitis, from the environment of the dairy sheep farm). a total of thirteen dairy sheep farms, producing cheeses manufactured with raw ewe's milk, were involved. the agr group were determined by multiplex and real-time pcr. the evaluation of adherence and slime production were assessed with methods previously described by christensen et al. ( ) . the susceptibility patterns to antibiotics were determined using the discdiffusion method on mueller-hinton agar plates. oxacillin susceptibility testing was performed on all the isolates. the others antibiotics susceptibility was only studied on the isolates recovered from subclinical mastitis as they represent the major source of cheese contamination. results: % ( / ) of the isolates belonged to agr group , regardless of clinical findings. % ( / ) were adherent, strongly adherent or with maximal adherence (biofilm producers). % ( / ) were slime producers (moderate or strong producers). all the isolates (n = ), but seven, were susceptible to all the antibiotics tested. two isolates recovered from subclinical mastitis were resistant to oxacillin and partly resistant to ampicillin and penicillin-g. the five other isolates were found: partly resistant to erythromycin (n = ), cefoperazone and penicillin-g (n = ), erythromycin (n = ), neomycin (n = ) or resistant to enrofloxaxin and partly resistant to ampicillin and penicillin (n = ). conclusions: s. aureus isolates recovered from sheep mastitis in the southeast of france are mainly related to agr group suggesting a role for agr-regulated proteins in the persistence of this bacteria in the sheep udders. biofilm and slime production may also be an important aspect for intracellular survival of s. aureus which could promote the development of persistent intramammary infections. finally, ewe's milk does not appear to represent a source of resistant s. aureus and specially methicillin (oxacillin)-resistant s. aureus (mrsa) for human health. detection of virulence genes in staphylococus aureus isolates from dairy sheep, goats and cows mastitis, using single-dye dna microarray e. vautor, v. magnone, g. rios, m. pepin, p. barbry (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy farms animals. although many putative virulence factors have been identified in s. aureus genomes (kuroda et al., ) , the differences in pathogenic potential between naturally occurring isolates remain largely unaddressed. the relative importance of host (tissue) factors versus bacterial virulence determinants in disease pathogenesis is not well known, but it is widely accepted that bacterial factors including toxins, cell wall-associated adhesions, and secreted exoproteins are involved in the process. in this study, we use a single-dye dna microarray assay to investigate the presence or absence of putatives virulence genes in s. aureus isolates recovered from cases of ovine, caprine and bovine mastitis. methods: mastitis s. aureus isolates: sheep (n = ), goats (n= ), cows (n = ).dna microarray: the arrays were spotted with long oligonucleotides ( -mer) representing known virulence genes and new candidates identified in mu genome (a human strain) and other s. aureus genomes. each gene were spotted four time. dna extracted from the strains were labelled with fluorescent cy using the bioprime Ò array cgh (invitrogen). control strains with known genetic and phenotypic characteristics were used to normalize the data. results: (i) the majority of the virulence gene was detected in all the isolates (e.g. coa, ica adbc operon, htra, hysa, nuc, sbi, sdre, ssp, feob, fnb, sib, spa). (ii) genes were not detected in the majority of the isolates (e.g. cna, edin, lukf-pv, sav ,…). (iii) genes were not found in isolates, depending on the herd (e.g. aur or sav absent in isolates from some dairy sheep farm), on the isolates whatever the species (i.g. bsap, caph, entk, eta, fnbb, hsds, lpl , lukd, …) . but we found gene mainly related to species (e.g. agriii, sav ,…) comprehensive results will be given in the poster. conclusions: the present study indicated that the prevalence of virulence genes among s. aureus isolates recovered from dairy farm species depends on the gene. these observations suggest a common occurrence of host-adapted (or tissueadapted) s. aureus strains in which particular virulence genes may play a significant role. when taken with complementary methods such as pcr or/and southern hybridisation, singledye dna microarrays may provide a powerful tool to type s. aureus strains for epidemiological and possibly pathogenesis studies. detection of dna sequences distinguishing two closely related genomes of staphylococcus aureus from subclinical versus gangrenous ewe mastitis strains n. chevalier, c. huard, r. thiery, e. vautor (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy sheep. the severity of mastitis ranges from subclinical to gangrenous forms. subclinical mastitis is an inflammation that is not readily detected clinically whereas gangrenous form is an acute necrotizing mastitis. with the ain to find genetic markers or virulence factors that are only present in gangrenous strains a suppression substractive hybridisation (ssh) method was used in the present study to compare two strains of s. aureus respectively recovered from subclinical or gangrenous mastitis in the same dairy sheep herd. methods: ewes were held in the investigated farm. the subclinical strain was recovered in january from the milk of ewes. the gangrenous strain was recovered in december from an primipare dairy sheep that subsequently died from this acute mastitis. dna extracted from the strains were first compared by pulsed field gel electrophoresis (pfge). then, ssh was performed by using dna from the subclinical strain (driver), as described in a commercial kit (clontech pcr-select bacterial genome substraction kit). results: using pfge, four band differences were found between the two strains. two dna fragments, presumably specific from the gangrenous strain were detected by ssh and sequenced: (i) a bp ( % of homology with the sulfide quinone reductase contained in orf pathogenicity island of the mrsa strain) (ii) bp ( % homology with a gene coding a bacteriophage holine contained in the s. aureus n genome). control pcr tests using primers designed from these specific gene candidates confirmed that they were only present in the s. aureus gangrenous strain. conclusions: according to tenover et al. ( ) , a band difference using pfge indicates that the strains may possibly be related genetically. although genes classically involved in the virulence of s. aureus were not detected in the present study, two putative virulence factors were detected. the sulfide quinone reductase allows s. aureus to growth on sulfide (found in animal manure). the holine protein breaks the internal membrane of s. aureus to release daughter phages suggesting that a mechanism of horizontal gene transfer could have been mediated by bacteriophages and could explain the acquisition of virulence factors. antimicrobial clinical trials p outpatient treatment of acute pyelonephritis in pregnancy after weeks. a randomised controlled trial z. ahmadinejad, s. hantooshzadeh (tehran, ir) objectives: the purpose of this study was to compare the safety and efficacy of outpatient and inpatient treatment of acute pyelonephritis in pregnancy. methods: this was a randomized controlled, clinical trial. one hundred twenty eight gravidas past weeks' gestation admitted in imam khomeini hospital, tehran & sahid dr bahonar hospital, kerman, divided by random blocks to outpatient or inpatient therapy, received two -g doses of intramuscular ceftriaxone at -hour intervals while hospitalized, then were discharged and reevaluated within - hours or remained hospitalized until afebrile for hours. all patients completed a -day course of oral cephalexin. we performed urine cultures on admission and - days after therapy. results: the two groups were similar with respect to age, parity, temperature, estimated gestational age, initial white blood cell count, and incidence of bacteremia. there were not any significant differences between two groups about the clinical improvement after - hours, bacteriuria - days after treatment, relapse of pyelonephritis, requirement to change in antibiotic, date of pregnancy at delivery and preterm labor. the relapse of bacteriuria and preterm labor in inpatients were significantly more than outpatients (pv = . and . respectively). the birth weight of neonate in outpatients were significantly more than inpatients (pv = . ). conclusion: outpatient antibiotic therapy is effective and safe in selected pregnant women with pyelonephritis. however in this study, the neonatal outcomes were better in outpatients and the maternal outcomes in inpatients. experience with daptomycin in patients with renal insufficiency investigators collected demographic, disease state, clinical and microbiological data; outcomes were defined using standard definitions. patients nonevaluable for outcome were excluded. core data were divided and data on cohorts of pts with a creatinine clearance (crcl) ‡ or < ml/min were examined. results: of the pts enrolled, ( %) had evaluable pt outcomes and either crcl ‡ ml/min (nml, n = ) or crcl < ml/min not yet requiring renal replacement therapy (ri, n = ). the distribution of males and females was equal in both groups. ri pts were older ( % ‡ yrs vs %, p < . ). the groups did not differ in the percent coming from the community setting prior to starting dap (nml %, ri %). nml had more frequent history of fractures/orthopaedic procedures ( vs %, p < . ) and haematological cancers ( vs %, p < . ) while ri had higher rates of any renal disease ( vs %, p < . ), chf ( vs %, p < . ) and other immunologic/ inflammatory disease ( vs %, p < . ). ri had higher rates of skin infections ( vs %, p < . ) and endocarditis ( vs %, p < . ). infections that were frequently reported for nml and ri were bacteremia, non-catheter-related ( vs %), bacteremia, catheter-related ( vs %), osteomyelitis ( vs %), and foreign body-orthopaedic ( vs %), all p > . . methicillin-resistant staphylococcus aureus was the most common pathogen; nml %, ri %. ri had higher rates of coagulase-negative staphylococci ( vs %, p < . ) and viridans streptococci ( . vs . %, p < . ). there was no difference in the percentage receiving antibiotics prior to dap; nml %, ri %. the mean dap dose and duration were similar; nml . mg/kg for d, ri . mg/kg for d. the most frequent dose was mg/kg; nml %, ri %. ri initial dap dosing was more frequent than recommended (q h) in %. the mean time to clinical response was similar; nml . d, ri . d. more pts in nml received concomitant antibiotics with dap; vs %, p < . ). the clinical success (cure and improved) rates were; nml %, ri %. conclusion: dap shows favourable clinical success rates in pts regardless of the presence of renal insufficiency. in vitro activity of second line antibiotics against helicobacter pylori infection objective: the aim of our study was to determine the in vitro activity of levofloxacin, ciprofloxacin and rifampicin in clinical strains of h. pylori. material and methods: isolates of h. pylori from biopsies of dyspeptic patients were obtained following standard methodology. in vitro activity of metronidazole, clarithromycin, levofloxacin, ciprofloxacin and rifampicin was determined by e-test using % sheep blood agar and incubated at ordm;c during - days in a co atmosphere. mic was determined as the point of complete inhibition of growth. breakpoint of the nccls for other microorganisms were considered for fluorquinolones: resistant if mic > mg/ l. for rifampicin we considered the strain susceptible if mic < mg/ l, as same studies reported. results: . % of the strains were resistant to metronidazole and % to clarithromycin. mic , mic and range (mg/l) was: . , . and . fi for levofloxacin, . , . and . fi for ciprofloxacin and . , . , and < . - for rifampicin. all the strains were susceptible to rifampicin and only % of them were resistant to fluorquinolones. conclusions: the fluorquinolones tested and rifampicin showed an excellent in vitro activity against h. pylori, despite the high resistance rate to metronidazole and clarithromycin. however, in vitro susceptibility test should be done before the use in clinical practice. vibrio antibodies in serum and breast milk samples of parturient women in calabar, nigeria objectives: serum and breast milk samples from parturient women and serum from non-parturient controls were analysed for prevalence and titres of vibrio antibodies. methods: v. cholerae agglutinins and vibriocidal antibodies in serum samples were analysed by direct agglutination and immune bacteriolysis techniques respectively, using well microtitre plates. the protective value of breast milk was evaluated by haemagglutination inhibition and rabbit intestinal mucosal attachment of v. cholerae cells. results: vibrio agglutinins were detected in serum samples of ( . %) parturient and ( . %) non-parturient subjects (p < . ). high prevalence rates of . % and . % occurred among parturient and control subjects of - years of age respectively. at : cut off titre to evaluate vibrio cholerae specific bacteriocidal antibodies, activity was detected in samples of ( . %) and ( . %) parturients and controls respectively aged - years. breast milk from ( . %) parturients contained vibrio agglutinins with titres ranging between : and : , while milk samples from subjects showed haemagglutination inhibition (hi) activity titres of p : . of the hi positive milk samples ( . %) showed inhibition of v. cholerae adherence to rabbit intestinal mucosa at titres p : , and - % reductions in cell attachment. conclusion: our study confirms that parturient women in calabar may benefit from significant serum titres of v. cholerae antibodies and provide immune protection for their babies through breast milk secretions. moxifloxacin vs clarithromycin for treatment of community-acquired pneumonia associated with common respiratory pathogens: a pooled analysis objectives: streptococcus pneumoniae and haemophilus influenzae are pathogens commonly associated with community-acquired pneumonia (cap). this study compared the clinical and bacteriologic efficacy of moxifloxacin (mxf) to clarithromycin (clar) in cap patients with these pathogens. patients and methods: data were pooled from three doubleblind, multicenter, phase iii trials comparing oral mxf mg qd to clar mg bid for days. all patients included had mild-to-moderate cap. clinical and bacteriologic success rates were identified for s. pneumoniae and h. influenzae isolated from these studies. data for the efficacy-valid population was recorded at the test-of-cure (toc) visit ( - days post-therapy). results: patients were entered, of which were microbiologically evaluable. infection with s. pneumoniae and/or h. influenzae was documented in ( %) of patients ( mxf and clar patients had mixed infection). within this cohort, the two treatment groups were well balanced based on demographic/baseline medical characteristics ( % male, mean age yrs, % smokers, % recent antimicrobial therapy). clinical success and bacteriologic eradication rates (one response per patient) at toc are presented in the table. conclusions: in cap associated with s. pneumoniae and h. influenzae there was a trend towards greater bacterial eradication for mxf vs clar. clinical success rates were significantly higher for mxf monotherapy vs clar. variability of creatinine clearance measurements in inpatients with community-acquired pneumonia r. grossman, s. choudhri, d. haverstock (mississauga, ca; west haven, us) objectives: moxifloxacin, levofloxacin and gatifloxacin have been recommended as empiric therapies for patients with community-acquired pneumonia (cap). levofloxacin and gatifloxacin require dose-adjustment for renal insufficiency while no dose adjustment is required for moxifloxacin. this study was designed to determine the frequency and underlying variability of renal insufficiency in patients with cap. methods: a pooled analysis of data from patients with mild to moderate or severe cap entered into one of six randomized, controlled clinical trials was undertaken. renal function (calculated creatinine clearance; crcl) was assessed in each patient prior to treatment with mxf and then again during and post-treatment. results: baseline crcl levels in this pooled population of patients with cap were: < ml/min in ( . %) of patients, - . ml/min in ( . %) and ‡ ml/min in ( . %) patients. after the pre-treatment crcl measurement patients ( %) were lost to follow-up, so there was no during or post treatment value. in patients with cap the crcl improved from baseline in many patients during or post-treatment, while some patients experienced a worsening of renal function (see table) . conclusions: renal function (crcl) is highly variable in cap patients with baseline evidence of renal insufficiency. renal function should be monitored closely to permit appropriate dose adjustments if levofloxacin or gatifloxacin is used in this patient population. moxifloxacin may be a better empiric choice in this setting as it does not require dose adjustment in patients with renal insufficiency or renal failure. a prospective, controlled, randomised, nonblind, comparative study of the efficacy and safety of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice-daily ceftazidime plus amikacin in the empirical therapy of febrile neutropenic patients objective: empirical antibiotic treatment for febrile neutropenia is well established. the best regimen is still controversial. the purpose of this study was to evaluate the efficacy, safety and cost of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice daily ceftazidime plus amikacin in neutropenic febrile patients. patients and methods: ninety-five patients with febrile neutropenia were included in a prospective, controlled, randomized, non-blind, comparative study. patients were randomly assigned to either treatment group ( in the ceftriaxone/ciprofloxacin group and in the ceftazidime/ amikacin group) and evaluated as successes or failures according to defined criteria. daily assessments were made on all patients all adverse events were record. results: the overall incidence of documented infections was . %: / ( . %) in the ceftriaxone/ciprofloxacin group and / ( %) in the ceftazidime/amikacin group. there was significant difference in clinical efficacy between groups (p = . ) at the end of therapy. ceftriaxone/ciprofloxacin group had an overall incidence of resolution and improvement of , % in comparison to the % of the ceftazidime/amikacin group. thirty-nine organisms were isolated, ( . %) gramnegative and ( , %) gram-positive. there was low incidence of adverse events in both groups. conclusion: the combination of high dose single daily ceftriaxone plus ciprofloxacin was more effective than the standard combination of thrice daily ceftazidime plus amikacn with no significant adverse events in either group. objective: in past studies of azithromycin in children, a posttreatment (pt) benefit was observed at day . in recent phase trials in adults, single-dose zmax was at least as effective as standard comparators for treatment of respiratory tract infections (rtis), including cap. our objective is to demonstrate a pt benefit in this adult population. methods: post-hoc analyses, including respiratory adverse event burden (raeb), were conducted on the all treated population (n = ; az-m, comparators) in the phase studies. the raeb is the sum of duration, in days, of all respiratory adverse events, divided by total number of observation days of all patients, normalized to year. the overall and per study day raeb were calculated for zmax and the pooled comparators for the studies combined. results: raeb, in days/patient year, was . for az-m patients vs . for comparator patients (p = . ). the difference in raeb consistently and progressively favoured zmax, beginning at day and achieving statistical significance between days and , when the upper limits of the % cis around the differences were below zero (figure). faropenem medoxomil (fm) is an oral penem with potent activity against streptococcus pneumoniae and haemophilus influenzae. this integrated analysis was conducted to summarize the efficacy of days of mg bid of fm compared with other beta lactams in the management of community acquired pneumonia (cap). methods: efficacy was determined in three multicenter randomized double-blind controlled trials (rct) and a single uncontrolled study of faropenem medoxomil. comparators were days of cefpodoxime (c), days of amoxicillinclavulanate (ac), or days of amoxicillin (a). the analysis allowed examination of treatment effects by age, race, gender and study site subgroups. results: a total of subjects were studied. studies and were conducted in n. america, studies and in europe, latin america, israel, and s. africa. n. american vs. other studies included subjects at least (vs. at least ) years of age and only out patient (vs. outpatient and hospitalized) subjects. the clinical responses for fm in both per protocol and intention-to-treat populations were non-inferior to comparator for each study and for the three trials combined. no differences were found in treatment effect by age, race, gender, or country. recovery of an etiologic agent from initial respiratory or blood culture varied between . and . % of cases in the studies for a total of microbiologically evaluable subjects. s. pneumoniae was eradicated or presumed eradicated in / ( . %) and / ( . %), h. influenzae in / ( . %) and / ( . %), s. aureus in / ( %) and / ( %), h. parainfluenzae in / ( %) and / ( %), and m. catarrhalis in / ( . %) and / ( %) fm and comparator recipients, respectively. clinical response for s. pneumoniae bacteremic patients was / ( . %) for fm. conclusions: fm efficacy was consistent across studies, within subgroups, and non-inferior to comparators. it is efficacious against the most common bacterial pathogens and in the most severe form (bacteremic) disease. fm is a good option for the treatment of cap. propionibacterium acnes strains isolated from acne vulgaris and severe infections c. oprica, c.e. nord (stockholm, se) propionibacterium acnes is a member of the resident flora of the skin and is an important factor involved in inflammatory reactions in acne patients. during the last years the prevalence of different severe infections due to p. acnes has increased. objectives: ) to detect the prevalence of resistant p. acnes strains isolated from acne patients in stockholm and different severe infections in europe; ) to identify the mechanisms of resistance and the genetic diversity among resistant strains. methods: p. acnes strains isolated from acne vulgaris and severe infections were tested against clindamycin, erythromycin, linezolid and tetracycline and pulsed-field gel electrophoresis was used for further characterization. pcr and sequencing of the genes encoding domain v of s rrna for clindamycin and erythromycin resistant strains and s rrna for tetracycline resistant strains were performed. results: i) antibiotic-resistant strains were more often isolated from antibiotic treated patients with moderate to severe acne area than from non-antibiotic treated acne patients. an individual might harbor different pulsotypes of p. acnes with various degrees of resistance. ii) among the clinical isolates from european countries were found resistant strains to tetracycline, clindamycin, and erythromycin. overall, in the southern europe a higher prevalence of erythromycin-resistant strains was noticed and in southern and eastern europe a higher prevalence of resistance to clindamycin. it was noticed a high genomic diversity and the geographical spread of some clones in related areas but also in geographically distant countries. most clindamycin or erythromycin resistant p. acnes isolates, were found to be members of a single clone that has spread in different geographically countries. iii) p. acnes clindamycin and erythromycin resistant strains carrying one of the described mutations within the s rrna were predominantly isolated from swedish acne patients compared to strains from other infections. forty-four per cent of tetracycline resistant strains were found to carry a mutation in the s rrna. these strains were isolated from swedish acne patients, were highly resistant and were clustered in one pulsotype. conclusion: surveillance of both the prevalence of resistant p. acnes strains and associated resistance mechanisms is important due to the rapid variation in resistance patterns, both in acne patients and other severe infections. antimicrobial activity of unisepta quick and deconex solarsept on the surface contamination and dental instrument in dental clinics in iran f. shahcheraghi (tehran, ir) objectives: quaternary ammonium compounds (qacs) are amphoteric surfactants that are widely used for the control of bacterial growth in clinical and industrial invironment.unisepta quick and deconex solarsept are new generation of qacs is widely used as adjuncts in iran to hygine in dental clinics.the aim of present study was to investigate clinical efficiency of these substances on the surface and instruments in dental clinics. material and methods: the following bacteria and fungi on the base of aoac standard were used.pseudomonas aeruginosa (atcc ), staphylococcus aureus (atcc ) bacillus. subtilis atcc ( ), mycobacterium bovis atcc ( ) and wild types of trichophyton mentagraphit, p. aeruginosa and salmonella typhimorium (a common fungi in iran). a stock solution of deconex solarsept (borer chemie) and unisepta quick (micro unident) was prepared as recommended by the manufacturer. the concentration of bacterial suspention was . macfarland and the results were reported on the base of decreasing in (cfu) colony forming unit from to . results: the results shows that both of these disinfectants have bactericidal and fungicidal activity on the standard p. aeruginosa, s. aureus, s. typhimurium and trichophyton mentagraphit, the number of bacteria decreased significantly (p < . ), but no significant difference was seen with b. subtilis, wild type of p. aeruginosa and m. bovis. conclusion: the results confirm that these qacs are not able to sterilize or disinfect medical and dental instruments, and they can not be used lonely, and it must be used with the other methods for sterilization of surface and dental instruments. macrolide as long-term treatment in patients with bronchiectasis colonised by p. aeruginosa background: a certain efficacy of macrolide against p. aeruginosa has been described in vitro, mainly through mechanisms such disruption of quorum sensing and suppression of inflammation. aim: to evaluate the efficacy of macrolide in patients with bronchiectasis colonised by p. aeruginosa. methods: the study prospectively included patients with bronchiectasis and p. aeruginosa isolated in sputum in stable state. all subjects received either azithromycin mg · days/week or clarithromycin mg daily on long term and completed daily diary cards for symptoms and pef values until the end of therapy. follow-up period was year. results: patients with bronchiectasis and p. aeruginosa evidence in sputum were included ( men, mean age . ± . yrs.). patients received azithromycin and patients clarithromycin, with a mean duration of . ± . months. five ( . %) patients discontinued treatment after less than weeks because of adverse events. at the end of therapy, ( . %) patients showed no evidence of p. aeruginosa in sputum while ( . %) patients still had ps. aeruginosa in sputum. an improvement in the following parameters could be observed in all patients: sputum volume ( . ml/day before therapy versus . ml/day after therapy, p = . ); pef ( . ± . l/min before therapy versus . ± . l/min after therapy, p = . ); number of exacerbations/year ( . in the previous year versus . in the follow-up year, p = . ). conclusion: the study shows that macrolide may be an effective therapy in patients with bronchiectasis colonised by p. aeruginosa. independently of the microbial eradication, an improvement of the clinical symptoms and a reduction of exacerbations were observed in all patients. fungal pathogens from haematoncology patients and their susceptibility to new and old antifungal drugs the expanding population of immunocompromised hosts has been infected with many established and emerging opportunistic fungi. most pathogens can be treated empirically whereas for an increasing number of species proper treatment starts once the mic becomes available. though invasive aspergillosis remains the principle life threatening complication in the haematoncology patients (hop) other pathogens cannot be ignored as selection and resistance during prophylaxis increases the risk of treatment failure.in order to understand the frequency of rare fungal pathogens, selection and emergence of resistance in our trust all fungi from hop were identified using standard mycological techniques and the mics to amphotericin b (amb), flucytosine ( fc), fluconazole (fcz), itraconazole (itz), voriconazole (vcz) and caspofungin (cfg) were determined using the nccls method. specimens were processed, % respiratory, . % blood, . % oral, . % other sterile (bile, csf, drains, lines and tissue biopsies) and . % nonsterile sites. yeasts accounted for % and filamentous fungi (ff) for %, representing candida sp, other types of yeast, aspergillus sp and other types of ff. c. albicans represented . %, c. glabrata . %, c. krusei . %, a. fumigatus % and other aspergillus sp % of all isolates. the mic s for all isolates were amb . , fc , fcz > , itz , vcz and cfg . mg/ l. with the exception of acremonium sp, a. versicolor, a. terreus and scedosporium apiospermum all isolates including the isolates of c. lusitaniae were sensitive to amb. most but not all ff and only one isolate of c. albicans from the yeasts were resistant to fc. all ff, rhodotorula sp, c. albicans %, c. glabrata % and c. krusei % were resistant to fcz. only absidia corymbifera, acremonium sp %, c. albicans %, c glabrata % and saccharomyces cerevisiae % were resistant to itz. for vcz a. corymbifera, acremonium sp %, c. albicans %, c. glabrata %, c. krusei %, c. tropicalis %, rhodotorula sp . % and p. aecilomyces variotii % had an mic ‡ mg/l. with cfg the effective concentration was ‡ . mg/l for a. corymbifera, fusarium solani, geotrichum capitatum, sporobolomyces salmonicolor, acremonium sp % and c. parapsilosis %.the data show that hop are exposed to many different fungal pathogens some of which are resistant to the old and the new antifungals and that amb is still the drug with the broader spectrum and less developed resistance for both yeasts and ff. faropenem medoxomil in the treatment of acute bacterial sinusitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial agent with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute bacterial sinusitis (abs) was evaluated in phase iii trials; , , and . study was conducted in n. america, study was conducted in europe and israel and study was conducted in the us and argentina. and were prospective, randomized, double-blind, active comparator trials and was an open-label ''sinus tap'' trial. the dose of fm was mg bid in all studies. the comparator in and was cefuroxime axetil (cfx) mg bid. the duration of fm treatment in was days and days vs cfx for days. in , fm or cfx were given for days. in , fm was administered for days. the primary efficacy variable in all studies was clinical response at the test-of-cure (toc). microbiologic response at the toc was a secondary efficacy variable in (sinus puncture and endoscopic collection) and (sinus puncture and aspiration). non-inferiority was defined as the difference in cure rates (fm minus comparator) where the lower boundary of the % ci was greater than - %. results: the cure rates at the toc are shown in the table for the valid per protocol (vpp) and the intent-to-treat (itt) populations. the frequency of isolation of key pathogens and the rate of eradication in samples obtained by endoscopicallyguided swab and in samples obtained by tap were consistent across studies. the eradication rates for s. pneumoniae, h. influenzae, and m. catarrhalis were . % vs. . % (fm / d vs. cfx / d), . % vs. . % (fm vs. cfx) and . % vs. . % (fm vs. cfx), respectively. conclusions: fm mg bid x days was shown to be noninferior to cfx in clinical efficacy in two prospective, doubleblind, comparative trials. a third, open-label trial, demonstrated similar efficacy in microbiologically documented abs caused by key pathogens. longer ( d treatment) with fm provided no additional efficacy. faropenem medoxomil in the treatment of acute exacerbation of chronic bronchitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute exacerbation of chronic bronchitis (aecb) was evaluated in phase iii trials. study was conducted in europe, israel, mexico, and south africa. study was conducted in the us and argentina. both were prospective, randomized, double-blind, active comparator trials. the dose of fm was mg bid for days in both studies. the comparators were clarithromycin (clr) mg bid for days and azithromycin (azi) qd for days ( mg on day and mg on . the primary efficacy variable was clinical response at the test-of-cure (toc). microbiologic response at the toc, in subjects with a baseline pathogen was a secondary variable. non-inferiority was defined as the difference in cure rate (fm minus comparator) where the lower boundary of the % ci was greater than ) %. results: the cure rates are shown below for the valid per protocol (vpp), intent-to-treat (itt) and modified itt populations (all itt subjects who met inclusion/exclusion criteria). in both the individual studies and the pooled analyses, for all populations, treatment with fm was not less effective than either comparator. % of treated subjects in and % of subjects in were evaluable for microbiological response. in , the eradication rates for the microbiologically evaluable population was higher in the clr group ( . %) compared with the fm group ( . %) ( % ci ) . , . ) . in contrast, the eradication rate in was similar in the fm ( . %) and azi ( . %) groups ( % ci - . , . ). when the data were pooled across studies, the response rates were similar with fm ( . %) and combined comparator ( . %) groups ( % ci - . , . ). the combined eradication/presumed eradication rates in the pooled fm and comparator groups were . % vs. . %, respectively for s. pneumoniae and . % vs. . %, respectively, for h. influenzae. conclusions: fm was shown to be non-inferior to either azi or clr in clinical efficacy in two adequate and well-controlled trials. pooled analysis further strengthened the clinical noninferiority conclusion. the difference in eradication rates observed in study (clr) was not supported by study (azi). an integrated safety analysis of faropenem medoxomil: results of , subjects from phase ii/iii clinical trials r. echols, r. tosiello (milford, us) objective: to evaluate the safety profile of faropenem medoxomil (fm), a novel oral penem antibiotic. methods: , subjects from phase ii and phase iii clinical trials received fm, mg bid for - days for treatment of acute bacterial infections. randomized controlled trials (rcts) included , fm and comparator treated subjects. analyses were conducted to identify possible disparate adverse event (ae) reporting based on type of infection, subject age ( - , - , , - , > ) and gender, duration of treatment ( / d v. / d), geography (na, eu, row), study design (open label v rct), relationship to treatment. comparisons were made to control treatment based on antibiotic class (b-lactam v. other), and individual antibiotic treatments. results: fm compared favourably to penicillins, cephalosporins and macrolides. fm was better tolerated than tmp/smx and co-amoxiclav. open labeled trials had higher aes reported v. rcts. aes reporting na = row > eu except serious aes and deaths where row = eu>na. aes for fm / d = fm / d. underlying infection did influence ae reporting. female gender had higher ae reporting than male gender. fm was tolerated equally well across age ranges, although deaths and saes were more common in > age group. common aes (> %/related from rcts) were diarrhoea, nausea, fungal vaginosis and headache and were generally less frequent with fm than control rx. no evidence of neuro or cardio toxicity was identified. laboratory tests identified no hepatic, renal or hematopoietic signals. conclusion: faropenem medoxomil, a novel oral penem antibiotic, has the safety profile expected of a b-lactam but is better tolerated than co-amoxiclav with approximately one-third the gi side effects. the efficacy of non-surgical and systemic antibiotic treatment regimens in smoking and non-smoking patients e. pähkla, k. lõ ivukene, p. naaber, m. saag (tartu, ee) periodontitis is a chronic infectious disease, which leads to the destruction of periodontal ligament fibres and alveolar bone until tooth loss. the objective of this study was to compare the longitudinal effect of combination of non-surgical periodontal therapy with systemic antibiotics in smoking (s) and nonsmoking (ns) patients. methods: there were total of patients with severe generalized chronic periodontitis involved in this study ( s, ns), who did not respond well to previous mechanical periodontal treatment. the clinical examination included recordings of visible plaque index (vpi), modified gingival index (mgi), bleeding on probing (bop) and suppuration after probing (sup), probing pocket depths (pd) and clinical attachment levels (cal). the non-surgical periodontal therapy was performed within weeks. clinical parameters were recorded at baseline, - weeks after the first mechanical treatment and months after combined treatment, during a regular check-up visit. as the patients did not respond to the conventional periodontal therapy, the microbiological analyses were taken and a combination of systemic amoxicillin mg · and metronidazole mg · for days, was prescribed. results: the results suggested that the combined systemic antibiotic therapy is effective in case of severe generalized chronic periodontitis, as vpi, bop, sup, cal, and mgi improved significantly after the treatment. in the ns group all parameters, except cal, improved significantly after the treatment. the s showed markedly smaller reduction in sup, mgi, and cal.after instrumentation, no periodontal pathogens were isolated in ( %) patients, while patients ( %) were infected with one to three different pathogens. among the pathogens, prevotella intermedia/nigrescens ( patients) and actinobacillus actinomycetemcomitans ( patients) were dominating. the total level of microbial load (log cfu/ml) as well as the spectrum of pathogens in s and ns patients remained similar. conclusions: despite of positive treatment effect in general, there were insignificant improvements in any clinical parameters in the smoking group. smoking has adverse effect on periodontal therapy; therefore the dentist should cooperate with patients in counselling of smoking cessation to achieve better results in the treatment of periodontitis. objectives: laminin (ln), which is a large multidomain glycoprotein of the extra cellular matrix, has attracted much attention because of its importance in many cellular functions, including induction of cell adhesion, growth promotion and mediation of cell communication. the target of this study was to find out whether there is any relation between the levels of serum ln and the inflammatory activity of a microbial infection. patients/methods: from june to october , immunocompetent adults, with confirmed bacterial infection were admitted to our hospital ( with pneumonia, with pyelonephritis and with cholecystitis) (group ). at the same time hospitalised patients for non-infectious causes (stroke, gastrointestinal bleeding, anaemia) were also studied (group ). the levels of serum ln and crp were measured on the day of admission in both groups. the levels of ln were measured using an enzyme immunoassay kit (takara laminin eia kit) and healthy volunteers were used to determine its normal limits ( - ng/ml). plasma crp concentration was assessed by immunoturbidometric method (using randox, uk kits). normal values were considered those below . results: the mean serum ln levels of patients of group were . ± . (much higher that the normal limits), while the mean crp value was . ± . . the mean corresponding values in group were . ± . for ln (within normal limits) and . ± . for crp. there is a statistically significant difference between the mean ln levels of the two groups (p < . ). additionally, there is a statistically significant correlation between the levels of ln and crp (a well studied serum inflammatory marker) in patients with bacterial infection (group ) (pearson correlation coefficient r = . , p = . ). conclusions: the definition of the ln levels could constitute a new reliable, simple, direct serum marker for the confirmation of an active bacterial infection. additionally, as the crp levels are above normal in group too (patients without infection) while ln lies within normal limits, maybe ln is even more specific than crp. more studies are required in the future, with more patients included, in order to confirm the outcome of this study. performance and clinical significance of a direct tube coagulase test using serum separator tubes for rapid identification of staphylococcus aureus from blood culture broth d. kwa, t. schü lin-casonato, p. sturm (nijmegen, nl) objective: blood cultures are important in the diagnosis of serious infections. early administration of effective antibiotics is associated with improved patient outcome. the performance of the direct tube coagulase (dtc) using serum separator tubes (ssts) for rapid identification of s. aureus from blood culture broth (bcb) was investigated. the clinical significance of rapid identification was assessed. methods: consecutive blood cultures with gram-positive cocci in clusters were tested. bcb was collected in ssts using a subculture-venting unit. after centrifugation, the supernatant was discarded and ml rabbit plasma was added to the remaining pellet of bacteria. coagulation was evaluated after and hours incubation at °c, and after overnight incubation at room temperature. in parallel, a direct tube coagulase test was performed using a : saline dilution of bcb as described previously. isolates were identified by standard microbiology procedures. clinical significance was measured by comparison of antimicrobial prescription based on gram stain results, direct coagulase results, and culture results. results: over a -week period, bcbs from patients were tested. s. aureus was present in bcbs. using the serum separator tube method and the saline dilution method, the sensitivity of the dtc after hours incubation was % and %, and after hours % and %, respectively. the specificity of both methods was %. rapid identification of s. aureus resulted in initiation (n = ) or streamlining (n = ) of antimicrobial therapy in of patients with s. aureus bacteremia. rapid identification of coagulase-negative staphylococci resulted in changes in antimicrobial therapy in of patients. conclusion: the dtc using ssts for bacterial enrichment is a very reliable, rapid, cheap and easy to perform method for identification of s. aureus from bcb. implementation of this test can improve antimicrobial therapy. evaluation of the results of the spanish seimc external quality control program for the diagnosis of enterococcus faecalis and klebsiella pneumoniae infections r. guna, j.l. pérez, n. orta, c. gimeno on behalf of seimc objectives: to evaluate the results obtained from four shipments of two different strains by the participants in the seimc external quality control program (eqcp). these controls were intended to analyse the percentages of correct species identification and the ability of the participants in detecting some special features of the control strains: vanb phenotype in the case of e. faecalis, and the production of extended spectrum betalactamase (esbl) in k. pneumoniae. methods: the same strain of each microorganism was sent in two different shipments. the vanb e. faecalis strain was sent both in a control of year as well as in other of , while the esbl-producing k. pneumoniae was sent in and in to an average of laboratories. the results obtained were compared with those of a reference laboratory that certified both the species identification and the resistance features. results: in the control, . % of participants identified correctly e. faecalis, while . % did it in . as for the glycopeptide resistance pattern of the enterococcal strain, . % and . % of participants detected the vanb phenotype in and , respectively. overall, the k. pneumoniae strain was correctly identified in both separate controls by most of the participants ( . % and . %, respectively). interestingly, the percentage of laboratories that detected the presence of the esbl in the k. pneumoniae strain sharply increased from . % in to . % in . the overall percentages of correct species identification were high for the two microorganisms and for both control points. most important, the ability of the spanish clinical laboratories in detecting the special resistance features of these strains clearly improved along the study period. these data confirm the importance of implement a continuous surveillance of the diagnostic training in the clinical laboratory, as well as the possible positive intervention of the seimc external quality control program in such improvement, since the analysis of results is accompanied of updated reviews on the subject of each control. a. bonnet-pierroz, a. resenterra, o. péter (sion, ch) objective: to evaluate new elisa ridascreen Ò borrelia igg and igm for antibody response in patients with confirmed lyme borreliosis and to compare to the results of vidas lyme (igg-igm) and in-house immunoblots (b. garinii igg and igm for early cases or b. burgdorferi sensu stricto, b. afzelii, b. garinii, b. valaisiana igg for late cases). methods: elisa ridascreen Ò borrelia igg and igm was used to screen sera from patients with clinically confirmed erythema migrans em (n = ). patients with confirmed neuroborreliosis by intrathecal antibody synthesis (n = ) were evaluated for igg antibodies to borrelia. sera from patients with acrodermatitis chronica atrophicans aca (n = ) and sera and synovial fluids from patients with lyme arthritis (n = ) were also evaluated for igg antibodies. patients with syphilis (n = ) and infectious mononucleosis (n = ) were screened for igg and igm antibodies to borrelia in order to estimate the specificity. conclusion: the elisa ridascreen Ò borrelia igg and igm have shown a good sensitivity for the serological diagnosis of lyme borreliosis. the short evaluation for the specificity of the igg test revealed a good assay with few false positive reactions, whereas the igm assay was, as expected more prompt to give false positive results with sera from patients with infectious mononucleosis. so far any equivocal or positive tests should be confirmed by immunoblots. is it necessary to incubate the bact/alert blood culture bottles more than days? objective: to assess the incubation time reduction of the aerobic and anaerobic bact/alert system bottles from to days. methods: from to we processed . blood culture sets and detected . ( %) positive blood cultures with clinical significance. we retrospectively examined the detection time of positive bottles and assessed the clinical significance of the bottles that were positive between the fourth and fifth day. results: out of positive blood cultures with clinical significance, ( . %) were detected within the first days of incubation. out of the positive blood cultures detected between the fourth and fifth incubation days, were recovered in concurrent cultures within the first days. chart reviews were conducted from patients with the remaining isolates. only in patients ( . % positive blood cultures) changes in antimicrobial therapy based upon the positive blood culture results on day to were made, in the other patients the empirical treatment was adequate. the isolated microorganisms in those patients were: gram-positive cocci ( staphylococcus spp. not s. aureus, staphylococcus aureus, streptococcus viridans and streptococcus pyogenes), anaerobes, enterobacteriaceae, pseudomonas aeruginosa, campylobacter spp., candida spp. cryptococcus neoformans, brucella spp. and haemophilus influenzae. conclusions: incubation of bact/alert blood cultures bottles only for days would have represented a detection loss of . % of the clinically significant isolates, which led to antimicrobial therapy changes. although we keep employing a -day incubation for routine blood cultures, we could reduce the incubation time to days depending on current instrument capacity. an enzyme immunoassay for anti-diphtheria antibodies: a practical alternative to the vero cell assay r. budd, e. harley, r. george, a. efstratiou, k. broughton, a. bradwell (birmingham, london, uk) introduction: in this extended study, results from an anti-diphtheria toxoid enzyme immunoassay (eia), specifically designed to detect higher affinity antibodies, were compared with those from a vero cell assay (vca). methods and results: serum samples with antibody concentrations ranging from . - . iu/ml on the vca from the respiratory and systemic infectious laboratory (rsil) were assayed by eia (the binding site ltd, uk). a further samples from rsil selected on the basis of being close to the protective level, were assayed to confirm the performance of the eia. the eia was calibrated, against the nibsc reference material / and the assay measuring range was . - . iu/ml results were compared using the who guidelines of . - . iu/ml as minimum protective level, and > . iu/ml as protective. relative agreement, sensitivity and specificity for the first samples were: . %, . % and . % respectively, for the second set of samples performance was: . %, . % and . %, and for the combined samples results were: . %, . % and . % respectively. roc analysis of the total samples confirmed the highest sensitivity . % and specificity . % occurred at a cut-off of precisely . iu/ml for the elisa assay. conclusion: of the total discrepant samples, had vca and eia values < . iu/ml, therefore we suggest the possibility of establishing an equivocal zone for the interpretation of the eia results. if the test is part of a general immune status assessment a grey zone is not required. if undertaken to determine the requirement for immunization, the use of the equivocal zone is recommended. by applying these criteria in the eia, only one sample would have suggested inappropriate immunization, as indicated by a vca result > . iu/ml. because of the > % agreement between the two assays, significant advantages of cost and speed, ease of use and the potential for automation, the eia could therefore be considered as an alternative to the vca. evaluation of accuracy limits of countable colony-forming units on agar plates j. arbique, a. rendell, k. forward (halifax, ca) objectives: accurate colony counts are an essential component of many microbiology research projects and clinical laboratory processes. the suggested range of accuracy of colony-forming units (cfu) extends from to (standard methods for the examination of water and wastewater). this recommendation dates to , and fails to adequately address the numerous sources of inter-and intra-variability. without more detailed analysis it is difficult to estimate the sample size and number of replicates necessary to ensure accurate results. the purpose of this study was to determine the validity of accuracy limits for quantifying cfus on agar plates. methods: escherichia coli (atcc ) and staphylococcus epidermidis (atcc ) were used to prepare series of four organism densities ranging from approximately - cfu, on three different days. on each day, each of the densities for both organisms was plated on sba and viable organisms were counted following incubation. an average of the margins of error obtained over the days of testing was used to determine the reproducibility of agar plate counts, and to estimate the optimum number of replicate plates (sample size) required for each organism at each concentration. results: margins of error for both organisms were greatest with suspensions yielding approximately cfu, and lowest for suspensions yielding and cfu. nine replicate plates were required for a suspension of s. epidermidis yielding cfu to achieve the same margin of error as obtained with replicate plates at concentrations yielding - cfu. seven replicates plates were required for a suspension of e. coli yielding and cfu to achieve similar margins of error to those obtained with replicate plates at concentrations yielding cfu, and replicate plates at concentrations yielding cfu. conclusion: we found that the greater the concentration ( and cfu), the fewer replicate plates necessary to reliably estimate organism concentrations. the lower the organism density ( cfu), the more plates necessary to reliably estimate cfus. contrary to the recommendations described in standard methods for the examination of water and wastewater, cfu of were reliably reproducible. for greatest accuracy, experiments should be conducted so as to assure that colony counts are in the range of - . direct microscopy: a valuable instrument for diagnosis and prognosis of periodontal disease objective: to appreciate the composition of micro flora from periodontal pockets, using light microscopy and to compare it with clinical status. introduction: it is generally accepted that periodontal disease occurs when anaerobic gram-negative flora increase in number with the subsequent decrease of facultative anaerobe grampositive bacteria. in other words, the switch from gram-positive to gram-negative of sub-gingival flora has a pathologic significance and could be observed using direct microscopy. materials and methods: specimens sampled with sterile paper points from periodontal pockets and samples from clinical healthy persons were included in this study. each sample was diluted in . ml saline solution and, with a calibrated loop, was taken ll aliquots in order to prepare a smear for microscopic examination and for inoculation on solid media (columbia with % sheep blood). the smears were gram stained and the culture plates were incubated in anaerobic conditions ( h, °c) and in air ( h, °c). results: in % of samples from patients with periodontal disease, easily notable, high number of gram-negative bacteria at direct microscopy, associated with abundant growth in anaerobic condition and poor growth in air. in from healthy patients, the gram-negative flora was almost absent and gram-positive bacteria were in high number, correlated with the absence of bacterial growth in anaerobiosis and some growth in air.the presence of treponema spp. at direct microscopy was associated with deep and bleeding periodontal pockets. after few days of proper therapy, the good clinical status was well correlated with an increasing number of gram-positive bacteria. conclusions: ) using a diluted sample for microscopic examination, the value of the method increase, offering important information about the composition of sub-gingival flora. ) the good correlation between the clinical status and microscopic finding recommend it as an easy to use diagnostic method in dentistry. identification of species and glycopeptide resistance among enterococcal isolates by bd phoenix objectives: vancomycin resistant enterococci are emerging in europe necessitating their fast and accurate identification by the laboratory. there was an attempt to evaluate the performance of the bd phoenix automated microbiology system (bd diagnostic systems, sparks, md.) for the correct identification of species and glycopeptide resistance in comparison to the gold standard of diagnosis, pcr, using a large collection of clinical strains. methods: a total of enterococcal isolates were tested by the bd phoenix sytem. these strains were isolated from faecal, urine, pus, blood and samples from other body sites cultures. a multiplex pcr was applied using different pairs of primers, specific for the identification of e. faecium, e. faecalis and the vana, vanb, vanc, vand, vang, vane glycopeptide resistance genotypes. susceptibility to the glycopeptides was also confirmed by the etest (ab biodisk, solna, sweden). results: according to the pcr, there were e. faecium (including vana-positive strains), e. faecalis (including vana-positive and vanb-positive strains) and e. cass/gall isolates. two strains were not identified and were excluded from the analysis. discrepant results between the multiplex pcr and the phoenix system were obtained for / isolates ( %) with similar rates amongst faecal ( / , . %) and the rest of the isolates ( / , . %). the most common discrepancies were the misidentification of e. faecium vana strains and e. faecalis strains as e. cass/gall by phoenix. two e. faecalis strains were incorrectly characterized as vancomycin resistant, two e. faecium strains were misidentified as e. hirae and e.cass/gall, respectively, and one e.cass/gall strain was reported as e. faecium resistant to both glycopeptides. thus, the sensitivity and specificity for the identification of e.cass/gall by phoenix were . % ( / strains) and . % ( / strains), respectively, while . % of vana strains ( / strains) were not recognized by this system. conclusion: this study demonstrates that the new identification system, phoenix, similarly to other automated or manual systems, presents with problems regarding correct identifica-tion of enterococcal species and glycopeptide resistance. specifically, laboratories should be aware that clinically significant isolates identified as e.cass/gall should be confirmed by another method. an audit of sputum requisition practices p. lal, i. balakrishnan (london, uk) objectives: to analyse the indications and rationale for the processing of sputum specimens in a london teaching hospital. methods: sputum samples received from / / - / / were included in this study. data were obtained from the patient requisition forms and the winpath systems and were analysed further as per the objectives. results: a total of specimens were received during this period. ( %) from hospital in-patients and ( %) from general practitioners. out of the total of samples received from hospital-in patients ( . %) had > epithelial cells/ lpf. no clinical details were mentioned in ( . %) and ( . %) were from patients already on antibiotics. repeat specimens within one week were sent in ( . %) cases and ( %) had atypical serology also sent. out of the hospital-in patient samples ( . %) had a significant isolate and ( . %) had normal respiratory tract flora isolated. others were reported as: ''gross oral contamination'' [ ( . %)], ''no growth'' [ ( . %)] and ''no significant growth'' [ ( . %)]. there were a few specimens reported as ''inappropriate specimen -two days old'' [ ( . %)] and ''leaking'' or ''saliva only' ' [ ( . %) ]. out of a total of samples received from gp patients ( . %) of samples had > epithelial cells/lpf, ( . %) had no clinical details provided, ( . %) samples were sent while patients were on antibiotics and ( . %) samples were repeated within one week. only ( . %) had atypical serology also sent. conclusions: -less than one-third of specimens yielded a significant pathogen.-adequate clinical details were lacking in about one-fifth of specimens.-nearly one-third of specimens were repeated within one week, without a clear indication.-about % of specimens were of poor quality.-atypical serology was only performed in . % of outpatients, as compared with % of in-patients.this audit brings forth the fact that the clinical indications for which sputa are being sent for culture need to be clearly defined and an educational campaign instituted amongst relevant healthcare professionals. sputum collection techniques need to be rigorously applied if good-quality specimens are to be obtained. indications for performing atypical serology need to be defined and reinforced, particularly in primary care. a new approach to laboratory diagnostic of infectious gastroenteritis -a follow-up objectives: in order to optimize use of laboratory facilities and ensure flexibility in relation to current epidemiology, a new approach to laboratory diagnosis of infectious gastroenteritis was applied: from an algorithm the decision of which organisms to test for was defined by the demographic, clinical and epidemiological information submitted to the laboratory on paper/electronic request forms. methods: from april , -june , , hospitals and general practitioners submitted a request form with the following information together with the stool sample (s): ( ) acute or persistent diarrhoea (duration > weeks); ( ) bloody stools; ( ) recent history of foreign travel; ( ) > patients within same epidemiological setting; and ( ) nosocomial infection. provision of data is mandatory when submitting electronically. based on these data, analyses were performed according to an algorithm. examination for salmonella, shigella, yersinia, campylobacter, and clostridium difficile was done by culturing. verotoxin producing e. coli (vtec), enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec) and enteroinvasive e. coli (eiec) were identified by pcr for virulence genes and serotyping. rota and adenovirus were detected by antigen tests and parasites by microscopy. results: in total we examined , samples from , patients. a pathogen was isolated in % of patients. in cases ( %) clinical/epidemiological data were missing. • , patients had diarrhoea < weeks: % with campylobacter, % with salmonella, % with etec, % with giardia, and % each with eiec and vtec • , patients had diarrhoea > weeks: % with campylobacter, % with giardia, % each with epec and vtec • patients had a history of foreign travel: % with campylobacter, % with etec, % with salmonella, and % with giardia • patients had bloody stools: % with campylobacter, % with salmonella, and % with vtec • patients were < years: % with campylobacter, % with epec, % each with giardia, vtec and salmonella, and % with etec conclusions: campylobacter was the most common bacterial pathogens in all groups and rotavirus was the most common pathogen in children < years. the new approach had a number of advantages: more relevant microbiological analysis, collection of data on defined patient groups, and flexibility regarding adaptation to current epidemiological knowledge. increasing use of electronic submission of request forms will optimize the approach used. objectives: small colony variants (scvs) are an emerging infectious disease problem, presenting as a naturally occurring, slow-growing subpopulation of staphylococcus aureus that are characterized by tiny colonies on solid media. studies on scvs recovered from patients with persistent infections are hampered due to their frequent unstable phenotype. in particular, scvs are not easily distinguishable from the normal phenotype in broth media and a reversion of scvs into the normal phenotype is not traceable. methods: a set of isogenic s. aureus isolates comprising the (i) normal and the (ii) scv phenotype (isogenic to the isolate with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding mutants mimicking the scv phenotype (knock-out of hemb), and (iv) their complemented mutants were used to investigate the feasibility of fourier-transform infrared (ftir) spectroscopy to trace the expressed phenotype in broth media. the respective isolates cultured on solid media served as controls. in addition, all isolates were genotyped by pulsed-field gel electrophoresis and spa typing. results: using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information in three different spectral ranges resulted in a dendrogram that showed a clear discrimination between both staphylococcal phenotypes. distinct clusters comprising the clinical and mutant scv phenotype on one hand and the normal phenotype (isolate with normal phenotype and complemented mutant) on the other hand were found. thus, scvs from different clonal lineages gave spectra that were more similar to one another than to their normal growth parent. ftir was also shown to be able to trace the switch of the phenotypes in broth when the medium was supplemented. conclusion: ftir spectroscopy allows a rapid, reproducible and clear discrimination of different phenotypes of s. aureus in fluid media for diagnostic and research purposes. in contrast to genotyping approaches, ftir staphylococcal fingerprinting is only reliable for typing purposes if the isolates exhibit the same phenotype. in future studies, this technique may also provide an approach for tracing the scv phenotype in infected tissues. objectives: triggering receptor expressed on myeloid cells- (trem- ) is a recently discovered cell surface molecule whose expression on phagocytes is up regulated by exposure to bacteria or fungi. a soluble form of trem- (strem- ) can be measured in various body fluids. we studied whether strem- in cerebrospinal fluid (csf) could serve as a biomarker for the presence and outcome in patients with bacterial meningitis. methods: in this retrospective study on diagnostic accuracy we used an elisa to determine levels of strem- in csf from adults with bacterial meningitis, confirmed by csf culture, who participated in the prospective dutch meningitis cohort study; patients with viral meningitis, confirmed by polymerase chain reaction of csf; and healthy control subjects, who underwent lumbar puncture to exclude the diagnosis of subarachnoid haemorrhage. the mann-whitney u test and the chi-square test were used to identify differences between groups. a receiveroperating-characteristic curve (roc) was constructed to illustrate various cut-off csf levels of strem- in differentiating between the presence and absence of bacterial meningitis and diagnostic accuracy was quantified by % confidence intervals ( % ci). results: levels of strem- in csf were higher in patients with bacterial meningitis as compared to those with viral meningitis [median, pg/ml (range, to pg/ml) versus . pg/ml (range, to pg/ml); p = . ] and controls [ pg/ml (range, to pg/ml); p < . ; fig]. patients with viral meningitis and controls had similar csf strem- levels. the area under the roc curve for discriminating between patients with and without bacterial meningitis was . ( % ci, . to . ; p < . ). at a cut-off level of pg/ml, strem- yielded a sensitivity of . ( % ci, . to . ) and a specificity of . ( % ci, . to . ). in patients with bacterial meningitis, csf strem- levels were associated with mortality [survivors versus nonsurvivors: median pg/ml (range, to pg/ml) versus pg/ml (range, to pg/ml); p = . ]. conclusions: measuring strem- in csf may be a valuable new approach to accurately diagnose bacterial meningitis and identify patients at high risk for adverse outcome. therefore, a prospective study on strem- as biomarker in bacterial meningitis is needed. systematic review of rapid diagnostic tests for enterohaemorrhagic e. coli i. abubakar, l. irvine, l. shepstone, s. schelenz, c. aldus, p. hunter (norwich, uk) objective: a variety of rapid tests for the detection of enterohaemorrhagic escherichia coli (ehec) have recently emerged. culture on sorbitol macconkey (smac) agar and biochemical identification, while easy to use and inexpensive, is slow and lacks sensitivity in the detection of non o :h serotypes. this study sought to determine the accuracy of rapid serological or polymerase chain reaction (pcr) assays which have been evaluated for the detection of all ehec serotypes compared to culture. methods: a systematic review and meta-analysis of articles, identified via searches of electronic databases, hand searching of selected journals, and through contact with experts and commercial test manufacturers. the majority of these needed to be excluded due to low quality or lack of accuracy data. sensitivity and specificity of each method was calculated using full biochemical identification as the reference standard. twenty-one studies met the inclusion criteria, of which used pcr methods and used serological assays and were based on culture. a summary receiver operator curve (sroc) was constructed from these data and the area under the curve (auc) calculated (using the trapezium rule). results: serological tests had individual sensitivities ranging from . to . and specificities ranging from . to . . pcr tests had individual sensitivities ranging from . to . and specificities ranging from . to . . additional analysis comparing smac agar culture with toxin detection methods showed poor sensitivity compared to pcr and serological tests (ranging from . to . ) yet the specificity was very good ( . for all studies considered). our results suggest that both molecular and serological tests may have a potential role in detecting ehec infection. whilst there is very little difference in the effectiveness of these techniques, both are faster and have improved sensitivity when compared to traditional culture methods. fast, reliable diagnosis could lead to more informed treatment choices and improved outbreak control measures. however, given the substantial extra cost of these assays, an assessment of economic feasibility is necessary prior to use in everyday practice. antibodies against bordetella pertussis detected by slow agglutination test and elisa in two agerelated groups of vaccinated people suspected of acute pertussis: a comparative study objective: the aim of presented study was to describe differences between results of two tests used for detection of antibodies against bordetella pertussis (slow agglutination and elisa) in two age-related groups of patients suffering from respiratory infection. each of the people has undergone vaccination against b. pertussis. methods: paired sera obtained from two age-related groups of patients [( ). age - years, n = ; ( ). age above years, n = ] suffering from acute respiratory infection were tested. the first group comprised the children who were vaccinated earlier than one year before testing; the second group was determined by longer interval between the vaccination and the testing. the criterion of positivity of the slow agglutination was based on quadruple increase/decrease of the titer of specific antibodies; the criterion of serodiagnosis of the illness was the same. each of the patients was tested by elisa iga,igg,igm (virotech) during the same period, positive results of each of class of immunoglobulins were evaluated as positive elisa. the differences of results obtained by the two tests were assessed inside and between the groups. results: there were found . % (respective . %) concordant positive and . % (respective . %) concordant negative results between the tests in the first (respective second) group. there were found the following discrepancies in the frame of non equal results: agglutination positive/elisa negative sera were present in . % (respective . %) persons and agglutination negative/elisa positive samples were present in . % (respective . %) persons. conclusion: ( ) . the frequency of serologically confirmed infection based on results of slow agglutination is higher in the group of people older six; the interpretation of the results in the younger group is limited by the influence of actual vaccination. ( ) . the elisa evaluated as described above shows extremely high frequency of positivity in both groups, thus, the usefulness for diagnostics of acute infection seems to be low. ( ) . the study will be continued to asses relationships between the positive results detected by slow agglutination and the positive ones detected by elisa in separate classes of specific immunglobulins. accuracy of the microscan walkaway system to identify coagulase-negative staphylococci a. sáez, b. ruiz, l. martínez-martínez (santander, es) objective: to determine the reliability of the identification of coagulase-negative staphylococci (cons) with the microscan walkaway (wa, dade behring) system at species level when a > = % probability is obtained, considering as a reference the results of molecular identification. methods: one hundred and sixty-eight isolates of cons from clinical samples (october -may for which the identification with the wa system was ‡ %, and atcc type strains were evaluated. bacteria were identified with the wa system using pos combo s panels. absence of coagulase was determined with a latex assay (pastorex Ò staph-plus, bio-rad). reference identification was established by sequencing of the s rrna; when identification with wa and s rrna disagree, definitive identification was defined after sequencing of the soda and tuf genes, as previously described (drancourt et al. jcm ; : - and heikens el al. jcm ; : - ) . for identification, the sequences of s rrna, soda and tuf were compared with those in genebank. homologies values above % were considered reliable. results: all type strains were correctly identified by s rrna sequencing as named by the atcc. among the clinical isolates, the molecular method identified the following species (number): s. hominis ( ), s. haemolyticus ( ), s. saprophyticus ( ), s. epidermidis ( ), s. lugdunensis ( ), s. schleiferi ( ), s. capitis ( ), s. simulans ( ), s. pasteuri ( ), s. warneri ( ), s. intermedius ( ) and s. equorum ( ). the wa system correctly identified out of the atcc strains. s. pasteuri is not included in the wa database, and the corresponding atcc strain was misidentified as s. warneri. one hundred and fiftyseven out of the ( . %) clinical isolates were correctly identified by the wa. five s. haemolyticus were identified by wa as s. auricularis ( ), s. simulans ( ) and s. warneri ( ) . other errors corresponded to: two s. pasteuri misidentified as s. warneri, one s. epidermidis as s. hominis, one s. lugdunensis as s. schleiferi, one s. hominis as s. haemolyticus and one s. equorum as s. cohnii. all isolates of s. saprophyticus, s. schleiferi, s. capitis, s. simulans, s. warneri and s. intermedius were correctly identified by the wa system. conclusions: the microscan walkaway is reliable to identify cons at species level when a probability of > = % is obtained. s. pasteuri should be incorporated to the wa database in order to improve its performance. objectives: the aim of this study was to analyse the results of proficiency testing obtained by polish microbiology laboratories participating in polmicro. haemophilus influenzae is an important pathogen causing a variety of community-acquired respiratory tract infections, acute otitis media and purulent meningitis. two mechanisms of ampicillin (amp) resistance in this organism are described. one is mediated by the production of beta-lactamases tem- and rob- ; these amp-resistant strains are termed beta-lactamase-producing, amp-resistant (blpar). the second mechanism involves development of altered penicillin-binding proteins (pbp) with decreased affinity to amp and other beta-lectam agents. strains with resistance mechanisms mediated by pbp alterations are termed beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae. methods: four hundred seventy eight laboratories participated in this part of the scheme. each participating laboratory received haemophilus influenzae (pm- )-beta-lactamase negative, ampicillin-resistant strain (blnar). the laboratories were asked to provide identification to the species level and of the susceptibility results and interpretation. results: correct identification to the species level of this strain was reported by laboratories ( . %) of the labs involved. thirteen laboratories reported the analysed strain as haemophilus parainfluenzae. three hundred ninety eight laboratories ( . %) of correctly detected the mechanism of resistance to beta-lactams. only three laboratories incorrectly reported the organism as beta-lactamase producer. the greatest dispersion of inhibition zone was observed in the susceptibility of h. influenzae to ampicillin, amoxicillin-clavulanic acid and clarithromycin. conclusions: over % of the laboratories correctly identified and interpreted beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae strain. purpose and methods.the architect syphilis tp assay is a chemiluminescent eia that employs three recombinant antigens of treponema pallidum on the solid phase and an anti-human igm and igg conjugate. we evaluated this assay in comparison with a conventional eia (diesse enzywell syphilis screen recombinant) on unselected routine serum samples and on repository specimens for whom the results for specific igg and igm and of the rapid plasma reagin (rpr) assay were already known. in both instances an immunoblot (ib: inno-liatm syphilis score, innogenetics) has been employed on discordant specimens as a confirmatory assay. the precision and robustness of the architect assay were also evaluated.results.on . routine samples ( from volunteer blood donors and from in and outpatients) the concordance between architect and eia was high ( . samples, or . %; positives, , negatives). one of the discordant, positive by architect and negative by eia, was confirmed by ib. the specificity of the architect assay was . % ( % confidence limits: . - . ). the repository samples assayed belonged to three groups: ) biological false positives from subjects: all negative by architect; ) true positives, all positive by architect, with a significantly stronger signal (average s/co: . vs. . ) on the igm positive samples, all of whom were also positive by rpr; ) samples positive by rpr and negative by eia igg: of them were negative by architect as well and for igm, while two specimens were strongly positive by architect and positive also for specific igm and with three specific bands by inno-lia, suggesting a pattern of recent infection. the reproducibility of the architect assay was good, with cvs of . %, . % and . % on replicates over weeks of the assay's negative and positive control and of an internal control; finally, the s/co distribution of negative specimens confirmed the robustness of the assay, with a mean of . , a median of . , standard deviations between the mean and the cut-off value and the th percentile at a s/co value of . .conclusion.the automated assay for anti-treponema pallidum antibodies on the architect system has an excellent sensitivity and a good specificity. the analytical performances, coupled with the elevated throughput and minimal samples handling, make this method a first-choice option for syphilis screening and diagnosis in medium and large volume laboratories. objective: quantitative urine culture is the gold standard for defining the diagnosis of urinary tract infection (uti), because it allows identification of the uropathogenic species. however, this method is time consuming and expensive. approximately, up to % of urine cultures are negative with high cost for unnecessary testing. thus, we have evaluated the usefulness of two automated analysers for uti screening to quickly identify the negative samples that can be prompt reported to the clinicians, improving in the quality of patient care and allowing the laboratory to direct more effort into positive samples. methods: . of midstream urine samples submitted for microbiological examination were analysed by conventional urine culture plates (mcconkey agar + trypticase soy agar + bile esculine azide), sysmex uf- (sysmex, japan) and coral uti screen (coral biotechnology, ca, usa) automated analysers. uti was defined positive as follows: one or two strains of bacteria with at least ufc/ml for the culture plates, more than . bacteria/ll and more than wbc/ll for the uf- and/or an rlu value grater than % of the calibrator value for the coral. when more than two strains of bacteria were found, the culture was classified as contamined. results: the diagnostic performance of sysmex uf- and coral uti screen are shown in table . the sensitivity ( . %) and negative predictive value ( . %) confirm that sysmex uf- and coral uti screen are an excellent screening for uti. after this evaluation, we decide the use of the sysmex uf- and coral uti screen on our routine workflow for uti screening. the results of both the analysers are sent to a software system (labfinity dasit, italy) connected to the lis. if the results are lower than the cut-off values, uti can be excluded and directly reported to the physician. positive results are submitted to microbiological culture and reported within or hours depending on negative or positive bacterial growth. in our experience, evaluated on further . samples, this means that % of samples are immediately reported within very few hours. of the % of positive samples, ( %) were confirmed by culture and reported within hours, ( %) were not confirmed and reported within hours. comparison of the blood and bone marrow culture positivity rates for the diagnosis of brucellosis objectives: brucellosis is a common disease, seen worldwide as well as in our country. the diagnosis of brucellosis is made with certainly when brucellae are recovered from blood, bone marrow. in our study, we aimed to compare the blood and bone marrow culture positivity rates in patient with brucellosis. methods: this study was performed in the infectious diseases and clinical microbiology department of ankara research and training hospital between and . the diagnosis of brucellosis was made on the history, physical findings, serologic findings and the isolation of the organism. the number of patients with brucellosis included to the study was . blood and bone marrow samples were taken from all of the patients on admission and cultured by using the bactec system. results: blood culture positivity for brucellosis was % ( / ), while bone marrow culture positivity was % ( / ). the difference between those positivity rates was found to be statistically significant (p < . ). the isolation ratio from blood cultures among acute cases was % ( / ) while it was % ( / ) among subacute cases. brucella isolation from blood was not detected in chronic cases. the isolation rates of the microorganism from bone marrow of acute, subacute and chronic cases were . %, . %, . % respectively. among our patients, had history of medical therapy for brucellosis before admission and of them was treated inadequately. of those cases, the organism was isolated in ( %) from blood and in ( %) from bone marrow.in the cases with high standard tube agglutination titers, the rate of positivity was also high both in blood and bone marrow cultures. however when compared with low standard tube agglutination titers, that difference was not statistically significant.the mean growing time for the positivity of cultures was . days for bone marrow and was . days for blood cultures. the difference between the mean growing times of two culture types was found statistically significant (t-test. p < . ). conclusion: premedication, subacute and especially chronic phases decrease the possibility of isolation of the microorganism from blood culture. therefore we suggest taking bone marrow culture only for these kinds of patients as it. is a traumatic process. serological findings in blood sera of patients with yersinia-triggered arthritis e. golkocheva, r. stoilov, h. najdenski (sofia, bg) objectives: immunoblot analysis of iga and igg antibody response of blood sera from patient with yersinia triggered reactive arthritis and with undifferentiated arthritis were made. patients and methods: serum samples were obtained from patients admitted to clinic of rheumatology at medical university, sofia, bulgaria with suspicion of yersinia triggered reactive arthritis, based on diagnostic criteria. a total of blood serum samples were analysed by immunoblot analysis with specific antigens-yops (yersinia outermembrane proteins). when y. enterocolitica is cultivated at o c under calcium restriction ( . mm ca + ), large amounts of yops are secreted into medium. these proteins were separated by d-sdselectrophoresis. results: immunoblot analysis of iga and igg antibody response against yops in blood sera from patients with arthralgias and polyarthropathies was carried out. yersinia enterocolitica, serotype o: , was used as source for yop. seven strong bands of the molecular weights kda-yope, kda-yopn, kda-yopd, kda -v-ag, kda-yopb, kda-yopm and kda-yoph were visualized. for immunoblot assay the optimal concentration of antigen was established by analytical electrophoresis. of the blood sera from the patients with yersinia triggered reactive arthritis igg antibodies were detected against yoph, yopm, yopb, yopd, yopn and yope. iga antibodies were established against yopm, yopb, yopd, yopn and yope. all sera from the patients with other rheumatic diseases were negative for the presence of anti-yersinia iga antibodies and two of them were positive for igg against yopd. antibodies from two classes were not detected in sera samples from healthy people. conclusions: yops are borne by the virulence plasmid, which mean that they are clearly associated with virulence properties of pathogenic strains. moreover, yops is not restricted to single serotype and this made them a specific antigen in diagnosis of different yersinia infections. conventional techniques such as culture and demonstration of serum agglutinins prove to be insufficient to demonstrate invasive or chronic yersiniosis in contrast with the determination of specific serum iga and igg antibodies by immunoblot analysis and antigen detection. the detection of anti-yops igg and iga antibodies by immunoblot can be used for diagnosis of yersinia triggered arthritis. acknowledgements: this work was sponsored by natoreintegration grant . objectives: to evaluate the identification and susceptibility results by using suspensions obtained directly from positive blood cultures. methods: during the period between st august and st october we selected all positive cultures grown in bact/ alert Ò sa and sn bottles (biomérieux) from gram-negative bacilli. only the first culture positive from each patient was included. we inoculated ml fluid from a positive bottle into a serum separator tube (bd vacutainer systems, plymouth, united kingdom) and centrifuged at x g for minutes and the supernatant was carefully aspirated. using a cotton swab the bacteria were removed from the top of the separator layer to be suspended in . % saline solution to get . mcfarland. the suspension was processed according to standard inoculation procedure for gn and ast-n vitek Ò cards. positive bact/alert d bottles were also sub cultured and after an overnight incubation several colonies were used to make a . mcfarland suspension in . % saline. the suspension was processed according to standard vitek Ò inoculation procedure for gn and ast-n cards. results: identification: a total gram-negative bacillus from positive blood cultures were investigated. fifty ( . %) strains were correctly identified to the species level, four ( . %) strains were not identified and two ( . %) strains were misidentified. antimicrobial susceptibility testing: in all, mics were determined for isolated by both methods. the unidentified strains ( ) were excluded. the overall mic agreement between direct and standard inoculation was . %. all individual antimicrobial agents scored > %. the overall minor error rate was . % ( of ). the overall major error rate was . % ( of ). the overall very major error rate was . % ( of ). the highest rate of mic agreement was for amikacin, norfloxacin ( %), meropenem ( %), gentamicin and ofloxacin ( . %). conclusion: the direct method from positive bact/ alert&# ; cultures cannot totally replace the approved methods of identification and susceptibility but in some cases provides earlier information which allows a better patient management and also reduce cost in patient care. investigation of listeria monocytogenes "o" antibodies in maternal and cord sera with the agglutination test e. us, a.t. cengiz, o. gelisen (ankara, tr) objectives: listeria monocytogenes is a gram-positive food borne pathogen that is responsible for listeriosis, a human infection with a mortality rate of %, which could cause severe motherto-child infections. this serious pathogen in pregnancy could be treated if diagnosed, but there is no routine screening test for susceptibility to listeriosis during pregnancy. therefore, we investigate different l monocytogenes serotype o antibodies for diagnosis of listeriosis in maternal sera with agglutination test. of them had spontaneous abortion, premature labour or stillbirth (group i), while had no obstetric patology (group ii) in their previous pregnancies. cord bloods were also obtained at the delivery and tested. methods: all sera were being tested against antigens with the o formulation of serotype / c, b, ab, c and d. the antigens were prepared by the method of osebold, and larsen et all. the bacterial suspensions were trypsinized for min at °c to prevent cross-reactions and contaminations. sera were diluted by doubling serially in saline followed by addition of an equal volume of antigen. a positive titre of greater than or equal to : was chosen as positive test result to maximize the sensitivity and specificity. results: . % of cases have ingested raw milk and diary products, . % ready-to-eat foods, and . % developed nonspecific febrile illness (nfi) during their pregnancies. % of group i were found positive ( . % developed nfi) while at group ii % had positive ( . % developed nfi) agglutination titres to one ore more serotypes. all the cord blood sera of group i were found negative, whereas two in group ii (all ab) were positive, with the positive maternal sera of the same serotype. it's evaluated as transmission of the antibody from mother to foetus. at group i the frequent serotypes were / c = ab, at group ii ab, / c, respectively. the newborns showed no symptoms or signs of listerial foeto-maternal infection. conclusion: the women encountered the antigens of l monocytogenes in any period of their life time (most - years of age) and produce antibodies against this pathogen. there is a relationship between nfi and positive titres. if the disease is recognized, it is possible to treat the mother and allow the birth of a healthy infant. we propose the less time consuming and easy to perform agglutination test as a routine screening test for susceptibility to listeriosis during pregnancy to prevent bad pregnancy outcomes. objectives: to evaluate the performance of a real time pcr assay (with a fluorogenic target-specific probe), mrsa-idi (geneohm sciences) for mrsa detection directly from mucocutaneous swabs in hospitalized patients. methods: clinical swabs ( to samples with a median of . samples per patient) from nares (n = ) and skin (n = ) were prospectively collected for mrsa screening from patients admitted to a -bed teaching hospital. swabs were inoculated onto selective mrsa agar (mrsa-id, biomérieux), into the buffer extraction solution for idi-mrsa pcr assay and into enrichment broth (bhi with . % nacl). after h, bhi broths were subcultured onto mrsa-id agar. selective agars were incubated for h at ordm;c and examinated daily. suspected colonies were identified by coagulase testing; oxacillin resistance was tested by cefoxitin disk diffusion according to clsi recommendations. the pcr assay was performed according to the manufacturer's instructions. pcr results were compared with phenotypic identification test results. in case of discordant results, the assay was repeated, but only results from first testing were considered for calculating test performance. results: mrsa was detected by culture in specimen ( . %) from patients. the sensitivity and specificity of the pcr compared with culture was . % and . %, respectively. positive predictive value and negative predictive value were . % and . %, respectively. the sensitivity of pcr ( %) was higher on nasal swabs than on swabs from other sites ( . %, p < . ). the pcr assay detected mrsa in patients ( . %). the pcr assay provided results in to versus to hours for conventional method. conclusion: in our hospital, the id-mrsa pcr assay detected . % mrsa carriers in less than hours when performed on multiple specimen. the assay appeared more sensitive in testing nasal swabs than other clinical specimens. prospective studies are needed to evaluate the impact of this assay for rapid implementation of infection control procedures and its global costs and benefits. the purpose of this study was to establish a rapid and sensitive real-time polymerase chain reaction (pcr) method for detection of methicillin-resistant staphylococcus aureus (mrsa) from blood culture bottle. as a result of over use of broad-spectrum antibiotics after the s in whole the world, an outbreak of mrsa infection has been seen. severe nosocomial infections with mrsa such as bacteraemia and sepsis may lead to multiple organ failure and high mortality in the hospital. although standard method took at least hours to identify mrsa by the blood culture method, the presence of meca and nuc genes which is specific for methicillin resistance and s. aureus was determined by real-time pcr method within only hours after blood culture signal positivity. nineteen s. aureus and coagulase negative staphylococci positive blood culture bottles were studied retrospectively for detection of s. aureus and methicillin resistance. staphylococci were identified with classical methods and mics of oxacillin were determined by etest (ab biodisk) on mueller-hinton agar supplemented with % nacl. real-time pcr was performed to all positive blood culture samples for s. aureus and methicillin resistance determination. nineteen ( %) s. aureus were determined correctly by real-time pcr method. forty-four methicillin resistant and methicillin sensitive staphylococci were detected by etest. using the real-time pcr method, the meca gene was detected in staphylococci except . when compared with etest and realtime pcr method gave sensitivity, specificity, and positive and negative predictive values of %, %, %, % for both positive and negative tests, respectively. agreements between two methods were high ( %); there were discrepant results among the strains were tested. detection of mrsa bacteraemia and methicillin resistance with real-time pcr definitely is useful for reducing mortality and morbidity of this type infection. in conclusion, this method, as many as sensitive and specific for detection of mrsa bacteraemia and clinically should be beneficial for prevention of unnecessary antibiotic use and determination of appropriate antibiotic treatments of mrsa infection. pcr detection of class b, c and d betalactamases in environmental and clinical aeromonas strains t. fosse, c. giraud-morin, f. la louze (nice, fr) objectives: aeromonas spp. strains are waterborne opportunistic pathogens. they are able to produce different types of beta-lactamases (class b, c and d). the determination of beta-lactamase content is not easy by phenotypic methods. we have developed a pcr tool to study diversity and distribution of class b, c and d beta-lactamases in a set of representative clinical and environmental aeromonas species. method: a total of references, environmental and clinical strains were tested. identification was realized by conventional tests and gyrb sequence analysis. beta-lactam antibiotic susceptibility was determined by diffusion agar and micro broth dilution methods. three sets of specific primers were defined for the pcr amplification of the internal region of class b beta-lactamase (mei and mei , bp size), class c beta-lactamase (aercp and aercp , bp) and class d betalactamase (aerd and aerd , bp). all pcr products were sequenced. results: class d pcr was positive with most strains except a. trota, a ticarcillin susceptible species ( strains) . class c pcr was positive with most cephalothin resistant strains (mic > mg/l; / strains, %) including a. hydrophila and a. caviae phenospecies. class b pcr was positive with most strains of a. hydrophila and a. veronii phenospecies ( / ; %) including three imipenem susceptible strains (mic < mg/l). beta-lactamase type distribution was species related and was particularly useful to better characterize environmental species such as a. bestiarum, a. popoffii and a. allosaccharophila. partial beta-lactamase gene sequence analysis allowed phylogenic studies. some cephalosporinase gene from environmental species was probable progenitor of ampc plasmidic beta-lactamase. conclusion: pcr with specific primers was a good method to detect class b, c and d beta-lactamase in aeromonas species. beta-lactamase type distribution and sequence analysis phylogeny were largely species related and could be helpful for molecular diagnostic and taxonomic purpose. objectives: the aim of this study was to develop a convenient dna extraction method and to optimise a pcr reaction in order to detect enterotoxin b producing s. aureus strains directly from milk. methods: we applied a chemical extraction method of bacterial dna from milk samples artificially inoculated with s. aureus. a pcr based method was used for the detection of seb gene (coding for enterotoxin b) and nuc gene (coding for termonuclease). a protocol for the multiplex pcr was developed and optimized. the sensitivity of the reaction was checked by determining the minimum number of organismsaeml - , which can be detected in the multiplex pcr and in each single pcr reaction. amplification specificity of the seb gene was verified by amplicon digestion with restriction endonucleases. results: the specific bands for both genes in the multiplex pcr were detected in samples containing a dna quantity corresponding to organismsaeml ) . in the same reaction, the amplicon for nuc gene was visible for as little as the dna concentration corresponding to organismsaeml ) . the sensitivity of each single pcr reaction was similar with those of multiplex pcr reaction. conclusion: the applied dna extraction method allowed us to obtain a good quality dna and can be used for a direct milk extraction. multiplex pcr reaction is a simple, rapid and reliable method for detecting enterotoxin b producing s. aureus strains from milk. objective: to detect the resistance to fluoroquinolones in acinetobacter baumannii strains by a pcr-rflp assay. methods: thirty a. baumannii clinical isolates were obtained from different specimens (bronchial aspirates, blood-cultures, catheters, etc.) . the mics (minimal inhibitory concentrations) for ofloxacin were determined by agar dilution following standard methodology.a pcr-rflp method using one primer pair for amplification of a bp fragment related to gyra gene (which codifies subunity a of dna-gyrase) and using one restriction enzyme hinf i was developed to study the resistance to ofloxacin in the different a. baumannii strains. when an a. baumannii strain is resistant to fluoroquinolones, a mutation in the position ser of the dna-gyrase has been detected, decreasing the affinity for the antimicrobial. agarosa gel was used to determine the dna pattern: fragments of bp and bp when there is not mutation and fragment of bp when the ser to leu mutation is present. results: the relationship between the pcr-rflp pattern and the mic to ofloxacin is shown in the table . the results of pcr-rflp analysis of most strains were in agreement with the results of mic. one isolate was susceptible to ofloxacin by agar dilution (mic = . mg/l) whereas by pcr-rflp this isolate seems to be resistant because it presents the mutation in gyra gene. two isolates with intermediate mic ( mg/l) showed mutation in gyra. the genotypic study by pcr-rflp proved that ofloxacin resistant a. baumannii strains showed a punctual mutation in gyra gene, in the same position inside the sequence of gene. evaluation of a rapid amplification-detection assay for the identification of vancomycinresistant enterococci j. fuller, l. turnbull, s. shokoples, b. lui, l. rosmus, r. rennie (edmonton, ca) objective: the routine identification of vancomycin-resistant enterococci (vre) in clinical laboratories often yields a lengthy turn-around-time that may impede infection control efforts, particularly in an outbreak situation. in search of an improved vre test, we evaluated the genotype Ò enterococcus assay (hain lifescience, germany), which provides both species and van gene identification for vre, and compared the results to conventional methods. methods: forty clinical enterococcal strains isolated on vrescreen agar media were selected for study. lactococcus and pediococcus were used as negative controls. conventional testing involved basic culture and identification tests, e-test susceptibility testing for vancomycin and teichoplanin, and pcr for vana, b, and c genes. the genotype Ò enterococcus assay involved multiplex dna amplification and reverse hybridization of amplified product on an immobilized dna strip-blot containing probes for e. faecium, e. faecalis, e. casseliflavus, e. gallinarum, vana, vanb, vanc , and vanc / . the genotype Ò enterococcus assay produced correct species and van gene identification for all ( %) vre isolates, including e. faecalis vanb, e. faecium vana, e. faecium vanb, e. gallinarum vanc , e. gallinarum vana-vanc , and e. casseliflavus vanc / . the only minor discrepancy was an e. casseliflavus that hybridized very weakly with the vanc probe in addition to the expected vanc /c probe. the costs per specimen were comparable for each test method. however, the genotype Ò enterococcus assay could be completed within a normal working day in contrast to conventional testing, which required a minimum of two days from the point of isolation on the vancomycin-screen media. conclusion: from this preliminary evaluation, the genotype Ò enterococcus amplification-detection assay provides vre species and van genotype identification in a rapid and costeffective manner, superior to conventional culture methods. although further study is required, this kit may have clinical utility during a vre outbreak. application of minimal sequence quality values prevents misidentification of blashv type in single bacterial isolates carrying different shv extended-spectrum beta-lactamase genes background: detection of extended spectrum beta-lactamase (esbl) genes by pcr and sequence analysis is the gold standard for detection of shv-type beta lactamases. usually, quality values of sequence analyses are not reported. during a study on esbl epidemiology, three strains for which the default sequence assembly showed an shv) or shv- gene, showed low quality values at certain positions in individual sequence traces. we investigated the reason for these lower values. methods: shv genes were amplified by pcr from three isolates (escherichia coli, enterobacter cloacae and pseudomonas aeruginosa). individual sequence traces were analysed with the computer programs phred and codon code. pcr products were ligated in vector pcr . and transformed to e. coli. sequence analysis was performed on eight individual clones from each transformation. results: visual inspection of the low quality positions in the sequence traces showed signals for two different nucleotides at three positions in the shv sequence: a or t at position , a or g at position and a or g at position . the polymorphisms at positions and lead to aminoacid substitutions, the four different combinations would give shv types , a, or . the double signals suggested that two or more blashv alleles were amplified. pcr amplicons were cloned in e. coli, in the sequences of individual clones only two combinations of the three polymorphisms were present: a g a and t a g . these two combinations correspond to shv- and shv- , respectively. conclusions: (i) in isolates of three different species, two different shv genes were present: shv- and shv- . (ii) genotypic detection with default sequence assembly parameters may lead to misidentification of the number and type of shv genes carried by a single strain. (iii) careful interpretation of sequence data of shv genes, including analysis of low quality positions, may further improve our understanding of the epidemiology and evolution of these esbl genes. antimicrobial susceptibilities and epidemiological analysis of salmonella typhimurium human isolates in slovakia by phage typing and pulsed-field gel electrophoresis v. majtán, l. majtánova, m. szabó ová (bratislava, sk) objectives: salmonella typhimurium is a common cause of salmonellosis among humans and animals in many countries. in the last few decades the incidence of multidrug-resistant s. typhimurium infections appears to pose a particular health risk. the objectives of this study were analysis by antibiotic susceptibility, phage typing and pulsed-field gel electrophoresis (pfge) of s. typhimurium human isolates. methods: a total of strains isolated during -september were analysed. the susceptibility of isolates to ten antibiotics was evaluated by a disk diffusion method. the phage types were identified according to anderson et al. ( ) in the national reference center for phage typing of salmonellae. pfge was used to resolve xbai macro restriction fragments from all strains. results: of human isolates ( . %) were resistant to more than two antibiotics. sixty-three of isolates ( . %) showed a classic dt resistance profile to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline (acssut). among this resistance type . % were dt , . % were dt and one strain was dt a. isolates encompassed phage types. the majority of isolates was found to be definitive phage type dt , representing . % of all isolates. other phage types were mainly dt , dt and dt a. nine pulsotypes and subpulsotypes were obtained using xbai restriction enzyme, but pattern x with its subtypes predominated ( . %). a major pulsotype x was represented by . % of dt isolates and was also found among dt isolates. conclusion: results indicated the spread of different clones of the multidrug-resistant s. typhimurium in the slovakia, but with predominance of one clone represented mainly by dt isolates. the phage typing as well as pfge may offer an improved level of discrimination for the epidemiological investigation of s. typhimurium human strains. novel reverse hybridisation assay to identify ctx-m genotype in cephalosporin-resistant isolates from uk and india to validate the assay results by dna sequencing. methods: isolate collection : enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in london and south-east england. these isolates were known to carry phylogenetic group blactx-m, but precise genotypes had not been determined. isolate collection : enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in aligarh, north india. resistance determinants had not been investigated previously. a novel multiplex pcr was used to amplify blactx-m. reverse hybridisation was carried out using biotinylated pcr amplicon and sequence-specific oligonucleotides designed to identify members of ctx-m phylogenetic group . hybridisation results were validated by dna sequencing for representative isolates from each collection. results: / london and se england isolates known to carry group blactx-m gave a consistent profile, corresponding to that for ctx-m- and ctx-m- ; / gave a profile corresponding to ctx-m- and ctx-m- . / indian isolates had blactx-m genes, all of which belonged to group , and all these gave a hybridisation profile corresponding to ctx-m- or ctx-m- . ctx-m- and ctx-m- are rare variants, suggesting that the enzymes present were more likely to be ctx-m- and ctx-m- , and this was confirmed by dna sequencing. conclusions: this is the first reported application of this novel reverse hybridisation assay to the analysis of large numbers of cephalosporin-resistant enterobacteriaceae. results were validated by dna sequencing. the assay is cheap and convenient, enables reasonable throughput, provides results within one day and can be used in place of dna sequencing. we believe it will be valuable for monitoring the prevalence and genotypes of blactx-m genes in enterobacteriaceae. detection of mexa and mexx efflux genes in p. aeruginosa: correlation between qc-rt-pcr and real-time pcr objectives: efflux systems are rarely identified as such in clinical microbiology laboratories. yet, over expression of transporters such as mexab-oprm and mexxy-oprm are likely to cause antibiotic multi-and cross-resistance in pseudomonas aeruginosa, leading to potential clinical treatment failures because of their inducible character. we have previously developed and validated with reference strains a qc-rt-pcr method to quantify mexa and mexx expression levels (eccmid . in the present study, we have developed a real-time-pcr assay and present here the correlation between both methods using control strains and clinical isolates. methods: expression levels of mexa and mexx were measured by both techniques in (i) reference strains expressing only one of these efflux mechanisms [mexa ( ) or mexx ( ) ]; and (ii) clinical isolates, in comparison with the wild-type strain pao (basal mexa and mexx expression levels). results: real-time pcr showed an inter-day reproducibility of ± . % (triplicates of strains). among the clinical strains, over expressed mexa and mexx. the table shows (i) the mean level of overexpression of mexa and mexx in comparison with the wild type strain pao (set at ), as detected by real-time pcr for all strains; (ii) the ratio of these values to those observed by qc-rt-pcr for the corresponding transporters. conclusions: both qc-rt-pcr and real-time-pcr are potentially useful in clinical laboratories as sensitive and rapid diagnostic tools to quantify the expression level of mexa and mexx in p. aeruginosa. combined with phenotypic characterization, this approach may help in a better understanding of the resistance mechanisms and epidemiology of resistance in this difficult-to-treat nosocomial pathogen. molecular detection of penicillin resistance in streptococcus pneumomiae n.g. rizk, n.a. abo khadr, s.m. abdel salam, n.m. gamil, m. hassan (alexandria, eg) objectives: the aim of the study was to detect penicillin resistant streptococcus pneumoniae by using seminested polymerase chain reaction (pcr) and to compare it with minimum inhibitory concentration (mic) of penicillin g. methods: fifty clinical isolates of streptococcus pneumoniae where isolated from patients admitted to alexandria main university hospital in egypt and were recovered from sputum ( strains), throat swabs ( strains), and pleural effusion ( strains) . two species-specific primers a- and a- , which amplified bp region of the pbp a penicillin-binding gene, were used for pneumococcal detection. two resistance primers, a-r and a-r , were used to bind to altered areas of pbp a gene which, together with the down stream primer a- , amplify dna sequences of bp and bp from isolates with penicillin mic > . objective: lipopolysaccharide-binding protein (lbp) is an acute phase protein produced in the liver. the objective of our study was to evaluate lbp as a marker of severity and prognosis in patients with bacteraemia. methods: adult patients with community-acquired bacteraemia were included in a prospective manner. daily blood sampling for lbp and interleukin- (il- ) was performed. the patients were classified according to the systemic inflammatory response syndrome (sirs) criteria. demographic data, co-morbidity, microbiological aaetiology, routine biochemical parameters, focus of infection, severity score and mortality on day were recorded. lbp and il- levels were analysed on plasma samples with a chemiluminescent immunometric assay (immulite- Ò ). results: the median age was yrs. the mortality rate on day was . %. patients had bacteraemia without sirs, patients had sepsis and patients had severe sepsis. lbp concentrations are presented as medians and range: . lg/ml ( . - . ) in patients without sirs, ) in patients with sepsis and . lg/ml ( . - . ) in patients with severe sepsis (p < . ). lbp levels correlated to levels of il- (rs . ), c-reactive protein (rs . ), leukocytes (rs . ) and neutrophils (rs . ) (p < . ). lbp did not predict the outcome of the patients with bacteraemia. conclusion: lbp levels increased with the severity of sepsis in patients with bacteraemia. lbp correlated to il- , c-reactive protein, leukocytes and neutrophils. lbp did not predict the outcome of the patients in this small cohort. pyrosequencing of the gra gene to discriminate type i, ii and iii toxoplasma gondii in clinical samples b. edvinsson, b. evengård on behalf of the esgt objectives: infection with toxoplasma gondii in immunocompromised transplant recipients is rare but often fatal. to increase our knowledge about the significance of the genotype of the parasite during infection, methods suitable for routine use need to be developed. pyrosequencing is a rapid sequencing-bysynthesis method performed in real-time. it is developed for detection of short nucleotide polymorphisms (snps), and is suitable for molecular genotyping of microorganisms. we here present a pyrosequencing assay for rapid and reliable discrimination of toxoplasma gondii type i, ii and iii in clinical samples. methods: twenty-two isolates of t. gondii were used for pyrosequencing analysis of the gra gene. real-time pcr was performed using a lightcycler . instrument to amplify a bp fragment of the gra gene. pyrosequencing analysis of two different snps contained within a bp fragment of the amplified product was preformed to identify t. gondii type i, by detection of nucleotides g and a at these respective positions. type ii was g and g, and type iii was a and a. to test the assay in a clinical context, blood samples and lung tissue from an immunocompromised patient was analysed. results: the detection limit of the assay is parasitic genomes in a sample. reproducibility (r) was calculated as r = nr/n (nr = the number of isolates assigned the same type on repeat testing and n = the number of isolates tested). r was determined using three independent runs, and was , suggesting clearly interpretable results with little variation. typeablility (t) of the assay was calculated as t = nt/n (nt = the number of typeable strains and n = the number of isolates tested). t was determined using three independent runs, each including four atypical isolates. t was . , suggesting that the assay discriminates correctly between the three main genotypes of t. gondii, but does not detect atypical strains. analysis of the clinical samples revealed type ii t. gondii in blood samples and lung tissue. conclusion: when preceded by real-time pcr, pyrosequencing is a rapid process with a high reproducibility and throughput. this makes it a good candidate for routine use. the method does, however, not detect atypical or recombinant strains. more than one gene may have to be analysed for that purpose. acknowledgement: in particular, we want to thank marie-laure dardé and hervé pelloux for provision of the t. gondii isolates. virulence genes in escherichia coli isolates from calves in shahrekord area, iran shiga toxin-producing escherichia coli (stec) strains, also called verotoxin-producing e. coli (vtec) strains, represent the most important recently emerged group of food-borne pathogens around the world. members of this group are a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (hc) or the hemolytic uremic syndrome (hus), which is the main cause of acute renal failure in children. domestic ruminants, mainly cattle, sheep, and goats, have been implicated as the principal reservoir. transmission occurs through consumption of undercooked meat, unpasteurized dairy products and vegetables, or water contaminated by feces of carriers because stec strains are found as part of the normal intestinal floras of the animals.we studied the prevalence of shiga toxinproducing escherichia coli (stec) in stool specimens of calves with diarrhoea or other gastrointestinal alterations from dairy cattle farms of shahrekord city (central of iran). the virulence genes, stx , stx , eae, intimin hly, enterohemolysin, st, lt, were detected by multiplex pcr method. stec strains were detected in ( . %) of e. coli from cases investigated. stec o was isolated in cases ( . %), whereas non-o stec strains were isolated from animals ( %). stec strains were the most frequently recovered enteropathogenic bacteria. pcr showed that ( . %) isolates carried st gene. none of isolates carried an ehxa, eae, and lt (labile toxin) genes. our results suggest that stec strains are a significant cause of calf infections in this area and confirm that, infections caused by stec non-o strains are more common than those caused by o :h isolates. the high prevalence of stec strains (both o and non-o strains) also found in human patients by other investigators, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of stec in spanish clinical microbiology laboratories. objectives: shiga toxins are a-b holotoxin including one enzymatically active a subunit associated non-covalently to five identical receptor binding b subunits. each subunit can cause different signalling pathways in different cells. to assess the effect of each single subunit the specific clones for expressing the single subunit was designed. periplasmic expression yielded native ab holotoxin or b pentamer. methods: o was used as bacterial strain for pcr amplification of shiga toxin gene. each subunit was amplified by specific primers and the amplified genes were cloned in pbad expression vector. the expression of the cloned genes was induced and optimized by different concentration of arabinose. the expressed proteins was assessed on sds-page and detected by elisa and western blotting. the expressed recombinant ab holotoxin and b subunit were purified and assessed for its biological activity on cells. cell cytotoxicity was shown by the expressed (ab ) holotoxin. moreover inhibition was observed by b subunit and antibody against it. results: e. coli clones expressing recombinant shiga toxin a and recombinant shiga toxin b subunits were established to release the toxin to periplasmic space. expressed toxin was examined by sds-page to visualize two subunits. the whole structure of these expressed subunits was checked in native gel. active ab structure expressed in periplasmic space was extracted by polymyxine b. the biological activity of the constructed recombinant shiga toxin showed both vero cell cytotoxicity and inhibition of in vitro protein synthesis. conclusion: in this study it was shown that for b subunit assembly and secretion to periplasmic space as b pentamer homologous leader sequence is not needed. although for biological active holotoxin (ab ) secretion to periplasmic space the presence of homologous leader sequence of gene is essential. these subunits can be used for studying on cell cytotoxicity and also as a vector for antigen presentation in immunotherapeutic approaches. characterisation of gram-positive anaerobic cocci by biochemical tests and partial s rrna sequencing a. bryk, a. kanervo-nordstrom, m. hyvonen, e. kononen (helsinki, fi) objective: gram-positive anaerobic cocci, which are common findings in various infections, are difficult to identify in clinical microbiology laboratories, where identification is based only on few phenotypic tests. in recent years, this group of organisms (traditionally known as peptostreptococci) has encountered several taxonomic changes. the aim of the present study was to compare the characterization made by a selection of key phenotypic tests to that by partial sequencing of the s rrna gene. methods: fifty-nine clinical isolates sent to our laboratory as gram-positive anaerobic cocci were examined for their colony and cell morphologies and biochemically characterized using spot catalase and indole reaction, enzyme reactions by individual diagnostic tablets (rosco), sodium polyanethol sulphate susceptibility, glucose fermentation, and determination of metabolic end products. in addition, commercial identification test kit (rapid id a) patterns were performed. the sequencing of the s rrna gene of the clinical isolates and reference strains comprised of about bp, and the sequences obtained were compared to those in genbank database by using the multisequence advanced blast comparison software from the national center of biotechnology information. results: the biochemical characteristics of the isolates were consistent with those of peptostreptococcus anaerobius (n = ), peptostreptococcus (micromonas) micros (n = ), finegoldia magna (n = ), peptoniphilus asaccharolyticus (n = ), peptoniphilus sp. (n = ) and anaerococcus sp. (n = ), whereas isolates remained as unidentified gram-positive anaerobic cocci. biochemical identification correlated with that obtained by partial s rrna sequencing in / ( %) isolates at genus level and in / ( %) isolates at species level. the agreement of the biochemical and sequence-based identification was % for p. micros and f. magna. of isolates biochemically identified as p. asaccharolyticus, isolates were identified as peptoniphilus harei and remained as peptoniphilus sp. by sequencing. according to the sequence data, the unidentified isolates were peptoniphilus ivorii. conclusion: most isolates from human infections proved to be f. magna. a relatively good agreement of identification was obtained using biochemical testing and partial s rrna sequencing. objectives: molecular methods for identification of infectious agents in patients with clinical infectious disease are increasingly being used. especially in cases where antibiotics have been given prior to sampling or when fastidious bacteria difficult to grow are the aaetiology of the infection. infectious arthritis is a serious disease where identification of the etiological agent is mandatory for optimal antibiotic treatment as well as indication of the primary focus if not the joint it self. methods: in the present prospective study, synovial fluids taken from patients in elucidation of affected joints and sent to a clinical microbiological laboratory in the copenhagen area, denmark, were examined by conventional (culture, phenotypic tests) and molecular methods (pcr/sequencing of s ribosomal genes). conventional methods included gramstaining and microscopy, aerobic and anaerobic culture and identification. pcr/sequencing included dna extraction, pcr assay which produced a bp fragment of s rdna, and sequencing of both dna strands of the amplicons. sequencing data were edited and a blast search in the ncbi database was done. results: overall a microorganism was identified in of the synovial fluids ( . %). in synovial fluids from nine patients bacteria were identified by either methods [staphylococcus aureus (n = ), streptococcus pneumoniae (n = ), streptococcus dysgalactiae (n = ), citrobacter freundii (n = )]. six synovial fluids were only culture positive; in four of those six specimens coagulase negative staphylococci were isolated. in three of the synovial fluids a microorganism was identified by s pcr only. in two synovial fluids s pcr identified only one microorganism, whereas culturing resulted in two isolates. conclusion: the present study indicates a significant contribution by molecular methods (pcr/sequencing of s ribosomal genes) in recognizing and identification of microorganisms from foci normally considered sterile like synovial fluids. continued suspicion of infected arthritis despite of negative cultures should result in use of molecular diagnostics. direct detection of cardiobacterium hominis by broad-range s rrna pcr and sequencing in the serum of a patient with infective endocarditis e. malli, d. klapsa, a. vasdeki, m. morava, m. pitsitaki, e. petinaki, a. maniatis (larissa, gr) objectives: to describe the detection of cardiobacterium hominis directly in the serum of a patient with infective endocarditis, by employment of broad-range s rrna pcr followed by sequencing. methods: a series of blood cultures were taken from the patient before starting empirical treatment. in addition, ml whole blood was collected in rubber sealed pyrogen-free tubes for direct detection of bacterial dna. bacterial dna was detected by a broad range pcr reaction and sequencing process allowed identification of bacteria species. results: cardiobacterium hominis was identified as the causative agent of infective endocarditis, on two days after the serum collection. blood cultures, simultaneously obtained with the serum sample, remained negative after days of routine incubation; however, after a prolonged incubation of twelve days a gram negative bacterium was isolated from the aerobic bottles, that was identified as c. hominis species, by the usual phenotypic studies (catalase, oxidase reaction, indole, nitrate, etc) which are time-consuming. conclusions: to our knowledge this is the first report of direct detection of c. hominis in the serum using molecular methods, emphasizing the need for the establishment of such methods especially for infections caused by fastidious organisms. identification of dangerous bacterial pathogens by s ribosomal rna gene sequence analysis w. ruppitsch, a. stoeger, a. indra, d. schmid, k. grif, c. schabereiter-gurtner, a. hirschl, f. allerberger (vienna, at) to assess the usefulness of partial s rrna sequence analysis for identification of dangerous bacterial pathogens, a total of isolates comprising bacillus anthracis, brucella melitensis, biovars melitensis, suis, abortus and bovis, burkholderia mallei, burkholderia pseudomallei, francisella tularensis, yersinia pestis, and genus-related and unrelated control strains were sequenced and analysed using the genbank database (blast . . , national institute of health, u.s.a), the microseq database (version . . and v . , applied biosystems, foster city, u.s.a.) , the ribosomal database project-ii database (rdp-ii, release , update , michigan state university, u.s.a), and the ribosomal differentiation of medical microorganisms database (ridom, university of wuerzburg, germany). on genus level all isolates were identified using genbank, rdp-ii, and microseq v . . the older microseq . . database identified % of the tested samples correctly on genus level. the ridom database did not include sequence data of the tested species even on genus level, the ridom database none (''there seems to be, at least currently, no close relative available''). genbank and rdp-ii identified all dangerous pathogens correctly. the microseq v . database identified four of the six species of dangerous pathogens. on species level none of the dangerous pathogens was correctly identified using microseq . . or ridom. as previously noted by various other authors, the most important reason for failure of databases in identifying a bacterium is a lack of the s rrna gene sequence of the particular bacterium in the database rather than misidentification because of poor sequence quality. one must also be aware that the following bacterial species or subspecies have the same s rrna gene sequence, which makes differentiation by sequence analysis impossible: b. anthracis and b. cereus, y. pestis and y. pseudotuberculosis, all brucella subspecies, and francisella tularensis ssp. holarctica and mediasiatica. in addition to s rrna gene analysis complementary methods are essential to discriminate between these bacteria on species or subspecies level. identification of nontuberculous mycobacteria by sequence analysis of the s ribosomal rna, the heat-shock protein and the rna polymerase beta-subunit genes s. shin, j.h. yoon, e.c. kim (seoul, kr) objectives: the diagnosis of diseases caused by nontuberculous mycobacteria (ntm) is difficult because ntm are prevalent in the environment such as soil and water and because they have fastidious properties. in this study, we investigated the distribution pattern of ntm clinical isolates and the identification to the species level. methods: among the presumptive ntm clinical isolates, cultured in a third referral hospital from -jan- to -jan- in seoul, south korea, which were negative by probe hybridization method for mycobacterium tuberculosis complex, we selected those of more than colonies or those cultured more than twice in a same patient. a total of isolates were studied for the distribution of ntm including isolates recruited for species identification by direct sequencing of s rrna, hsp and rpob gene segments. ( . %) were also identified in the presumptive ntm isolates. the identification rate by sequencing of s rrna, rpob, and hsp were %, % and %, respectively. hsp or rpob gene was more efficient than s rrna in identification of ntm by sequencing. conclusions: some ntm are considered to be the causative organisms of clinical diseases even in the countries with intermediate burden of tuberculosis, so accurate identification method by direct sequencing can be adapted to clinical laboratories. evaluation of the genotype mtbdr assay for the simultaneous detection of resistance to rifampicin and isoniazid of mycobacterium tuberculosis clinical strains f. brossier, c. truffot-pernot, n. veziris, v. jarlier, w. sougakoff (paris, fr) objectives: the rapid determination of drug resistance in mycobacterium tuberculosis is an important challenge to ensure a rapid effective chemotherapy. the genotype mtbdr test is a commercially available dna strip assay enabling the molecular genetic identification of the m. tuberculosis complex and its resistance to rifampicin (rif-r) and isoniazid (inh-r) by detecting the most commonly found mutations in the genes rpob (asp val, his tyr, his asp, ser leu) and katg (ser thr). here, we report the evaluation of the genotype mtbdr assay from a set of clinical isolates of m. tuberculosis. methods: clinical isolates were collected in france over a years period ( ) ( ) and were included in the study: were rif-r, were inh-r (of which were also rif-r) and were susceptible to both drugs. the susceptibility tests were carried out by the standard proportion method. the mutations involved in rif-r and inh-r in rpob, katg, inha and his promoter region, were characterized by dna sequencing. results: the genotype mtbdr assay identified % of the rif-r strains harbouring mutations in the rpob gene, of which ( %) showed a ser leu mutation and ( %) a his asp or tyr mutation. of the inh-r strains ( %) harboured a ser thr mutation in katg, all identified by the genotype mtbdr assay. of this strains displayed a high level of inh-r. among the other inh-r strains, showed a katg mutation at the level of the regions, which was different from ser thr ( of which showing a low level of inh-r), and one harboured a deletion in katg (with a high level of inh-r). these mutations were also detected by the strip. finally, among the remaining inh-r strains not detected by the mtbdr assay, were characterized by a mutation in position - of the promoter region for the maba-inha regulon ( with a low level of inh-r), by a ser ala mutation in inha (all with a low level of resistance) and by other mutations. conclusions: the mtbdr assay, which can readily be included in a routine laboratory workflow, identified % and % of the strains resistant to rif and inh, respectively. interestingly, of the inh-r strains showing a high level of resistance ( %), but only of the inh-r strains with a low level of resistance ( %), were detected by the mtbdr assay, indicating that complementary tests are necessary for detection of the m. tuberculosis strains having a low level of resistance to inh. variation in the streptococcal s rdna detected by pyrosequencing m. haanperä, p. huovinen, j. jalava (turku, fi) originally the aim of this study was to identify alpha-haemolytic streptococcal isolates to the species level by pyrosequencing the v and v regions of the s rdna and comparing the results to the sequences of type strains that have been determined earlier. however, the isolates could not be unambiguously identified due to sequence variations detected in the alpha-haemolytic isolates. materials and methods: invasive s. pneumoniae isolates (n = ), alpha-haemolytic streptococcal blood culture isolates (n = ) and alpha-haemolytic streptococcal isolates from the normal pharyngeal microbiota (n = ) of six elderly persons were analysed by pyrosequencing the v and v regions. results: varying degree of genetic variation was found in different types of streptococcal isolates. in the pneumococcal isolates, no sequence variation was detected as all the isolates contained the sequence specific for s. pneumoniae in both regions. also the sequences of the alpha-haemolytic blood culture isolates were well in agreement with the sequences of the streptococcal type strains. however, most of these isolates could not be unambiguously identified, as they contained sequences belonging to different species in the v and v region. consequently, only five of the isolates could be unequivocally identified as s. gallolyticus (n = ), s. anginosus (n = ), s. mitis (n = ) and s. sanguinis (n = ). the commensal streptococci contained numerous sequences to which an identical type sequence could not be found. also sequences identical to type strains were found; but similarly to the blood culture isolates, the results enabled the identification of only four isolates: s. mitis (n = ), s. parasanguinis (n = ), and s. salivarius or s. vestibularis (n = ). moreover, the pyrograms of three blood culture isolates and ten pharyngeal isolates indicated heterogeneous s rdna alleles. one such pyrogram of the v region is presented in the figure. interestingly, four of the eight different nonheterogeneous v and v sequence combinations of the blood culture isolates were also present among the pharyngeal isolates. the results of this study indicate that the variation in commensal streptococci is greater than that of the streptococcal type strains and pathogenic isolates. the presence of identical sequence combinations among the blood culture and pharyngeal isolates supports the assumption that potentially pathogenic isolates are present in the normal microbiota. evaluation of partial s rrna gene sequencing for identification of clinical isolates of nocardia species m. marín, m. sánchez, m. del rosal, e. cercenado, p. martín-rabadán, e. bouza (madrid, es) new species of nocardia are being described. conventional identification based on biochemical characteristics and pcrrestriction enzyme analysis is frequently unable to distinguish them. partial sequencing of s rrna gene has proven useful in the identification of bacteria. objective: to evaluate the utility of 'end s rrna gene pcr and sequencing in the identification of clinical isolates of nocardia sp. compared with conventional methods and pcr-rflp of hsp . methods: clinical isolates of nocardia sp. were characterized by biochemical reactions and disk diffusion susceptibility testing. molecular identification was performed by hsp pcr-rflp and pcr of 'end of s rrna gene followed by sequencing. the sequences obtained were compared with those included in genebank. only alignments with similarities higher than % were considered. a comparison of sequences of our nocardia isolates with those deposited in genebank and well characterized phenotypically was performed using clustal x . software. results: distribution of species after pcr-rflp of hsp was n. asteroides vi ( ), n. farcinica ( ), n. nova ( ), n. asteroides i ( ), n. otitidiscaviarum ( ) and n. asteroides iv ( ) . partial sequence analysis of s rrna revealed a great heterogeneity between the isolates of n. asteroides vi, as follows: n. cyriacigeorgica ( isolates), n. abscessus ( isolates) and n. carnea ( isolate). for isolates, no genebank sequence was found with more than % similarity. all n. farcinica isolates had the same sequence and showed % similarity with those deposited in genebank. n. nova, n. asteroides i and n. otitidiscaviarum also showed sequence heterogeneity. three n. nova isolates matched with the recently described n. veterana and with n. nova. n. asteroides i isolates were identified as n. abscessus ( ) and n. beijingensis ( ) . all n. otitidiscaviarum were identified properly. the isolate of n. asteroides iv was identified as n. transvalensis. conclusions: sequencing of 'end s rrna gene is a useful and rapid molecular tool for the identification of nocardia clinical isolates. this method could provide more accurate results than the conventional ones used routinely in our laboratory. sequence analysis of the 'end s rrna has enabled us to recognize great diversity and new species among our nocardia isolates. several species would have gone unnoticed using non-sequencing-based methods. antibacterial susceptibility studies-iii p anaerobic bacteraemia due to fusobacterium necrophorum and clostiridium cadaveris: a case report m. panopoulou, e. alepopoulou, e. chrisafidou, a. tsaroucha, c. simopoulos, s. kartali (alexandroupolis, gr) introduction: anaerobic bacteremia is uncommon accounting . - % of bacteremias and it is associated with a high mortality rate, which is strongly and independently associated with underlying liver disease. case report: a year-old man presented to our hospital with a -day fever and rigor. he had a history of cancer of the extrahepatic biliary tree, which was found incidentally during an operation for the treatment of echinococcal cyst of the liver. physical examination reveals high fever ( c) and tachycardia. blood tests showed the following results: hb: . gr/dl, wbc: . /ul, plt: . /ul, tprot: . each colony type subcultured to blood agar plates and incubated aerobically and anaerobically (aerotolerance test). after hours of incubation the two organisms grew only in anaerobic conditions. they identified by the api a system (bio-merieux-france) as fusobacterium necrophorum and clostiridium cadaveris. the patient's treatment started with metronidazole, amikacin and ceftriaxone and followed by metronidazole and imipenem. he was discharged after weeks in a good condition. conclusions: although anaerobic bacteremia is rare, there is value in performing separate anaerobic blood cultures. the early recognition of anaerobic bacteremia and administration of the appropriate antimicrobial therapy play a major role in preventing mortality especially in patients with underlying disease. fluoroquinolone resistance among enterobacteriaceae strains isolated from urinary tract infections v. skandami-epitropaki, p. fostira, a. tsiringa, a. xanthaki, k. zampitha, m. toutouza (athens, gr) objectives: to study the frequency and antibiotic susceptibility of quinolone resistant bacterial stains isolated from patients with community-aquired bacteriuria and compare it with urinary pathogens from hospitalized patients. methods: during a -month period (october -october a total of bacterial strains were isolated out of urine samples submitted for culture in our hospital laboratory from the community and from hospitalized patients with urinary tract infection symptoms. cultures and bacterial identification were obtained by conventional methods. antibiotic susceptibility testing was done by kirby-bauer disk diffusion method according nccls criteria. results: of the bactrial strains studied (escherichia coli , klebsiella pneumoniae , proteus mirabilis ), . % of them were found to be quinolone resistant. the percentage of quinolone resistance was . % for hospitalized patients (hp) and . % for community patients (cp). the quinolone resistance for e. coli was . % ( . % for hp and . % for cp), for k. pneumoniae . % ( . % for hp and . % for cp) and for p. mirabilis . % ( . % for hp, . % for cp). susceptibility pattern of the quinolone resistant isolates to other antimicrobial agents was for hospitalized patients and community patients respectively as following: for e. coli ampicillin (am) %- . %, amoxicillinclavulanate (amc) . %- . %, piperacillin-tazobactam (tzp) . %- . %, cefuroxime (cxm) . %- . %, trimethoprimsulfamethoxazole (sxt) . %- . %, ceftazidime (caz) . %- . %, cefepime (fep) . %- . %, gentamicin (gm) . %- . %. for k. pneumoniae am %- %, amc %- %, tzp . %- %, cxm . %- %, sxt . %- %, caz . %- %, fep . %- %, gm . %- %. for p. mirabilis am . %- %, amc . %- %, tzp . %- %, cxm %- %, sxt . %- %, caz . %- %, fep %- %, gm . %- %. seven strains of k. pneumoniae ( . %) were carbapenem resistant and metallo-beta lactamase producing. conclusions: high resistance rates to fluoroquinolones were observed in uropathogen bacteria isolated not only from hospitalized patients but also from patients with communityacquired urinary tract infections in greece. increasing resistance rates to the rest antibiotic agents make the treatment of urinary tract infections a very difficult problem. susceptibility of pseudomonas aeruginosa isolated from the mystic programme to the carbapenems: meropenem and imipenem p.j. turner (macclesfield, uk) objectives: the meropenem yearly susceptibility test information collection programme (mystic) was initiated in in order to track the susceptibility of organisms in centres that were prescribing meropenem. this poster seeks to examine the susceptibility of pseudomonas aeruginosa isolates over this period to the carbapenems; meropenem and imipenem and, in particular, records the susceptibility of imipenem-resistant isolates to meropenem and vice versa. methods: pseudomonas aeruginosa isolates were speciated by the methods in current use at the participating centres. minimum inhibitory concentrations of meropenem and imipenem were determined using reference methods described by clsi. results: a total of isolates of pseudomonas aeruginosa have been tested globally, of these . % were susceptible to meropenem at the breakpoint of < mg/l and . % to imipenem. globally, susceptibility to the two carbapenems has remained stable over the period - , however when imipenem-resistant isolates were examined (n = ) . % proved to be susceptible to meropenem, conversely of the meropenem-resistant isolates only . % proved to be susceptible to imipenem. a similar pattern was seen when isolates were separated into global regions:usa imipenem-resistant isolates, . % susceptible to meropenemusa meropenem- results: bacteroides fragilis group (bafg) accounted for % of the isolates, fusobacterium spp. for %, other gram negative bacilli (ognb) for %, clostridia (clos) for %, nonsporeforming gram-positive bacilli (nsfgpb) for % and cocci for %. beta-lactamases (bl) were detected in % of isolates. most bl + strains belonged to bafg ( %) and ognb ( %). at nccls-recommended breakpoints, more than % of isolates were susceptible to tzp, mtz, chl and mem, % to amc but only %, %, % and % to fox, ctt, cli and pen respectively. no nccls-breakpoints for anaerobes are available for mxf, lzd and tig. mic and mic for mxf were and mg/l, for lzd and mg/l and for tig . - mg/l. in comparison with similar surveys conducted in and - susceptibility of bafg to clindamycin decreased from % in , to % in - and % in in bafg % of b. fragilis and % of non-b. fragilis were susceptible to amc in this study; in - susceptibility in these groups was % and % and in - % and % respectively. all isolates, except bafg and clos, were susceptible to mem. % of the isolates were susceptible to chl. susceptibility to mtz remains stable and is high in all groups except nsfgpb where mtz is active on merely % of the isolates. conclusions: tzp, mem and mtz remain very potent antimicrobial agents in the treatment of anaerobic infections. although still rare, resistant organisms were detected to each of them. therefore susceptibility testing of anaerobic isolates is indicated in severe infections to confirm appropriateness of antimicrobial therapy. further monitoring of background susceptibility is necessary to guide empiric treatment. comparative in vitro activity of levofloxacin against escherichia coli isolated from acute pyelonephritis in france in c.j. soussy, c. lascols, c. dib-smahi and the multicenter group study. objectives: the objective of this study was to evaluate the in vitro activity of levofloxacin (lvx) comparatively to other antibiotics against escherichia coli strains isolated from acute pyelonephritis in women consulting emerging rooms by french hospitals in . methods: mics of lvx, ofloxacin (ofx), ciprofloxacin (cip), nalidixic acid (nal), amoxicillin-clavulanic acid (amc), ceftriaxone (cro), cefixime (cfm), amikacin (an), gentamicin (gm) and cotrimoxazole (sxt) were determined by agar dilution according to the eucast breakpoints approved by recommendations of the comité de l'antibiogramme de la société française de microbiologie. quality control was performed with e. coli strain atcc . results: a total of strains were collected. . % of strains were isolated from urinary samples, . % from blood culture and . % from the two specimens. mics / (mg/l), the range of mics and the percentage of susceptibility (%) are presented in the following table: concerning the fluoroquinolones, mics / of lvx were one/two dilution lower than those of ofx and two/one dilution higher than those of cip. for the other antibiotics, a higher percentage of susceptibility was observed with cro and an, when a lower percentage of susceptibility was observed with amc and sxt. conclusions: levofloxacin exhibited good in vitro activity against e. coli strains isolated from acute pyelonephritis with . % of susceptible strains. in vitro activity of double and triple combinations of colistin, imipenem, rifampicin and linezolid against epidemic strains of multidrug-resistant acinetobacter baumannii producing oxa carbapenamases d.w. wareham, d.c. bean (london, uk) objectives: a. baumannii has emerged as an important cause of nosocomial infection in critically ill patients worldwide. in the uk three strains in particular exhibiting multi-drug resistance and producing oxa carbapenamases have been responsible for ongoing outbreaks. treatment options for infection with these organisms are limited as only colistin and tigecycline retaining significant activity in vitro. animal models and in vitro studies using other multi-resistant strains suggest that drugs in combination with colistin may be effective. we assessed the activity of colistin in combinations including imipenem, rifampicin and linezolid against epidemic strains from a recent uk outbreak. methods: isolates of a. baumannii exhibiting resistance to carbapenems were recovered from patients at barts and the london nhs. isolates were referred to the health protection agency and confirmed as belonging to clones producing oxa carbapenemases. activities of polymyxin, imipenem, rifampicin and linezolid alone and in double and triple combinations were determined using standard chequerboard assays with increasing concentrations of drug on the x axis, drug on the y axis and drug three in multiple replicate plates. after incubation at hours wells were examined for growth and mic's determined for each combination. synergy between agents was defined as a fixed inhibitory concentration index (fici) of < . . results: the isolates tested belonged to the oxa- clone , oxa- clone and the south east clone, as confired by the hpa. colistin was the most active agent alone with mics from - mg/l. imipenem mic's varied from - mg/l. the most active combinations were colistin plus rifampicin (fici = . ) and colistin, rifampicin and imipenem (fici = . ). synergy was not seen with colistin in combination with imipenem alone. linezolid in combination with colistin (fici = . ), or imipenem (fici = . ) was synergistic but at therapeutically unobtainable linezolid concentrations ( mg/l). conclusion: multidrug resistant strains of a. baumannii from the uk producing oxa carbapenemases remain susceptible to polymyxin in vitro. polymyxin exerts its effect on the bacterial cell wall; theoretically assisting other antibiotics to reach their respective targets, and seems a logical choice for inclusion in combination therapy. we have shown that rifampicin is synergistic with polymyxin against these isolates in vitro and may be effective in treating severe a. baumannii infections in man. a comparative in vitro evaluation of resistance development after exposure to teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin in staphylococcus spp. and enterococcus spp. mssa, mrse, msse, e. faecium and e. faecalis strains was determined on agar plates containing each antibiotic at clsi resistance breakpoints and at peak blood concentrations. after incubation at °c for h colonies were counted and compared to the inoculum to calculate frequency of mutation. colonies grown in plates containing antibiotics were sampled for determination of mic values. results: frequency of mutation was less than - for all the tested antibiotics at peak blood concentrations. same results were obtained when breakpoint concentrations for each drug were used. conclusion: this one-step in vitro study demonstrated the ability of teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin to prevent growth of resistant mutants of staphylococci and enterococci, thus suggesting no occurrence of mutational events leading to resistance when bacteria are exposed to blood concentrations of these drugs. in order to establish the development of resistance after in vitro serial exposure to the same antibiotics simulating different in vivo concentrations, further studies are needed and are now in progress (multi step induction of resistance). in vitro activity of antimicrobial agents against legionella obtained from hotel water systems in turkey objectives: the aim of this study was to evaluate the in vitro activity of colistin against endemic pan-resistant acinetobacter baumannii (including resistance to imipenem) isolated during a year period in a university hospital. methods: imipenem-resistant acinetobacter spp. isolates were collected between january and october , from a variety of clinical specimens of different patients attending distinct wards in a university teaching hospital. isolates were identified by api gn and by sequencing the s rrna gene. mics of colistin were determined by agar dilution method, according to nccls susceptible breakpoint (£ mg/l). pfge (apai restriction enzyme) was performed. results: of a. baumannii isolates ( %) were susceptible to colistin. colistin resistance (mic ‡ mg/l) was observed in isolates ( isolates with a mic of ‡ mg/l and isolates with a mic of mg/l) recovered from different patients in distinct wards. among these imipenem-and colistin-resistant isolates, distinct pfge patterns were identified (clones a, b, and c). resistance to almost all beta-lactams (including carbapenems) and variable susceptibility to aztreonam, amikacin and tobramycin was a common feature of clone a. isolates belonging to clone b showed resistance to imipenem, amoxicillin and its association with clavulanic acid (amc), ureidopenicillins and their associations; susceptibility to ceftazidime; and variable behaviour to meropenem, cefepime, cefpirome and aztreonam. the susceptibility profile to aminoglycosides was variable, differing from clone a in its susceptibility to netilmicin and minocycline. clone c was resistant to imipenem, amoxicillin, amc, piperacillin, piperacillin + tazobactam, ticarcillin and ticarcillin + clavulanic acid, but remained susceptible to meropenem, aztreonam, cefpirome, ceftazidime and cefepime. conclusion: only colistin, one of the few effective drugs available against multi-drug-resistant acinetobacter infections, showed in vitro activity against the majority of acinetobacter spp. strains isolated within the sampled hospital. the observed % a. baumannii resistance to the recently re-introduced colistin seems like the first chapter of a novel repeatedly told for several antibiotics. emergence of high-level gentamicin resistance in clinical enterococcal isolates of companion animals in portugal objectives: to characterize in vitro gentamicin susceptibility among enterococci causing infections in cats and dogs, in order to evaluate the impact of high-level gentamicin resistance in small animal therapeutics. methods: the samples were collected at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. from january until november , a total of enterococci were isolated from dogs and cats with urinary tract infection (uti), otitis externa (oe) and pioderma. bbl crystal gram positive id system was used for identification at the species level. minimal inhibitory concentrations (mic) were determined by the microdilution method according to nccls ( ) . the bifuntional enzyme gene that confers high-level gentamicin resistance (hlgr) was detected using pcr ( ) . results: enterococcus faecalis was the predominant isolate (n = ), followed in frequency by enterococcus faecium (n = ). mic cumulative data analysis showed that mic values were lg/ ml and mic lg/ml. six ( %) hlgr clinical enterococcal isolates were detected, with mic ranges between - lg/ml. four of these enterococci were isolated from uti and from oe. four of the phenotypically high-level gentamicin resistant isolates carried the aac( ')-ie-aph( '')-ia gene. conclusions: the importance of enterococcal infection in small animal clinical samples has increased over the last years. mic cumulative data points out low-level gentamicin resistance among clinical enterococci isolates of veterinary origin and the emergence of high-level isolates, as previously detected ( ). this fact compromises cell-wall active agents (such as ampicillin or vancomicin) and aminoglicoside in vivo synergy. the aac ( ')-ieaph( '')-ia gene carriage is of concern because its expression confers resistance also to tobramicin, netilmicin, amikacin and kanamicin. our findings are of critical importance, as they may have a direct impact in therapeutic decision in the management of companion animal's infections by enterococci. furthermore, transfer of resistance genes and resistance strains between animals and owners/caretakers by direct contact is a concerning probability. references: ( ) results: interpretative criteria were used according to nccls .during the study period penicillin resistant strains of s. pneumoniae was noted as follows: % in sputum or ta, % in blood, %in csf,and % in others,against cefuroxim resistant strains: % in sputum or ta, % in blood, % in csf.regarding the susceptibility to ofx,penicillin resistant s. pneumoniae strains from sputum or ta revealed . %.the penicillin resistant strains coming from sputum or ta showed resistance as follows; % to em and % to sxt,against strains isolated from others: % to em and % to sxt.no resistant strain to va was found. conclusion: the percentage of the penicillin resistant s. pneumoniae isolates from the lower respiratory tract, middle ear fluid, eye fluid and sinus was markedly higher than that of the isolates from blood and csf. the most efficient drugs against penicillin resistant pneumococci were cefuroxim and ofloxacin. these results from romania also underline the previous observations regarding the higher emerging rates of resistance in s. pneumoniae worldwide. penicillin resistance in streptococcus agalactiae objectives: streptococcus agalactiae has become recognized as a cause of serious illness in newborns, pregnant women, and adults with chronic medical conditions. heavy colonization of the genital tract with streptococcus agalactiae also increases the risk that a woman will deliver a preterm low-birthweight infant. early-onset infections (occurring at < days of age) are associated with much lower fatality than when they were first described, and their incidence is finally decreasing as the use of preventive antibiotics during childbirth increases among women at risk. penicillin or ampicillin remains the drug of choice for intrapartum antibiotic prophylaxis for streptococcus agalactiae colonization in pregnant women. erythromycin and clindamycin are the drugs of choice for women with serious penicillin allergy who are colonized with streptococcus agalactiae. the objective of this study is to estimate the insorgence of penicillin resistance among streptococcus agalactiae. methods: all streptococcus agalactiae were tested against penicillin by agar dilution method according to clinical and laboratory standards institute (clsi); breakpoints for resistance were those recommended by the clsi. antimicrobial agents were obtained from their manufacture as laboratory grade powder. results and discussion: four hundred seven ( ) clinical isolated were analysed during . streptococcus agalactiae resulted resistant to penicillin in case; and about % resulted borderlines.the present findings indicate a probable evolution in s. agalactiae toward penicillin resistance this finding suggest the need a continuous national and international surveillance programs to provide timely data on the evolution of incidence of penicillin resistance in this pathogen. ciprofloxacin susceptibility of the most common isolates at bacterial conuctivitis conclusion: according to the average numerals we concluded that all the isolated strains are highly susceptible at ciprofloxacin. its application in the conuctivial saccus is especially important in curing the conuctivial infections with resistent strains like pseudomonas aeruginosa. we successfully cure the bacteria chronic conuctivitis with the adequately used therapy according to antibiogram. antimicrobial resistance patterns of acinetobacter baumannii in clinical isolates g.t. tsilika, v.p. pliatsika, m.t. tsivitanidou, d.s. sofianou (thessaloniki, gr) objectives: a. baumannii is a nosocomial pathogen, commonly isolated from critically ill and immunocompromised patients. the aim of the present study was to evaluate the antimicrobial resistance of a. baumannii strains isolated in a tertiary care hospital througout a three-year period. methods: a total of a. baumannii strains were selected from january to december .the specimens were obtained from inpatients hospitalized in intensive care unit (icu) and pediatric intensive care unit (picu) and other departments of our hospital.the identification and the antimicrobial susceptibility testing were performed using the vitek automated system(biomerieux,france conclusions: the emergance and rapid spread of multidrug resistant a. baumannii isolates are of a great concern worldwide.imipenem was one of the most potent agents for treatment of those infections caused by multiresistant strains.the increasing prevalence of imipenem resistance limits therapeutic options and leads to outbreaks of carbapenems resistant strains. tigecycline in vivo studies objectives: antibacterial agents disrupt the ecological balance of the normal human microflora. disturbances may lead to the emergence of antibiotic resistance and/or to infections by potentially pathogenic bacteria. tigecycline, a member of a new class of antibiotics (glycylcyclines), has been shown to have a potent expanded broad-spectrum activity against most grampositive and gram-negative aerobic and anaerobic bacteria. the aim of the study was to investigate the ecological effects of tigecycline on the normal oropharyngeal and intestinal microflora in healthy subjects. methods: thirteen ( ) white subjects ( women, men) aged to years, received mg of tigecycline in the morning on day as a -minute intravenous (iv) infusion, followed by mg doses of tigecycline given every hours as a -minute infusion for days. one ( ) subject was withdrawn on day because of an adverse event. serum, saliva, and faecal samples were collected before, during, and after administration for microbiologic cultivation and for assays of tigecycline. all new colonizing bacteria were tested for susceptibility (resistance > mg/l) during the investigation period. results: the serum concentrations on day , hours after dosing, were . to . mg/l (mean value . mg/l, median value . mg/l, and sd . mg/l). the faecal concentrations on day were . to . mg/kg (mean value . mg/kg, median value . mg/kg, and sd . mg/l). saliva concentrations were generally low, with highest mean value . mg/l, median value . mg/l, on day , hours after dosing. a minor effect on the oropharyngeal microflora was observed. the numbers of enterococci and escherichia coli in the intestinal microflora were reduced at day , while other enterobacteria and yeasts increased. there was a marked reduction of lactobacilli and bifidobacteria but no impact on bacteroides. no clostridium difficile strains were isolated. two ( ) klebsiella strains and enterobacter strains resistant to tigecycline were found. conclusion: tigecycline had a minor effect on the oropharyngeal microflora. tigecycline's effect on the intestinal microflora was due to its spectrum of antibacterial activity and intestinal concentrations. objectives: to examine and report the use of tigecycline (wyeth) in the treatment of multidrug resistant acinetobacter (mdra) culture positive sepsis in patients requiring mutiorgan support. methods: all patients were managed within the liver intensive care unit. physiological data was collected prospectively and entered onto a specialist database. patients received standard intensive care management; antibiotic and antifungal therapy administered as indicated by microbiological cultures. systemic inflammatory response (sirs) features initiated blood cultures (vascular lines and peripheral), drain fluid culture and broncoalveolar lavage (bal). screening swabs were undertaken weekly and samples sent for culture at laparotomy. mdra positive cultures from blood, bal, drain fluid or samples taken at laparotomy in the context of sirs resulted in the initiation of tigecycline treatment. results: patients received tigecycline treatment for mdra infections. the underlying disease states were necrotizing pancreatitis ( ), post hepatectomy ( ), polytrauma ( ), all with postive intra-abdominal cultures. acute and acute on chronic liver failure ( ), mdra +ive broncho-alveolar lavage ± blood cultures and post liver transplant patients (necrotising pancreatitis in one, with recurrent small bowel perforation and with retroperitoneal haemorrhage) all with positive blood cultures and in positive intra-abdominal tissue/clot. mean time from admission to treatment for mdra was days. mean duration of treatment was days (range - ). mean apache ii score at initiation of therapy was (range - ); / patients survived to intensive care discharge and / to hospital discharge. microbiological clearance of mdra was observed in / cases. in those who did not achieve microbiological clearance cause of death was intra-abdominal haemorrhage, recalcitrant organ failure with recurrent small bowel perforation and vasopressor resistant shock. in these patients one remained culture positive for intraabdominal sepsis despite full treatment (small bowel perforation x ). the drug was well tolerated with the only side effect being that of hypercalcaemia observed in / patients, mean corrected calcium . mmol/l, range . - . . in all cases this resolved on drug discontinuation. conclusion: tigecycline appears to be an efficacious agent in the treatment of deep seated mdra infections. objectives: nausea (n) and vomiting (v) have been reported with tigecycline, a new glycylcycline with expanded broad spectrum activity. exposure-response relationships and patient covariates predictive of the first n and v occurrence were evaluated in patients with complicated intra-abdominal infections (ciai). methods: data from patients from ciai trials (one phase and two phase ), receiving mg loading dose and mg every hours, were pooled for analysis. n and v (definitely, possibly, or probably related to tigecycline) reported from the start of infusion until hours after the last dose were included. individual exposure measures [auc - and cmax] were calculated using a previously developed population pk model. logistic regression was used to evaluate predictors of first n and v occurrence. covariates included age, weight, sex, region of treatment, and baseline n and v. results: the dataset included patients ( with pk). mean (sd) age and weight were ( ) years and ( ) kg. % of patients were men and %, %, and % were enrolled in north america, europe, and latin america, respectively. baseline nausea or vomiting was reported in % and %. overall, n and v occurred in % and % of patients receiving tigecycline, however most ( %; %) of first n and v events were mild in nature. women had more n and v ( %; %) than men ( %; %). n and v were lower in europe ( %; %) than in other regions. auc - and cmax were not predictive. the final nausea model included weight, sex, region, baseline nausea, and the interaction of weight/region as predictors of the first nausea occurrence (p = . , . , . , . , & . , respectively objective: because hospitalisation for community-acquired pneumonia (cap) is associated with substantial morbidity and health resource utilisation, we evaluated the predictors of prolonged hospital length of stay (los) and treatment duration. methods: we conducted a retrospective analysis of data from a double-blind, randomised, multicentre clinical study that compared the efficacy and safety of tigecycline with that of levofloxacin in the treatment of patients with cap requiring hospitalisation. patients were stratified by the fine pneumonia severity index and randomly assigned to receive tigecycline or levofloxacin via iv administration for at least days. treatment duration and hospital discharge were based on physician assessment of signs and symptoms of infection and patient condition. we used cox proportional hazards modelling with stepwise selection to identify statistically significant predictors (p < . ) of treatment duration and hospital los. results: among patients with cap in the clinical intent-totreat population with complete hospitalisation data, mean age was . years (range - ) and . % of patients were aged ‡ years. diabetes ( . %), chronic obstructive pulmonary disease ( . %), and congestive heart failure ( . %) were leading co-morbidities. about . % of patients were smokers and . % were characterised by alcohol abuse. median fine pneumonia severity index score was ; . % of patients had a score > . . there were no significant differences between the groups in treatment duration or los. conclusions: tigecycline, a first-in-class glycylcycline, was associated with treatment duration and los similar to that of levofloxacin, adjusting for several identified risk factors. tigecycline effective in treating patients with intra-abdominal or skin/skin structure infections who have bacteraemia e.j. ellis-grosse, r. maroko (collegeville, us) objectives: the treatment of bacteraemia, which is a potentially fatal complication of infections originating at other body sites, is complicated by increasing resistance. tigecycline, a first-in-class glycylcycline, has an expanded spectrum of activity against gram-positive, gram-negative, anaerobic, and atypical bacteria including resistant strains. tigecycline is safe and effective in treating complicated skin and skin structure (csssi) and intraabdominal infections (ciai). this analysis examines tigecycline clinical trial experience in patients with ciai or csssi who had bacteraemia (presence of bacteria in blood) at baseline. objectives: treatment of complicated intra-abdominal infections (ciai) is challenging due to diverse bacteriology and bacterial resistance. the efficacy and safety of tigecycline (tgc), a first-in-class glycylcycline approved in mexico, brazil, peru, colombia and usa for treating ciai and complicated skin and skin structure infections, was compared with imipenem/cilastatin (imi/cis) in adult hospitalised patients with ciai in two double-blind, phase multinational trials. this analysis evaluated tgc efficacy and safety in the european region of the integrated results of these two trials. methods: one study was conducted in centres ( countries) and the other study was conducted in centres ( countries). patients were stratified by disease severity (apache ii score £ vs > but £ ), and randomly assigned to iv tgc ( mg loading, then mg q h) or iv imi/cis ( / mg q h) for - days. clinical response at test-of-cure (toc, - days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) were co-primary efficacy endpoints where cure/failure responses were determined. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, patients were mitt (received ‡ dose), m-mitt ( tgc, imi/cis) and me ( tgc, imi/cis). treatment groups were balanced with respect to demographics. patients were mostly white ( . %) men ( %) with a mean age of years. for me, clinical cure rates at toc were . % ( / ) for tgc vs . % ( / ) for imi/cis ( % ci = ) . , . ; test for non-inferiority p < . ). clinical cure rates for m-mitt were . % ( / ) for tgc vs . % ( / ) for imi/cis ( % ci = ) . , . ; test for non-inferiority p < . ). most commonly reported treatment emergent aes (teaes, mitt) for tgc and imi/cis were nausea ( . % and . %, p = . ) and vomiting ( . % and . %, p = . ). the imi/cis group had significantly higher teaes of fever ( . % imi/cis vs . % tgc, p = . ), hyperglycaemia ( . % imi/cis vs tgc, p = . ) and dyspnoea ( . % imi/cis vs . % tgc, p = . ) where tgc had significantly higher amylase increase ( . % tgc vs . % imi/cis, p = . ) and bun increase ( . % tgc vs imi/cis, p = . ). conclusions: similar to the overall integrated analysis of the two phase trails, in the european analysis, tgc was safe and effective in the treatment of hospitalised patients with ciai in comparison with imi/cis. tigecycline is safe and effective in the treatment of complicated skin and skin structure infections: european experience of two double-blind phase comparison studies with vancomycin/aztreonam r. maroko, n. dartois, d. sarkozy, j. goodrich, e.j. ellis-grosse on behalf of the tigecycline and study groups objectives: tigecycline (tgc) a first-in-class expanded spectrum glycylcycline, has been approved in mexico, brazil, peru, colombia and usa for treating complicated skin and skin structure infections (csssi) and complicated intraabdominal infections. two phase , randomised, double-blind studies were conducted in hospitalised men and women with csssi to determine tgc safety and efficacy compared with vancomycin/aztreonam (v/a). the objective of this analysis was to evaluate the efficacy and safety seen in the european population of the integrated analysis of these phase trials. methods: one study was conducted in centres in countries while the other study was conducted in centres in countries. patients were randomly assigned ( : ) to receive either tgc ( mg, followed by mg iv twice daily) or vancomycin ( g iv twice daily) plus aztreonam ( g iv twice daily) for up to days. clinical response at test-of-cure (toc, - days after therapy) for clinically evaluable (ce) and clinical modified intent-to-treat (c-mitt) populations were coprimary efficacy endpoints in which cure/failure responses were determined. secondary objectives included determination of in vitro susceptibility to tgc of a range of bacteria that cause csssi and microbiological efficacy. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, patients comprise mitt (received ‡ dose of study drug), comprised ce ( tgc, v/a/cis) and comprised c-mitt ( tgc, v/a/ cis). treatment groups were balanced with respect to demographics. patients were mostly white ( . %) men ( . %) with a mean age of years. in the european region, clinical responses to tgc and v/a at test-of-cure were similar: c-mitt, . % ( / ) versus . % ( / ), difference tgc-v/a was - . % ( % ci - . , . ). similar results were noted in the ce population with tgc curing . % ( / ) and v/a curing . % ( / ), difference tgc-v/a was - . % ( % ci - . , . ).most commonly reported treatment emergent aes (teaes, mitt) for tgc and v/a were nausea ( . % and . %, p < . ) and vomiting ( . % and . %, p = . ). the v/a group had significantly higher teaes of sgpt increase ( . % v/a vs . % tgc, p = . ) and rash ( . % v/a vs tgc, p = . ). conclusion: in the european analysis of the integrated phase worldwide clinical studies, tgc monotherapy is as safe and efficacious as the combination of v/a in the treatment of patients with csssi. safety and tolerability of tigecycline r. maroko, n. dartois, g. rose, e.j. ellis-grosse (collegeville, us; paris, fr) objectives: tigecycline (tgc), a glycylcycline, is a first-in-class, extended, broad-spectrum iv antibiotic that has demonstrated clinical activity in patients with complicated intra-abdominal infections (ciai) and complicated skin and skin-structure infections (csssi). the safety of tigecycline was evaluated in four phase iii trials. methods: a total of hospitalized patients from these trials were pooled and evaluable for safety analysis. in the ciai trial, patients received tgc mg q hrs (following a -mg loading dose) or imipenem mg and cilastin mg q hrs. those in the csssi study were treated with either tgc (same dose/schedule) or vancomycin gm with or without aztreonam gm q hrs. results: the most frequently reported adverse events (aes) in both tgc-treated groups were nausea (n) and vomiting (v). the incidence of n was . % while v was approximately . %; these were generally mild to moderate in severity. infection-related serious aes were slightly more frequent with tgc versus comparators ( . % vs . %). discontinuations due to treatment-emergent aes (including n/v) occurred at similar rates with tgc and comparators ( . % vs . %). six patients ( . %) treated with tgc presented with intestinal perforations and developed sepsis/septic shock compared with ( . %) for imipenem/cilastatin, with higher baseline apache ii scores in the tgc group; the relationship to treatment could not be determined. in the overall efficacy analysis, subjects with ''perforation of the intestines'' were balanced between the two groups, and overall efficacy was not statistically different. no clinically significant renal, hepatic, cardiac (qtc), bone marrow, or cns toxicities were noted with tgc. conclusion: tgc appears to be safe and tolerable for patients with ciai and csssi. n/v were generally mild to moderate in severity, self-limiting, and did not result in increased overall drug discontinuation. there did not appear to be clinically significant renal, hepatic, cardiac, bone marrow, or neurological toxicities related to tgc treatment. all-cause mortality rates did not statistically differ between those treated with tgc and the comparators. its demonstrated efficacy and favourable toxicity profile make tgc a good monotherapy option for selected serious infections. tigecycline as effective as imipenem/cilastatin in the treatment of complicated intra-abdominal infections: experience in india objective: due to diverse bacteriology and bacterial resistance, treatment of complicated intra-abdominal infections (ciai) is a challenge. in a double-blind, phase , multinational trial, the efficacy of tigecycline, a first-in-class glycylcycline, was compared with imipenem/cilastatin (imi/cis) in hospitalised patients with ciai. this subanalysis evaluated tigecycline safety and efficacy from investigational sites in india. methods: patients were stratified by disease severity (apache ii score £ vs > but < ), and randomly assigned to iv tigecycline ( mg loading, mg q h) or iv imi/cis adjusted for body weight ( / mg q h for ‡ kg) for - days. clinical response at test-of-cure (toc, - days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) populations were co-primary efficacy endpoints where cure/failure responses were determined. safety evaluations included vital signs, laboratory tests and record of adverse events (aes). results: in india, patients received at least dose (mitt, tigecycline, imi/cis), patients were clinically evaluable (ce), were me, were m-mitt. treatment groups were balanced with respect to demographic/baseline medical characteristics. primary diagnoses (mitt) were complicated appendicitis ( %), gastric/duodenal perforation ( %), perforation of intestine ( %), cholecystitis ( %), peritonitis ( %), and intraabdominal abscess ( %). cure rates at toc in me in india were / ( . %) tigecycline and / ( . %) imi/ cis, which are consistent with overall me results [ . % ( / ) tigecycline vs . % ( / ) imi/cis ( % ci = ) . , . ; non-inferiority p < . )]. in india m-mitt, cure rates at toc were / ( . %) tigecycline and / ( . %) imi/cis, similar to the overall m-mitt results [ . % ( / ) tigecycline vs . % ( / ) imi/cis ( % ci = ) . , . ; non-inferiority p < . )]. noninferiority of tigecycline among india patients could not be statistically demonstrated because of insufficient sample sizes, however, magnitude of response to study drugs in patients treated in india was comparable to that in overall patients. in india, treatment aes were similar with significantly higher incidence of dyspnoea in tigecycline ( . %) vs imi/cis ( . %), p = . . conclusions: efficacy results in india are consistent with findings from the overall study and results at other centres, suggesting tigecycline is noninferior to comparator in treating ciai. nosocomial infection: control of environment, viral infections p bacterial flora contamination of blood pressure cuffs in use on hospital wards n. walker, r. gupta, j. cheesbrough (preston, uk) blood pressure cuffs are a plausbile vehicle for the transmission of nosocomial infection between patients. despite this, few studies have examined the level of bacterial contamination and tested for the presence of common nosocomial pathogens on their surface. we swabbed cuffs currently in use on hospital wards. using sterile gloves, a disposable template measuring · cms was placed onto the cuff and a moistened sterile swab was rubbed onto the defined area for minute and then transported in mls of buffer medium. from each sample, . mls of the buffer was plated onto different media which included a non-selective agar medium for total viable count (tvc) and selective media for s. aureus, mrsa, c. difficile, coliforms and vancomycin resistant enterococci (vre.) bacterial growth was recovered from all cuffs. pathogenic organisms were isolated from cuffs ( %). mssa from , mrsa from and c. difficile from . the remaining three cuffs grew more than one pathogenic organism; mssa + mrsa + c. difficile from one and mssa + c. difficile from cuffs. colifroms and vre were not isolated from any of the cuffs. the range of total viable counts recovered per cm area of the cuff varied from > cfu and the cuffs with the highest counts tended to have more pathogens present. mssa and c. difficile were isolated from % of the cuffs sampled and mrsa from %. while the actual importance of this potential route of transmission for nosocomial pathogens remains unclear, it can not be dismissed. the impracticality of decontaminating blood pressure cuffs between patients suggests that single patient use cuffs or a barrier between cuff and skin would be a more viable option on a busy general ward. needlestick and sharp injuries of health care personnel in a newly founded tertiary hospital: a prospective study m. falagas, i. karydis, g. georgoulias, p. hatzopoulou, d. nikita, i. kostogiannou (athens, gr) objectives: needlestick and sharp injuries of health care workers are a major cause of anxiety and may expose susceptible employees to the risk of infectious diseases. however, the incidence of such injuries has not been examined in a newly founded hospital while preventive programmes are taking place. methods: we prospectively studied the needlestick and sharp injuries of employees in a newly founded tertiary hospital in athens, greece while a vaccination program against hepatitis b virus as well as educational activities for avoidance of injuries were taking place. serologic studies for hepatitis b and c virus as well as human immunodeficiency virus (hiv) were performed in all injured employees and the source patients (when known). results: sixty-eight needlestick, sharp injuries, and splashes were reported during the study period ( / / to / / ) in nurses, housekeepers, technicians, and ambulance workers. the overall incidence (percutaneous injuries and splashes) per full-time employment-years ( fteys) was . % whereas the incidence of percutaneous injuries alone per fteys was . %. a higher incidence of injuries was noted during the first than the second half of the study period ( . % versus . %, p = . ). no source patient was found positive for hepatitis c or hiv. the use of high-titre immunoglobulin after adjustment for the incidence of injuries was higher in the first than the second half of the study period ( . % vs . %, p = . ). conclusion: although we did not adjust for possible confounders, our data show that educational and vaccination preventive programs for needlestick and sharp injuries led to a statistically significant decrease in the incidence of such injuries and use of high-titre immunoglobulin. epidemiology of occupational needlestick and sharps injury among healthcare-workers in turkey s. hosoglu on behalf of the occupational infections study group, turkey background: health care workers (hcws) are frequently exposed to the danger of infectious agents through needle stick and sharps injury (nssi) in their occupational efforts. in turkey, the hepatitis b and c viruses cause an essential threat to the hcws because of their prevalence rate ( %- % and . %- %, respectively). a cross-sectional countrywide survey study was performed on the epidemiology of nssi among hcws at hospitals in cities throughout the country. data relating to the epidemiology of nssis were collected using a standard questionnaire in . results: totally hcws completed the questionnaire forms. nurses are the leading group ( persons) that joined into the study were followed by doctors ( persons) andlaboratory technicians ( ). totally of them ( . %) declared an occupational exposure or nssi in the last months related their job. needle stick injury was reported in of them ( . %), splash into the eye in ( . %), sharp injury in ( . %), and the other injuries in ( . %). the hepatitis positivity was reported in cases ( . %) objectives: to assess the microbiological status of reprocessed single-use devices for interventional cardiology by testing bioburden, sterility and pyrogenic load. methods: a total amount of electrophysiology non-lumen catheters (ep) were collected after the first clinical use on patient. devices were contaminated with bacteria spiked human blood and underwent four different pre-sterilization protocols including chlorine, polyphenol, and enzymatic agents. treated samples were assayed by cultural quantitative methods (cqm) for bactericidal properties and electron microscopy (em) for biologic residuals. ep were tested for sterility. by the repetition of simulated-use (bacteria spiked blood) and regeneration (enzymatic and chlorine treatment, gas plasma sterilization) we obtained , , , , , samples respectively reprocessed , , , , , times. devices were cultured for days in trypticase soy broth. the pyrogenic status of ep was monitored after clinical use, after decontamination-cleaning treatments and after complete reprocessing by lal test. results: high-resolution em and cqm confirmed the superior properties of chlorine releasing agent added to enzymatic detergent for devices treatment before sterilization. hypochlorous acid based protocols were more biocide (> . log cfu reduction) than polyphenolic ( . - . log cfu reduction). sterility tests showed no positive sample to inoculated strain until the fourth cycle of reprocessing. catheters showed the growth of the inoculated strain, bacillus subtilis in / and / samples after five cycles and six cycles respectively. every reprocessed device was non-pyrogenic (< eu/catheter). in addition, tests conducted on in-vitro spiked catheters showed that pyrogenic loads of eu/device were reduced to less than eu/device. conclusions: reprocessing procedures following the adopted regeneration protocol were able to satisfy the fundamental microbiological requirements until five in-vitro reuses. sterility tests showed that devices' sterility was not guaranteed after five reuses. pre-sterilization treatments including enzymatic solutions and chlorine revealed high cleaning properties with effective bioburden reduction. storage intervals among reprocessing steps longer than hours should be avoided in order to limit contamination and pyrogenic load. technical considerations suggest to consider the introduction of reprocessing procedure only in hospitals with a considerable workload. room disinfection in the hospital setting using akacid plus Ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: akacid plus Ò , a novel polymeric guanidine with broad antimicrobial activity also against multi-resistant bacterial strains, was used in the present study as room disinfectant. methods: disinfection of closed rooms experimentally contaminated with antibiotic-susceptible and multi-resistant staphylococcus aureus (mrsa), pseudomonas aeruginosa and escherichia coli was performed using akacid plus Ò at concentrations of . %, . % and . % for minutes. bacterial suspensions were distributed on stainless steel plates and placed in a test and control room. recovery of the test bacteria was determinedbefore nebulizing, and minutes after the beginning and hours after the end of room disinfection by a modified simple swab-rinse technique. for the detection of mrsa in isolation units, surface samples were collected by direct swab and enrichment culture. results: the swab-rinse method demonstrated a dose-and time-dependent effectiveness of akacid plus Ò in eradicating s. aureus, e. coli and p. aeruginosa on stainless steel plates. nebulizing of . % akacid plus was successful in eliminating all hospital pathogens in min contact time, while mrsa was still detectable after use of . % akacid plus Ò . . % akacid plus Ò achieved a reduction > cfu of s. aureus and p. aeruginosa, but was only able to eradicate e. coli during the observation time. the results suggest that nebulized akacid plus Ò at a concentration of . % is a potent substance for eradication of pathogenic organisms in the hospital setting. study on the antiviral efficacy of citrofresh Ò , a flavonoid based organic acid complex sanitizer z. nack (north-geelong, au) objective: determine the antiviral efficacy of this organic sanitizer against enveloped and non-enveloped viruses using a carrier based method. seeking registration for citrofresh Ò in australia and in the eu as a hospital grade antiviral sanitizer. methods: the study was performed according to the american society of testing and materials (astm) designation (e - ) recommended by the australian therapeutic goods administration (tga) to determine the efficacy of a disinfectant intended to use on inanimate, environmental surfaces. we tested citrofresh Ò (diluted in standard hard water) in three different concentrations: %, % and % on adherent cell lines (pk- , mrc- , mdck, a , l ) in four replicates against five different viruses including: porcine parvovirus (non-enveloped, high resistant against sanitizer); human rhinovirus- (non-enveloped, high resistant against sanitizer); human adenovirus- (non-enveloped, moderate resistant against sanitizer); human influenza type a (h n ) virus (enveloped, moderate resistant against sanitizer); human herpes simplex virus type (enveloped, low resistant against sanitizers). prior to the viral testings, acute toxicity assay was carried out to determine the adherent cells viability against citrofresh Ò . results: cell lines exhibited > % viability after exposure to all three concentration. herpes simplex type , human influenza type a and human adenovirus- exhibited the most significant viral log reduction of log to at % concentration of citrofresh Ò followed by the human rhinovirus- and porcine parvovirus log reduction at % concentration. the reduction of viable virus load was exhibited after minute exposure time to citrofresh Ò , which means no time-dependant activity. citrofresh Ò clearly exhibited concentration and ph dependent viral load reduction activity against influenza type a and the human adenovirus - and human herpes simplex type virus. the reduction in viral titre for porcine parvovirus and human rhinovirus- is probably ph dependent (the ph of % citrofresh Ò is . , % is . and % is . ). conclusion: our investigation shows that citrofresh Ò is an effective disinfectant on environmental surfaces, eliminating enveloped and non-enveloped viruses and sufficient to achieve the minimum -log reduction with complete viral inactivation which is prerequisite for registration. rapid environmental recontamination of an intensive care unit after decontamination with hydrogen peroxide vapour objectives: to evaluate the effectiveness of hydrogen peroxide vapour (hpv) to reduce the levels of total bacterial and methicillin resistant staphylococcus aureus (mrsa) environmental contamination on an intensive care unit (icu), and to establish the rate of environmental recontamination. methods: the study took place on a bed open plan icu. on each environmental screen sites in each bed space (under the bed, the workstation and the monitor) were examined using broth enrichment for the detection of mrsa. in addition total bacterial counts were determined for under the bed and workstation using rodac plates. environmental screening was carried out monthly for the months preceding the usage of hpv, increasing to weekly for the weeks prior to usage. additional sampling was carried out immediately before patients were discharged from icu, following the subsequent terminal clean and then immediately after hpv use. after readmission of patients sampling was carried out at h, h and then weekly for a period of weeks. patients were screened for mrsa on admission and then weekly. results: sampling of the environment prior to the usage of hpv revealed contamination of the environment with mrsa on / occasions, with mrsa colonised patients being present on only / occasions. after discharge of the patients and terminal cleaning of the environment, mrsa was isolated from ( %) environmental sites. after the use of hpv, mrsa was not isolated from any environmental sites upon immediate sampling, but h after patients were readmitted, including patients known to be colonised with mrsa, mrsa was isolated from sites. these sites were not clustered around the colonised patients but were widespread across the icu. in the weeks post hpv usage mrsa has been isolated every week. the mean total bacterial counts prior to the use of hpv were . / cm underneath the beds and . / cm on the workstations, this was reduced after hpv to . / cm and . / cm respectively. after patients readmission the counts were . / cm underneath the beds and . / cm on the workstations after h and returned to pre-hpv levels of . / cm and . / cm at each site respectively after week. conclusion: hydrogen peroxide vapour is effective in eliminating bacteria from the environment. the rapid rate of recontamination of the environment suggests that the use of hpv is not an effective means of maintaining low levels of environmental contamination on an open plan icu. objectives: the nosocomial infections are more serious and dangerous than community acquired infections since they have high rate of morbidity and mortality as well as they increase the cost of therapy. recently many precautions have been taken to prevent these infections. one of these applications is that covering of the floor of the wards, clinics, intensive care units and operating rooms of the hospitals with vinyl flooring material, which is believed to be cleaned easily and effectively. in this study it was aimed to determine the duration of survive of the staphylococcus aureus, enterococcus feacalis, escherichia coli and pseudomonas aeruginosa, which were most common encountered as nosocomial infection agents, on the surface of flooring materials such as vinyl flooring, ceramic laminated wood and galvanized sheet at room temperature. methods: four kinds of flooring materials were prepared approximately in - cm coupons and sterilized. separate bacterial suspensions equal to mc farland turbidity were swapped to the surface of each flooring materials by sterile cotton swabs. all contaminated test materials were put in sterile petri dishes with cover and kept at room temperature without subjecting to the direct sunlight. on the third day, culture samples were taken from the surface of each material by sterile cotton swaps soaked with sterile saline and streaked on the blood agar surface. culturing procedure was repeated every other day until no growth detected. in case of three consequently, negative culture results obtained culturing was ended. results: overall results of the study were presented on table . conclusions: among the four flooring materials, galvanised sheet seemed to be the most unsuitable one for the bacteria to survive long period. in other words this material should be preferred as to laminated wood for covering benches and laboratory tables. as for the flooring of the floors the vinyl flooring material is better than ceramic. covering the complete cmv ie- and pp proteins.results: cmv seropositive transplant recipients had significantly hightened ie- and pp specific t cell frequencies compared to seronegative individuals. patients withevidence of cmv antigenemia or dnaemia could not be discriminated based on cmv-and donor-reactive t cells or serum creatinine. however, recipients of seropositive grafts with low ie response showed a tendency towards more frequent cmv infection. cmv disease was observed in only / individuals. had no detectable ie or pp -t cell response, the third presented with a dominant pp response. interestingly, ie -specific t cells correlated inversely with early post-tx donor-reactive t cell frequencies during weeks - post-tx. most importantly, ie -specific t cell frequencies correlated inversely with serum creatinine at and months at several times post-tx. in patients without acute rejection, even pre-transplant ie- specific t cells correlated inversely with and months creatinine. conclusion: these data suggest subclinical control of cmv infection by ie- specific t cells and subsequently less graft injury by (cmv-induced) alloimmunity. universal precautions: knowledge, attitude and practice of healthcare workers regarding hiv, hepatitis b and c v. gupta, s. bhoi, a. goel, p. aggarwal (new delhi, in) objectives: increasing incidence of hiv, hepatitis b (hbv) and hepatitis c (hcv) in the patients expose the healthcare professionals of acquiring these infections during occupational exposure. we studied the knowledge, attitudes and practices of healthcare workers regarding hiv, hbv, hcv and the risk of occupational transmission of these diseases. methods: an interview survey was conducted among all the health care workers (hcw) using a standardised questionnaire comprising of items in english and local language, as suitable, by an expert in the emergency ward of a tertiary care teaching hospital of a developing nation. data analysis (bivariate and multivariate analysis) was done using spss version . results: (response rate: %) hcw participated in the study. the mean age was ± years, were females. the study population comprised of % doctors, % nurses, % lab technicians and % support staff. respondents had adequate knowledge about causative ( %) usual transmission ( %), symptoms ( %) of aids but poor knowledge about hbv and hcv ( %, % and % respectively). inadequate knowledge was also revealed about the infectious bodyfluids ( %), disinfection of equipments ( %), pregnancy in hcw as a susceptibility factor ( %), post exposure prophylaxis ( %) and comparative infectivity of hiv and hepatitis ( %). % of hcw became anxious while treating these patients. poor compliance with universal precautions was noticed. high compliance was reported for wearing masks ( %) and wearing gloves ( %). doctors were more likely to suffer needlestick injury (p = . ) occupational exposures was found to be high ( %) with poor declaration rate ( %). guidelines adherence was influenced by profession (p < . ), availability or adequacy of protective equipments but not by work experience as hcw (p = . ). all of the respondents urged for an interactive information session. conclusions: results from this study reveal that there is a fair level of knowledge about hiv/aids but hepatitis b and c have not generated adequate concern among the hcw. incongruity between perceived knowledge and reported practice suggests that there is a need for an interactive awareness course about the universal precautions. the educational programmes need to consider attitudes in conjunction with empirical knowledge. objectives: the sero-prevalence of hepatitis a (hav) antibodies are known to be low in young adults in korea. recently, seventeen cases of hepatitis a have been reported in health-care workers (hcw) of icu in a university hospital from may to july . we performed surveillance, and determined molecular identification of outbreaks. methods: . we checked the hav igm from all the patients of sicu with elevated ast/alt retrospectively and screened ast/alt level from all the nurses and the doctors in contact with suspicious index case. . when we determined the existence of outbreak, the molecular subtypes of hav from a blood of hcw were determined to provide the data for epidemiologic study. we determined the index case, a transmission route and the intervention for control an outbreak were planned. results: . seventeen hcw including nurses and doctors who are to years old, suffered from acute hav over weeks period. . the possible transmission of hav was fecaloral route from the bed-ridden patients with diarrhea to the exposed hcw. . seventeen hcw were identified with a positive anti-hav igm. the eight hcw had a positive hav rna. analysis of the vp -p a region of each isolate showed genotype a in five strains and co-circulation of a and b in others. conclusions: the occurrence of hav outbreak highlights the importance of standard precaution in a hospital. the hav vaccination is considered in young aged-hcw. the genotype identification of blood would be useful for the epidemiologic study of suspicious hav outbreak in a hospital. management of a norovirus-associated gastroenteritis outbreak on two psychiatric wards a. buehling, u. arnold (magdeburg, de) objectives: we report a norovirus-associated outbreak of gastroenteritis on a closed psychiatric and a gerontopsychiatric wards from december to february . during this time patients and healthcare workers (hcws) were affected. introduction and results of hygiene measures based on published guidelines on psychiatric wards are described. methods: effective and adapted measures had to be implemented to stop the outbreak and to prevent the spread of disease to other areas of our hospital. isolation or cohorting of the psychiatric patients was excluded for therapeutic reasons. regular hand disinfection in patient rooms was impossible because of the high risk of abuse. the following measures have been introduced: use of gowns, masks and gloves by hcws during care of infected patients-frequent hand disinfections with alcohol-based disinfectants by hcws using ''pocket bottles''; recommendation for all persons entering the station to use gowns, gloves and masks and to disinfect their hands frequently, distribution of handouts describing the measures; hand disinfection by all patients after using toilet, before and after taking meals (distribution of disinfectants by hcw); increased frequency of routine surface disinfection ( times daily) instead of routine cleaning once daily; routine disinfection of door handles, handrails, wash-basins and -fittings and light switches - times a shift; avoidance of patient transfer via hospital; visitor restriction during outbreak time; daily evaluation of recommended measures and adaptation to the current situation; exclusion of affected staff from the ward until h symptom free. results: the hygienic measures have been explained to the local hcws in daily meetings. they have been fully accepted only after a severe staff shortage in the fifth week of outbreak because of new cases of gastroenteritis during hcws and newly infected patients. because of the restrictive application of the adapted guidelines for these special wards the outbreak has been stopped within further weeks. conclusion: in case of norovirus-based gastroenteritis outbreaks on closed psychiatric wards hygienic measures which are adapted to the concrete situation are necessary. especially in these cases the compliance with guidelines can be increased by daily meetings and daily evaluation of recommendations. staff shortage during the outbreak forced the strict compliance with the recommended measures. regional spread of antibiotic resistance methods: we performed surveillance of patients, healthcare stuff and icu environment and we registered the infections of ab during periods of days each one. the interval between st- nd period was months and nd- rd period was year. rectal, oropharyngeal swabs tracheal aspirates from patients, handswabs from stuff and samples from environment were taken weekly. the identification of ab was performed using vitek ii system the susceptibility was tested by kirby-bauer and mic methods and the <<dna fingerprints>>obtained by pulsed field gel electrophoresis (pfge). results: during the st nd and rd period, patients ( men, women), patients ( men, women) and patients ( men, women) were hospitalized in icu respectively. ab was isolated in from samples ( . %) at the st period, from ( . %) at the nd and from ( %) at the rd period. totally ab was isolated in from specimens ( %) at the st nd and rd period among the patients carrying ab, / ( %), / ( %) and / ( %) were infected respectively. the infections observed during the study period were: sepsis ( ), urinary tract infection ( ), pneumonia ( ), meningitis ( ), thrombophlebitis ( ) . all the isolated ab strains were multiresistant to antimicrobial agents. molecular analysis of isolated strains by pfge distinguished the following types: a ( , subtypes a -a ), b ( ) at the st period a( ), c( ), d( ), e( ), f( ), g( ), h( ), i( ). j( ) at the nd period a( ), b ( ), d( ), h( ), k( ). l( ) at the rd period. infections were caused mainly by a and d types while the same types were isolated from the environment and the hands of the icu stuff. conclusion: there was a high rate of colonization and infection of icu patients by multiresistant clones of ab. the persistence of clone a of a. baumannii and the appearance of b type at the rd period after its disappearance at the nd period despite the application hygiene measures, indicates the need for more strict reinforced infection control in icu. the transmission via the hands of stuff to patients has become the most important contributor factor in patient colonization and/or infection. objectives: the antibiotic resistance and its mechanism of group a streptococci (gas) varies according to nations or study period. we have investigated antibiotic resistance and mechanism of macrolide resistance for the strains isolated from korean children and compared to the previous ( ) results. methods: throat cultures were taken from elementary school children in jinju, korea from october to december, to isolate gas. antibiotic susceptibility test to erythromycin (em), clindamycin (cc), and tetracycline (tc) was performed by disk diffusion method. macrolide resistance phenotype and genotype as well as emm genotype were studied. results: isolation rate of gas was . % ( / ). resistance rates of em, cc, and tc were . %, . %, and . % respectively, which were dramatically decreased from %, %, and % in at the same area. emm / was prevalent ( %), while emm was the most common type ( %) in . cmlsb, m, and imlsb were observed in . %, . %, and . % respectively, compared to %, %, and % in . the strains with cmlsb and imlsb had ermb gene and the ones with m phenotype were positive with mefa gene. conclusion: the resistance rates to em and cc were dramatically decreased compared to the past ( ). education to the public and physicians, decreased consumption of antibiotics, acquisition of immunity to the resistant strains, or change of prevalent emm types could be considered to explain the reason of decrease of antibiotic resistance. although antibiotic resistance rate was decreased, cmlsb type which has high mic was prevalent suggesting treatment failure for those children carrying these resistant strains in jinju, korea. analysis of skin and soft tissue infections in european medical centres: report from the sentry antimicrobial surveillance program ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) g. moet, p. strabala, h. sader, t. fritsche, r. jones (north liberty, us) objective: to analyse the skin and soft tissue infections (ssti) or wound infections in hospitalized patients in the sentry program for pathogen prevalence and resistance (r) variations in european (eu) medical centres for the years to ( years). this program also included north america (na) and latin america (la) for the same years, except . methods: consecutively isolated pathogens/site were collected from each centre per year and varied in number of sites each year in eu: eu: ( ), eu: ( ), eu: ( ), eu: ( ), eu: ( ), eu: ( ), and . susceptibility testing was determined by clsi (formerly the nccls) broth microdilution methods and interpreted by current ( ) breakpoints. results: table of all years total of ssti pathogens. (see table) sa was the predominant pathogen in eu ranging from . % of ssti isolates in to . % in . the top most prevalent organisms accounted for . % of isolates in all years with psa and ec ranking second and third, respectively with . % combined, and enc and ent ranked fourth and fifth with . % of the total isolates. compared to the americas, mrsa and vre isolation was at a lower occurrence rate in the eu; but between the rates of other monitored continents for ctz-r psa, cipro-r ec and ctz-r ent (ampc). vre increased in eu over the year. conclusions: pathogen prevalence in ssti for eu has been consistent over the monitored years although sa (with mrsa) appears to be increasing. eu is not a world leader in any key r marker compared to the americas. however, the r rates are evolving which suggests continued need for surveillance programs at regular intervals to detect mobile genetic r elements. objectives: carbapenems play an important role in the therapy of pseudomonas aeruginosa infections. the aim of our study was to characterize the molecular alteration responsible for changed susceptibility towards carbapenems in multiresistant p. aeruginosa strains from germany. methods: multiresistant p. aeruginosa strains from cystic fibrosis and non cystic fibrosis patients were collected in german hospitals in . the strains showed reduced susceptibility (intermediate or resistant; din guidelines) to imipenem, piperacillin, ciprofloxacin and gentamicin. clonality was tested using pfge. a pcr screening for vim and imp was carried out. effluxpump overexpression was detected using an effluxpump inhibitor (epi) test. oprd and for strains with positive results in the epi test the effluxpump repressorgenes mexr and nfxb were sequenced. results: pfge patterns revealed no clonal relationship among the multiresistant strains. neither vim nor imp was detected. the geno-and phenotypes found are depicted in table . defective oprd genes caused by premature stopcodons or frameshifts were found in strains. among those had no mutations in mexr or nfxb and showed the highest mics found ranging from to > and to mg/l of imipenem and meropenem, respectively. additionally had defective mexr genes, but intact nfxb genes, also had modifications in mexr and nfxb, and showed only in nfxb additional alterations. for strains no alterations in oprd but in mexr were proven. conclusions: the predominating mechanism of carbapenem resistance in multiresistant p. aeruginosa strains from germany was the loss of oprd. accessory overexpression of mexaboprm due to modifications in mexr did not result in significantly elevated mics of meropenem. moreover, the additional overexpression of mexcdoprj did not lower the mic of imipenem. in strains with modifications only in mexr only elevated mics of imipenem indicate a reduced expression of oprd accompanied by overexpression of mexefoprn as conferred by nfxc-type mutants. objective: mart (study for monitoring antimicrobial resistance trends) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intraabdominal infections (iai). the aim of this sub-analysis was to assess antimicrobial susceptibility patterns among gramnegative bacilli from different regions of the world during . methods: pa total of major medical centres in north america, latin america, europe, middle east/africa, & asia/ pacific tested the in vitro activity of antimicrobial agents commonly used to treat iai against consecutive unique aerobic and facultative gram-negative bacilli from iai using microdilution techniques according to clsi guidelines & breakpoints. results: enterobacteriaceae were recovered from ( %) & non-enterobacteriaceae were recovered from ( %) of the patients in the study worldwide, constituting ( %) & ( %) of the total isolates, respectively. e. coli (n = ; %) and klebsiella spp. (n = ; %) were the most commonly isolated enterobacteriaceae. pseudomonas spp. (n = ; %) and acinetobacter spp. (n = ; %) were the most commonly isolated non-enterobacteriaceae. isolates from asia/pacific and latin america were generally more resistant. ( %) of the enterobacteriaceae & ( %) of the non-enterobacteriaceae were recovered < hours after hospitalization. the % susceptible isolates are reported below: conclusion: in this study, enterobacteriaceae were the predominant intraabdominal isolates recovered both < h and > h after hospitalization. carbapenems were overall the most active agents against enterobacteriaceae worldwide. resistance rates varied among geographic regions, with the asia/pacific and latin america regions generally having the most resistance. characterisation of streptogramin resistance genes among enterococcus faecium isolates from austrian animal husbandry a. eisner, g. gorkiewicz, g. feierl, f. dieber, e. marth, j. kö fer (graz, at) objectives: the streptogramin virginiamycin has been widely used as a growth promoter in animal husbandry in the european union but was banned in because of concerns about evolving cross-resistance to the streptogramin quinupristin-dalfopristin used in human medicine. the aim of the present study was to investigate the prevalence of streptogramin resistance genes of enterococcus faecium recovered from animal faecal specimens collected in southeast austria. methods: we analysed e. faecium isolates of cattle (n = ), pig (n = ), and poultry (n = ) for the presence of streptogramin resistance genes. we used selective enterococcal broth for isolation. species identification was done on basis of gram stain, catalase and pyrrolidonyl arylamidase activity, motility, lancefield group d antigen typing, and by the strep apitest (biomérieux). detection of the resistance genes vat(e), vat(d), and erm(b) was done by pcr. results: the erm(b) gene encoding macrolide, lincosamine, and streptogramin b (mlsb) resistance was found in each e. faecium isolates recovered from pig and cattle, none of the isolates from these animals carried genes coding for streptogramin a resistance. on the contrary, e. faecium isolates from broiler specimens contained the vat(e) gene and one isolate contained the vat(d) gene. all of these isolates also contained the erm(b) gene. conclusion: our data indicate that the use of the meanwhile banned antimicrobial feed additive virginiamycin has created a reservoir of streptogramin-resistant e. faecium in southeast austrian poultry. characterisation of macrolide-resistant streptococcus pneumoniae isolates from russia objectives: streptococcus pneumoniae (spn) resistance to macrolide antibiotics continues to be of major concern. the aim of present study was to analyse phenotypic and genotypic characteristics of macrolide resistant spn isolates. methods: eighty one macrolide resistant spn isolates were collected in moscow, moscow, - . the susceptibility testing was performed according to the clsi guidelines. macrolide resistance phenotypes were characterized by triple-disk diffusion test, using erythromycin, clindamycin and rokitamycin disks. detection of genes, coding resistance to macrolides, was done by rt-pcr. sequencing for qrdr mutations was performed on levofloxacin resistant spn isolates. selected isolates were analysed by mlst. results: by the triple-disk test, isolates were assigned to the m phenotype, of them were carrying mefa gene, and one was negative. twenty eight isolates were cmlsb-phenotype, of them were carrying both ermb and mefa genes, in two isolates only ermb was detected, and one isolate was negative for both genes. imclsb phenotype was demonstrated by isolates, both ermb and mefa genes were detected in of them, and only ermb in . three isolates didn't demonstrated blunting of zone of inhibition around rokitamycin disk. associated resistance to penicillin g, tetracycline, chloramphenicol and co-trimoxazole was observed in . %, . %, . % and . % of spn isolates respectively. nine multidrug resistant isolates, harbouring both mefa and ermb genes, were subjected to mlst. among them one isolate was found to share the allelic profile st (spain f- clone), and four isolates were single-allele variants of st . in four isolates new allelic profiles were detected. three isolates were resistant to levofloxacin (mic ‡ mg/l), in two of them with levofloxacin mic > mg/l (st single-allele variants) e k, s f and i v substitutions were detected in gyra, parc and pare, respectively. d n and i v substitutions were detected in parc and pare of one isolate with new allelic profile. conclusion: high prevalence of macrolide resistant spn, harboring both ermb and mefa genes is observed in moscow, macrolide resistance is associated with resistance to other groups of antibacterials. some multidrug resistant isolates are highly related to internationally disseminated multiresistant clone spain f- . strains with fluoroquinolone resistance in moscow were all single locus variants of the spain f- clone. occurrence of tet(w) gene in a clostridium difficile clinical isolate p. mastrantonio, f. barbanti, p. spigaglia (rome, it) objectives: to investigate the presence of tet(w), a tetracycline resistance gene recently identified in anaerobic commensal bacteria from animals and humans, in c. difficile clinical isolates. methods: several c. difficile clinical isolates from different italian hospitals were analysed for the presence of a tet(w) gene by pcr assays. the primers used were designed on the tet(w) sequences available in genbank. pcr fragments obtained by these amplifications were sequenced. tet(w) dna flanking regions were also examined with a set of pcrs constructed on the sequence of the conjugative transposon tnb of butyrivibrio fibrisolvens . , that is the only element carrying a tet(w) partially characterized so far. tet(w) positive isolates were also examined for the tet(m) gene and for the presence of int and tndx, markers for the tn and the tn like elements, respectively. the tn -like elements were further characterized by pcrs designed on enteroccus faecalis tn sequence. tetracycline mic values were determined by the e-test method. tetracycline resistance gene transfer was evaluated by filter mating experiments, using c. difficile p r strain as recipient. results: a tet(w) gene was found in only one isolate, c. difficile cd , also positive for the tet(m) gene. this isolate was resistant to tetracycline with a mic of mg/l. sequence analysis of the tet(w) pcr fragment (about bp) showed that this gene had an identity of % with the genes found in clostridium spp strain k , mitsuokella multacida and butyrivibrio fibrisolvens. no amplifications were obtained with the primers designed on tnb , indicating the presence of a different genetic support for tet(w) in c. difficile. tet(m) gene of c. difficile cd was carried by a tn -like element that showed nucleotide sequence mutations in the region containing orf - compared to the element of e. faecalis. conjugative transfer of tet(w) was not observed, whereas the tet(m) gene was transferred to the recipient strain. c. difficile transconjugants were resistant to tetracycline with a mic of mg/l. conclusion: the results obtained in this study demonstrate for the first time the presence of a tet(w) gene in a clinical isolate of c. difficile, providing further evidence of the spread of this resistance determinant among gastrointestinal bacteria. macrolide resistance determinants are prevalent and readily selected for in viridans group streptococci among healthy norwegian adults background: norway has a low prevalence of antimicrobialresistant bacteria including macrolide resistant (mr) respiratory tract pathogens. we have observed an increase in macrolide consumption in norway and there is a lack of knowledge on the reservoir of macrolide resistance determinants among viridans group of streptococci (vgs) in the pharyngeal flora. objectives: examine the occurrence, selection and persistence of macrolide resistance determinants in vgs pharyngeal flora in healthy norwegian adults before and after treatment with azithromycin. methods: throat samples were collected before (day ), after treatment (day ) and after months (day ) from healthy volunteers. the samples were plated directly as a lawn on pdmii agar plates with % defibrinated blood with an erythromycin etest strip. photos were used as quantitative comparisons. up to morphological different colonies with erythromycin etest mic ‡ lg/ml from each specimen were collected; day (n = ), day (n = ) and day (n = ). in total representatives mr, vgs-isolates were selected for further studies: (i) mics of erythromycin, tetracycline and penicillin were determined by etest. (ii) pcr's for erm(b), erm(tr), and mef(a/e), and subsequent sequence-typing of mef. species identification was performed by soda sequencing. results: a total of / persons carried a low number (< ) of mr vgs in day specimens, while / had a significant higher number (> ) of mr strains in day specimens. in day specimens, / carried a low number of mr, resembling day . reduced susceptibility to penicillin was observed in / ( %) isolates. tetracycline resistance was found in / ( %), and mainly in erm(b)-positive strains. mef(a/e)-positive dominated day ( %) and erm(b) day specimens %. sequence typing revealed mef(e) (n = ) and mef(a) (n = ). soda sequence; s. mitis (n = ), s. oralis (n = ), s. parasanguinis (n = ), s. salivarius (n = ), and s. sanguinis (n = ). conclusion: there is a pool of vgs carrying macrolide resistance determinants in the normal pharyngeal flora of healthy adults that are readily selected for during azithromycin exposure. the mef(e) and erm(b) were the most prevalent resistance genes and co-resistance to tetracycline was frequently observed, resembling the findings in norwegian clinical isolates of s. pneumoniae. these vgs may provide a pool of resistant bacteria that may transfer resistance determinants to more pathogenic organisms. relationships in genotype, phenotype, t type and pfge type among macrolide-resistant streptococcus pyogenes strains isolated in the czech republic v. jakubù , p. urbášková, l. straková (prague, cz) objectives: to determine relationships between phenotypic and genotypic methods among erythromycin-resistant s. pyogenes strains. methods: a total of clinical isolates of s. pyogenes resistant to erythromycin were collected in microbiology laboratories during - . erythromycin susceptibility was tested by the disk diffusion method. strains with an inhibition zone < mm around the erythromycin disk ( lg) were sent to the national reference laboratory for antibiotics (nrl). presences of mlsb resistance genes (ermtr, ermb and mefa) were tested by pcr. t serotypes were determined in randomly selected representatives of each phenotype (n = ). pfge type were determined in strains from year only (n = ). results: the rate of the most prevalent phenotype (constitutive mlsb resistance) was %, % in the year and , respectively. the major prevalent t types among the analysed strains were serotype t ( %), t ( %), t ( %) and t b ( %). gene ermb was the most frequent ( %). the results of pcr method was highly congruent with observed phenotype of resistance. pfge patterns of strains with constitutive mlsb resistance were highly identical. conclusion: m phenotypes, constitutive and inducible resistance to mlsb antibiotics were found and ermtr, ermb and mefa genes were detected among the analysed strains. the t serotype was identified the mainly prevalent in our collection. the majority of strains harbouring t serotype were constitutively resistant to macrolides. the study showed close relationships among genotypes, t types, specific resistotypes (phenotype) and pfge types. objectives: since recognition of transferable clindamycin and tetracycline resistance in bacteroides, we have undertaken a us national survey on the susceptibility of b. fragilis group to analyse emergence of resistance and trends, since these species are not routinely tested for susceptibility in hospital clinical laboratories. methods: agar dilution mics were determined for isolates from - for b. fragilis and related species from geographically diverse centers in the us. antibiotics included carbapenems, b-lactam/b-lactamase inhibitors, quinolones, a tetracycline, clindamycin, metronidazole, chloramphenicol, a glycylcycline and linezolid. isolate identity was confirmed by api a. results: analysis of resistance trends from - showed a decrease in geometric mean mic's (geomic) for imipenem ( . mcg/ml to . mcg/ml, p < . ) and meropenem ( . mcg/ml- . mcg/ml, p = . ) for the bacteroides species. ertapenem geomic remained unchanged ( . mcg/ ml). for the b-lactamase inhibitors, piperacillin-tazobactam geomic declined from . mcg/ml to . mcg/ml (p < . ). ampicillin-sulbactam geomic did not change. few isolates were resistant to any carbapenem or b-lactamase inhibitor combination. clindamycin resistance increased, especially for b. fragilis, b. ovatus and b. thetaiotaomicron (all p < . ). among quinolones, resistance of bacteroides to moxifloxacin increased (geomic went from mcg/ml to . mcg/ml, p < . ). b. fragilis remains the most sensitive bacteroides species to moxifloxacin, although approximately % of stains have mic's ‡to mcg/ml in . tigecycline susceptibility, tested over years, did not change. the first confirmed metronidazole-resistant isolate (mic = mcg/ml) obtained in the us was noted in but none were noted in or . conclusion: improved susceptibility of bacteroides species to some carbapenems and the b-lactamase inhibitor combinations is unexplained but significant. clindamycin resistance continues to increase, especially for b. fragilis. moxifloxacin susceptibility for the non fragilis species shows that the majority of strains are resistant. the first metronidazole resistant isolate has been reported from the us. since resistance trends are associated with species, the differentiation within the species is of extreme importance, since it may impact the choice of antimicrobial agent for the treatment of infections caused by this group of anaerobes. observed duration of nasopharyngeal carriage of penicillin-resistant pneumococci: relations to age and serogroup p. geli, l. hö gberg, h. ringberg, e. melander, m. lipsitch, k. ekdahl (solna, malmö, lund, stockholm, se; boston, us) background and objectives: knowledge of how the duration of pneumococcal carriage varies with age and serogroup is essential to understanding how immunity to carriage arises throughout the course of life, and designing appropriate models for the effects of vaccination or other public health initiatives aiming to reduce the pneumococcal transmission in the community. using data from an ongoing swedish intervention project, the duration of nasopharyngeal carriage of penicillinresistant pneumococci (mic pcg > . mg/l) stratified by both serogroup and age of the carrier were estimated. methods: the mean duration and corresponding % confidence interval was estimated by fitting a gamma distribution to the observed duration of carriage for each serogroup and age stratum. results: the mean duration of carriage for all cases was days ( % ci - ). children below the age of years carried prp for significantly longer periods ( days, % ci - ) compared with older individuals ( days, % ci - ). there were also differences within the group of cases below the age of years, as the duration of carriage became significantly shorter for each year older the cases were. serogroup and were carried for significantly shorter periods compared with serogroup . serogroup also had significantly shorter carriage duration compared with serogroups and for cases - years. for cases years or older, no significant difference in carriage duration for different ages or serogroups could be noted. conclusions: even though the estimate does not cover any correction for the censored carriage duration and therefore not yield an estimate of the total length of carriage, the results highlight the importance to take both serogroup and age of the p exploring the molecular basis for differences in phenotype of salmonella enteritidis typing phage n. delappe, d. morris, m. cormican (galway, ie) objectives: the salmonella enteritidis phage tying scheme of the laboratory of enteric pathogens, health protection agency, uk, is a widely used method for subtyping this important pathogen. the method is rapid and highly discriminatory. interpretation of results can be subjective and the typing phage which are central to the method have not been well characterised. complete sequence data is available for the salmonella typhimurium podovirus phage p . methods: the typing phage were propagated on s. enteritidis pt b (pb ). phage were visualised by electron microscopy. phage dna was extracted and digested with hindiii. consensus pcr primers were designed based on sequences of p and other s. typhimurium phage. additional primers were designed based on the sequence of the s. enterititidis typing phage (a siphovirus). amplification, sequencing and dna probe hybridisation of various phage genes were performed using standard techniques. results: on em the typing phage comprise podoviridae (phage , , , , and ) , siphoviridae (phage , , , , and ) and myoviridae (phage , , and ). digestion with hindiii subdivided each morphotype into groups. the podoviridae contained genes homologous to p while the siphoviridae contained genes homologous to the sequenced s. enteritidis typing phage . some sequence variation was detected in podovirus and siphovirus genes however in some cases phage, which differ in their phenotype had no difference detected in hindiii digestion pattern or partial sequence. conclusions: the s. enteritidis typing phage set comprise distinct phage morphotypes. in some instances distinct phage that contribute to differentiation between s. enteritidis phage types had no dna sequence variation detected. variations in phage typing reactions may in part be due to epigenetic difference in typing phage, e.g. due to methylation of phage dna. salmonella enteritidis typing phage biology could provide a model for developing approaches to phage therapy. tularemia is a zoonotic bacterial disease. the causative agent, francisella tularensis, is spread to humans by direct contact with infected rodents, inhalation, ingestion of contaminated water or by arthropod bites. in some endemic regions, outbreaks occur frequently, whereas nearby rural parts may be completely free. we presented two cases of tularemia in non endemic region of the turkey. case : a year old female patient referred to tertiary hospital due to swollen on the neck for months. before admission beta lactam antibiotics had been prescribed to her for tonsilopharyngitidis. but her complaints had been continued. so she admitted to our hospital. she had been suffered fever sore throat and neck pain. she had a palpable and painfull cervical lymphadenopathy which was not suppurated. leukocytosis and elevated c reactive protein were predominant. at screening there were not any lymphadenopathy detected elsewhere. she had been examined about cytomegalovirus epstein barr virus and brucellosis. they were negative. fine needle aspiration from neck was negative considered as malignancy. cultures were negative for routine bacteriologic examination. microagglutination test for tularemia was / positive. then we decided to treat her with gentamycin for days. after treatment cervical lymphadenopathy became small. leukocyte count and c reactive protein levels were reach normal range. case : a year old female patient referred to university hospital due to cervical lymphadenopathy and fever and sore throat. before admission beta lactam antibiotics were prescribed to her for weeks. but no apparent benefits had been detected. there was a palpable and fistulated cervical lymphdenopathy. drainage was examined microscopically and cultured for bacteria, mycobacteria and fungi. on routine cultures no microorganisms were grown. fine needle aspiration was done. it was reported that suppurative granulamatous lympadenitis. so we were examined for tularemia, cat scratch disease. microaggltunation test for tularemia was / positive. then streptomycin had been given for days and excision of lymphadenopathy had been done. no complications or recurrence occur. results: both patients were applied to us from non endemic and different regions of the turkey. they had no known insect bite history. both of them were diagnosed by serological tests. conclusions: in the differential diagnosis of tonsillopharyngitidis, tularemia also must be considered in the non endemic regions. tularaemia presenting with tonsillopharyngitis and cervical lymphadenitis: two case reports b. kandemir, i. erayman, m. bitirgen, e. turk aribas, a.c. inkaya, s. guler (konya, tr) tularemia is a zoonotic disease caused by francisella tularensis. francisella tularensis is transmitted to humans by direct contact or ingestion of infected animal tissues, through the bite of infected arthropods, by consumption of contaminated food or water, or from inhalation of aerosolized bacteria. in this report we describe two cases of oropharyngeal tularemia who presented with tonsillopharyngitis and cervical lymphadenitis. case i: a years old woman with multiple cervical lymphadenitis has been admitted to our clinic. her complaints started months ago with signs and symptoms of tonsillopharyngitis. she had received non specific treatment (ampicillin+sulbactam) and ten days later cervical lymph nodes appeared. the diagnosis was made serologically. the antimicrobial therapy (streptomycin · g im) was given for fourteen days. the patient recovered completely. case ii: a years old girl with multiple cervical lymphadenitis was admitted to hospital. her complaints started months ago with throat ache after which multiple cervical lymphadenitis appeared. she was admitted to our out patient clinics and diagnosed to have tularemia. anti-microbial therapy (streptomycin · g im+doxycyciline · mg) was given for four weeks but no clinical response was achieved. patient was admitted to the hospital and surgical drainage was performed. treatment against tularemia was prolonged. patient was finally recovered at the end of nine weeks of therapy. it can be concluded that early diagnosis and treatment of tularemia are important. some patients may benefit from surgical drainage and prolonged therapy. a case of nonclostridial crepitant cellulitis which is due to escherichia coli c. ayaz, m. ulug, m.k. celen, m.f. geyik, s. hosoglu (diyarbakir, tr) objectives: this condition is caused by gas forming bacteria that involve the skin, either or as an extension from deeper structures. the origin of infection is an abdominal wound, perianal disease, or operative incisions that have become secondarily infected. tracking of gas-forming organisms from deeper sites of infection may also present as crepitant cellulitis without a break in the skin. diabetics are more likely to acquire such infections, especially in the lower extremites. among the bacteria isolated are anaerobic organisms such as bacteriodes or anaerobic streptococci, or coliform bacteria, especially escherichia coli and klebsiella. because of this reason we reported a case of a nonclostridial crepitant cellulitis which is due to escherichia coli. case: a year old man who was previously healthy, has come with fever, pain, oedema, erythema, crepitant and limitation of movement at the right lower extremity. in his history he had no complaint until weeks ago. perianal abscess has developed at this time and it has drainged spontaneously days later. than his complaints has comprised day duration. on physical examination, the temperature was . °c, pulse rate / minute, respiratory rate /minute and blood pressure was / mmhg. laboratory evaluation showed a haemoglobin . g/dl, leucocyte count of /mm (neutrophils %). serum electrolytes, renal and liver function tests were within normal limits. c reactive protein was elevated up to mg/dl, esr was mm/h. escherichia coli was isolated from wound and blood cultures. he was treated initially with ampicilinsulbactam ( g/day) and required attempt. even with optimal surgical and medical therapy, he dies at the third day of the treatment from septic shock. conclusion: the onset is generally gradual, and there is usually mild local pain and systemic toxicity, allowing clinical differentiation from the more fulminant clostridial myonecrosis. the surgical approach should be aggressive, but tailored specifically to the underlying cause of infection. antibiotic therapy is directed at a mixed aerobic-anaerobic flora, until culture reports are available. a case of iliopsoas abscess which is due to pseudomonas aeruginosa objectives: pyogenic psoas abscess, a rare but life-threatening infection, results from primary suppuration or is secondary to the spread of infection from an adjacent structure. primary iliopsoas abscess occurs probably as a result of hematogenous spread of an infectious process from an occult source in the body. primary iliopsoas abscess can occur in diabetus mellitus, intravenous drug abuse, aids, renal failure and immunosupression. ultrasound is diagnostic in only % of the cases. computed tomography should be done for definitive diagnosis and is considered the gold standard. stapylococcus aureus is the causative organism in patients with primary iliopsoas abscess, but pyogenic psoas abscess caused by pseudomonas aeruginosa is uncommon. because of this reason we reported this case. case: a previously well year old woman presented with a month history of right loin to groin pain, limping or limitation of hip movement, fever and nausea. she was a diabetus mellitus patient for years. on her physical examination, the temperature was . °c, pulse rate /minute, respiratory rate /minute and blood pressure was / mmhg. examination of the respiratory system, cardiovascular system and abdomen were found to be normal. laboratory investigations revealed total leucocyte count of /mm (polymorphs %), c reactive protein was elevated up to mg/dl, esr was mm/h. serum electrolytes, renal and liver function tests were within normal limits, but serum glucose level was elevated to mg/dl. her blood cultures were sterile, but abscess culture yielded pseudomonas aeruginosa which was taken during the surgery. she was treated imipenem ( g/ day) + amicasin ( . g/day) and required surgical drainage. she was treated and followed up days, and discharged at the end of the treatment. conclusion: in these patients treatment involves the use of appropriate antibiotics along with drainage of the abscess. an adequate knowledge of the causative organisms should guide the initial choice of antibiotics. depending on the results of the abscess fluid culture and sensitivity, adjustments should be made. percutaneous drainage or surgical drainage may be done in them. in conclusion early recognition, empiric antimicrobial coverage and aggressive drainage or debriment are indicated in these patients. cervical lymphadenitis in a diabetic woman f. Ç okça, a. azap, s. gö çmen, h. erdi sanli, s. gü l (ankara, kirikkale, tr) objective: rhodococcus equi infections are commonly seen in immunocompromised patients. exposure to domestic animals, such as horses and pigs may play a role in some cases. two thirds of the r. equi infections in immunocompromised were reported in hiv infected patients, and the rest divided between transplant recipients, immunosupressive medications and other kinds of immunosupression. the clinical picture presents with pulmonary infection in % of patients. here, we report a rare case of cervical lymphadenitis in a diabetic women due to r. equi. case: a sixty-year-old diabetic woman was admitted with the complaints of fever, right cervical erythematous swelling with tenderness and warmth. on physical examination; inflammation beginning from the right submandibular region and descending to the upper chest was detected. a tender mass of · · cm. was palpated on the right cervical region. ampicillin/sulbactam g/day was given emprically for a week with no improvement. the ct scan of the neck showed conglomerated lymphadenopathy extending from the submandibular area to the supraclaviculary region with . · cm in size. the mass began to fluctuate and cc abscess material was drained surgically. gram's stain of the purulent material showed polymorphonuclear leukocytes with pleomorphic gram positive coccobacilli. the cultures of the material grew r. equi. therapy was changed to teicoplanin and ciprofloxacin combination and surgical care of the wound with antiseptics was performed. after a month, intrevenous medical therapy was changed to oral route with roxythromycin and ciprofloxacin and was continued to months with complete resolution. conclusion: increased awareness and improved laboratory techniques help for the early diagnosis of rhodococcal infections. timely diagnosis is important because the microorganism is usually resistant to penicillin g, oxacillin, ampicillin, carbenicillin and cefazolin. the use of at least one antibiotic with intracellular activity is necessary in the treatment of r. equi infections. empirical two drug regimens with erythromycin, rifampin and/or ciprofloxacin are recommended. objectives: to analysed the features of spondylodiscitis (sd), their clinical presentation, the commonest diagnostic methods and the kind of treatment applied according to the different groups of the study. methods: a retrospective and descriptive study taking place amongst the patients diagnosed as having sd from till . in each case we studied the presence of underlying disease, primary infectious sources in the prior months, the way symptoms started, location, diagnostic methods, treatment and evolution, comparing between different aetiologies. results: patients with sd were studied. of them had pyogenic sd,( had spontaneous sd and had an sd after spinal surgery) and patients had tuberculous sd. were men ( to years; mean . ). patients with postoperative sd were the youngest (mean . y, p = . ). underlying diseases were found in % of patients, mainly in postoperative sd ( % of cases) (p = . ). an episode of previous bacteremia or infectious source was found in % and % respectively of patients with spontaneous pyogenic sd, significantly higher than in surgical sd ( % had bacteremia and % other infectious source, p < . ). the most common presenting symptoms were back pain ( . %) and neurological deficits ( %). frank fever occurred in % of cases, being more frequent in spontaneous sd ( %) than in postoperative sd ( %) or tuberculous sd ( %), p £ . . leukocytosis was found only in % of patients. postoperative sd presented the lowest levels of esr (p = . ). s. aureus was the most frequent bacteria isolated ( %) in pyogenic spontaneous sd, as coagulase negative staphylococci was in surgical sd. lumbosacral localization was detected in % of spontaneous pyogenic sd and in % of postoperative sd. tuberculous sd predominate in dorsolumbar region. paravertebral abscess formation was observed in % of pyogenic sd and in % of tuberculous sd (p = . ). surgical treatment was required in . % of tuberculous sd and in % of pyogenic sd (p = . ). outcome of patients with spontaneous sd was worse (sequelae in %), than in patients with surgical sd ( . %) or tuberculous sd ( %) (p = . ). conclusions: ) spontaneous sd was the most frequent and it occurred mainly in patients suffering from underlying diseases; ) nearly all patients had pain but only in / of them was accompanied by fever; ) the lumbar zone was the most frequent location; ) the majority of patients had a complete resolution of their symptoms only with medical treatment. background: the ethiopathogenesis of cns abscess includes a broad spectrum of pathogens and predisposing conditions, so that a polymicrobial flora is a quite frequent event. capnocytophaga spp. includes fastidious gram-negative organisms, usually underestimated in the common clinical practice, and poorly tested in vitro for antimicrobial susceptibility. surprisingly, also agents usually active on gram-positive pathogens demonstrated some efficacy against capnocytophaga spp. (i.e. erythromycin, rifampin, tetracyclines, cotrimoxazole, chloramphenicol, and glycopeptides), which is usually responsible of anecdotal episodes of cns infection (meningitis, brain abscess, and subdural empyema). methods and results: the fourth case report of capnocytophaga spp. brain abscess is herewith reported. a probable origin from a recent cat bite and a mandibular granuloma is suspected. due to the lack of clinical and neuroradiological response to neurosurgical debridement and an association therapy including imipenem, amikacin, clindamycin and fluconazole, empiric administration of linezolid ( mg/day) was attempted, and a rapidly favorable clinical, microbiological, and neuroradiological response was achieved. notwithstanding the identification of capnocytophaga spp. as the sole microorganism yielded by purulent drainage of a cns abscess, patients with multiple risk factors and recent surgery are expected to suffer from a polymicrobial cns infection. due to its favourable cns penetration and its dual mode of administration (both i.v. and oral), linezolid may represent an alternative option in the event of cns diseases borne by numerous risk factors and a suspected polymicrobial origin, especially when a lack of response to first therapeutic attempts is of concern. in the management of a cns abscess where the role of microorganisms with an unpredictable sensitivity profile remains of concern, chemotherapy should be directed also against potentially multiresistant organisms. considering also the relevant limitations given by the often poor cns penetration, the activity of glycopeptide agents is limited, compared with that of linezolid. aetiologies and antimicrobial resistance profiles of purulent meningitis study carried out in a hospital of infectious diseases, algiers objectives: bacterial meningitis is a serious clinical and medicolegal consequences if management is incorrect. meningitis protocols have recently been published by the british infection society/meningitis research foundation and are widely disseminated in our institution. local guidelines are also available on the hospital intranet and in the emergency department and acute medical admissions wards. this study investigated the level of understanding about meningitis and knowledge of the guidelines in medical staff of different grades working in the emergency department and the acute medical admissions unit in a large teaching and emergency hospital. methods: medical staff were interviewed faced to face and asked a series of questions on the management of meningitis. results were stored on a database and responses were analysed. results: general knowledge about meningitis was variable. although % knew that bacterial meningitis was a notifiable disease only % knew the procedure for informing the health protection agency and only % would notify viral meningitis. only % of responders were aware that guidelines could be viewed on the hospital intranet. only % correctly identified the indications and cautions for lumbar puncture. although the majority recognised the need for urgent administration of antibiotics % would omit antibiotics until further assessment and lumbar puncture results. only % were aware of the need to consider adding ampicillin to cover listeria in patients over years of age and there was uncertainty about the management of patients with penicillin resistance. conclusions: although protocols and guidelines for meningitis have been produced and are easily accessible the majority of medical staff were uncertain how to access and utilise this information. the level of knowledge and expertise in managing meningitis amongst medical staff working in a and e and the acute medical unit was poor and there is a need for further education to improve patient management. guidelines are of no value if they are not disseminated to front-line medical staff. objectives: the aim of this study was to evaluate the prevalence of penicillin resistant and multi-drug resistant pneumococci isolates in streptococcus pneumoniae meningitis. methods: a retrospective study was carried out on clinical records between january and october . among the csf samples the pneumococcal ethiology was confirmed by % positive cultures and % latex agglutination. antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. isolates of pneumococci with oxacillin zone sides of > mm are susceptible (mic < . microg/ml) to penicillin, while at those of < mm the mic has to be determined (by e -test). results: isolates from patients ( %) were found with penicillin-resistance (prp) -of which % were multi-drug resistant-and ( %) with penicillin susceptibility -of which % were resistant to other drugs. an abrupt onset of disease was found in % prp patients and % from non-prp ones. chest x ray pulmonary determinations were found in % prp patients and % non-prp ones. sixty-six per cent of prp patients and % of non-prp ones had a prior hospitalization. only % of non prp patients had a positive blood culture. antibiotic switch was made in % cases with prp isolates and % cases with non prp ones. the overall rate of mortality was %, with % for prp patients and % for non-prp ones. conclusions: non-prp isolates were the prevalent ethiology of s. pneumoniae meningitis. % of non -prp strains developed other drug resistance, and % prp strains were multi -drug resistant. prp meningites evolved more as a hospital-related pathology, with an abrupt onset, frequently associated with pulmonary determinations and higher mortality rate. background: although vaccination strategies have shifted the age distribution of meningitis to older age groups, few studies have specifically examined bacterial meningitis in the older adult. methods: from october to april , we prospectively included episodes of community-acquired bacterial meningitis, confirmed by culture of cerebrospinal fluid, which occurred in patients aged > years. we dichotomized the cohort with respect to age: patients aged ‡ years were defined as older adults and patients aged - years as younger adults. predictors for an unfavourable outcome (defined as score - on the glasgow outcome scale) were determined by logistic regression. we tested for statistical interaction between age group and potential prognostic factors by adding multiplicative interaction terms to the model. the mann-whitney u test and the chi-square test were used to identify differences between groups. results: of episodes ( %) occurred in older adults and episodes in younger adults ( %). streptococcus pneumoniae was the most common pathogen in older adults ( %). meningitis in younger adults was caused by neisseria meningitidis and s. pneumoniae in % and % of the episodes, respectively. older adults were more likely to present with the classic triad of bacterial meningitis (fever, neck stiffness and altered mental status) than younger adults ( % versus %; p < . ). the prognostic value of independent risk factors for unfavourable outcome was similar in both age groups. older adults had more complications during clinical course, resulting in a higher mortality rate than in younger adults ( % versus %; p < . ). sepsis was the most common cause of death in both age groups ( % in older adults versus % in younger adults; fig) . whereas older adults tended to die more often due to cardiorespiratory failure ( % versus %; p = . ), younger adults more often died due to brain herniation ( % versus %; p = . ). conclusions: bacterial meningitis in older adults is associated with high morbidity and mortality rates. elderly patients often present with classic symptoms and s. pneumoniae is the most common pathogen within this age group. whereas older adults often die due to cardiorespiratory failure, younger adults more often die due to brain herniation. incidence of serogroups and penicillin susceptibility in neisseria meningitidis isolates ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) objective: the aim of this study was to analyse the serogroup incidence and penicillin susceptibility in n. meningitidis before and after spanish epidemic outbreak in . in this year the public health service decided a massive vaccination in our sanitary area (galicia, north-west of spain, . inhabitants) and in autum of the inclusion of vaccine against n. meningitidis serogroup c in vaccination programme. methods: retrospective study of all cases of meningococcal disease confirmed by culture and/or pcr in the health care area of santiago de compostela (galicia) from to . results: in the period - we identified meningococcal disease episodes by microbiologic diagnosis ( . %, . % and . % to b, c and w respectively). in , serogroup c were the % of the isolates. in and the serogroup incidence was almost the same (b = . %, c = . %). from an increase in b serogroup cases were detected, in ( . %), in ( . %) and in ( . %). the most frecuent phenotype has been b p : . in c serogroup. during this period an increase in penicillin susceptibility was observed (in b serogroup % in and % in of the isolates were susceptible and in c serogroup % in and % in ). conclusions: the b serogroup is the most frecuent isolate during this period except in the years and . the strain that cause the epidemic outbreak in (c: b p : . ) was not isolated since . in our health care area, c: a serotype, was isolated for first time in , and since then, is the unique serotype isolated in c serogroup. incidence rate in c serogroup has changed from . / . in to . / . in . this decrease was caused by the drop of incidence rate on the youngest groups (< years and - years). the incidence rate in b serogroup during these years was modified from . / . in to . / . in . a four-year retrospective analysis of infective endocarditis in a belgian university hospital n. de visscher, b. delaere, b. krug, y. glupczynski (yvoir, be) objectives: to establish the epidemiology of infective endocarditis (ie) and determine the prognostic factors for adverse outcome in patients admitted to a university hospital with a cardiovascular surgery department. methods: between / and / , the clinical and laboratory features of all consecutive adult patients with a definite diagnosis of ie (duke criteria) were evaluated retrospectively by two infectious diseases physicians on the basis of clinical data charts and microbiological laboratory. results: patients ( men, women) presented with a definite diagnosis of ie. mean age was , yrs; cases ( %) were native valve endocarditis (nve) and ( %) were prosthetic valve endocarditis (pve); % of patients with nve had underlying valvular abnormalities ( bicuspidies, mitral prolapsus or regurgitation, others). ten out of cases of pve were late-onset episodes (> year after surgery). global mortality was % ( / pts), including patients ( %) still under antibiotic therapy. a higher mortality rate was observed in pve [ / ; ( %)] than in nve [ / ; ( %)]. overall, pts ( %) underwent surgery (mean: days following admission). valvular replacement was contra-indicated in pts because of critical status and/or major co-morbidities. the distribution of isolated pathogens was: streptococci: cases ( %) including cases of s. bovis, s. aureus: cases ( %, including mrsa), enterococci: cases ( %), miscellaneous: cases. the affected valves were: only aortic: ( %), only mitral: ( %), only tricuspidal: , aortic and mitral: , mitral and tricuspidal: , aortic, mitral and tricuspidal: . a high mortality rate was observed in s. aureus ie ( / [ %]), especially in the subgroup of patients with a pve ( / pts [ %] ). the mortality rate in patients with ie episodes caused by streptococci amounted % ( / pts). clinical microbiology and infection, volume , supplement , related, % (n = ) had previous surgery and % (n = ) were related to urinary or digestive tract procedures. only patients had illegal substance abuse. the most frequent predisposing acquired cardiac condition for native valve endocarditis was degenerative valvular disease in % ( / ). twelve percentage (n = ) had prior ie. the most frequent predisposing congenital cardiac condition was a bicuspid aortic valve in % (n = ). in % (n = ), no predisposing heart disease was discernible. causative microorganisms included: staphylococci in % (n = ) with s. aureus in % (n = ), cons in % (n = ), streptococci in % (n = ) with s. viridans in % (n = ), s. bovis in % (n = ), enterococci in % (n = ) and other pathogens in % (n = ). culture negative ie was reported in % (n = ). both in community-acquired and nosocomial ie, s. aureus was the most frequent causative agent. twenty-three percentage ( / ) were methicillin-resistant s. aureus. s. viridans ie was mainly community-acquired while enterococcal ie was nearly equally distributed between community and nosocomial origin. conclusion: compared to older series, we observed a high proportion of nosocomial ie and of prosthetic valve ie. s. aureus and e. faecalis were the most prevalent causative microorganisms. enterococci were nearly equally distributed between community and nosocomial origin, suggesting that nosocomial enterococcemia should be added as a major criterion, as proposed before for s. aureus. the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis: a meta-analysis of comparative trials m. falagas, d. matthaiou, p. papastamataki, i. bliziotis (athens, gr) objectives: the addition of an aminoglycoside to a beta-lactam for the treatment of patients with infective endocarditis has been supported mainly from data from laboratory and animal studies. we sought to review the evidence from the available comparative clinical trials regarding the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis due to gram-positive cocci. methods: the studies for our meta-analysis were retrieved from searches of the pubmed database and from references of relevant articles. included studies were trials that provided comparative data regarding the effectiveness of the treatment and/or mortality in patients receiving monotherapy with a betalactam or beta-lactam/aminoglycoside combination therapy. two independent reviewers performed the literature search, study selection, and extraction of data from relevant studies published in english during the period / - / . results: no clinical trial comparing beta-lactam monotherapy to beta-lactam/aminoglycoside combination therapy for the treatment of enterococcal endocarditis was found. we performed a meta-analysis of available comparative trials ( randomized controlled trials and comparative prospective trial) that included patients with bacterial endocarditis in native valves due to staphylococcus. aureus ( studies) or streptococcus viridans ( study). there was no statistically significant difference between the compared arms regarding mortality (or . , ci % . - . ), treatment success (or = . , ci % . - . ), treatment success without surgery (or = . , , and relapse of endocarditis (or = . , . nephrotoxicity was less common in the beta-lactam monotherapy arm compared to the beta-lactam/aminoglycoside combination therapy (or = . , ci % . - . , p = . ). conclusion: the limited evidence from the available prospective comparative studies does not offer support for the addition of an aminoglycoside to beta-lactam treatment of patients with endocarditis due to gram-positive cocci. a large multicenter randomized controlled trial may be necessary to reach a definitive conclusion on this issue. outpatient antimicrobial therapy for infective endocarditis. single-centre experience objectives: to evaluate the characteristics and outcome of infective endocarditis (ie) patients included in a outpatient antimicrobial therapy (opat) program. methods: from january to may all patients who received opat therapy for an ie were prospectively evaluated. inclusion in opat program require clinical stability and agreement of patients. active drug addiction was contraindicated for inclusion. antibiotic treatment was administered in bolus for once-daily antibiotics regimens. we used cadd-legacy tm plus (deltec, inc. st paul. usa) portable infusion system for either continuous or intermittentprogrammed bolus infusion. results: we included patients, male ( %), mean age years old (sd: . years). the diagnostic of ie was definite in cases ( with pathologic diagnosis), probable and possible. mostly of the cases were community-acquired ie ( %). mitral valve ie was the most frequent anatomical site involved ( %), followed by aortic ( %). native-valve ie represent the majority of cases ( %), but % were prostheticvalve and % were pacemaker lead ie. viridans group streptococci was the most frequent isolate ( patients, %) with cases of s. bovis ie. eleven patients had s. aureus ie ( %). at the time of the diagnosis, patients had valve rupture and patients had periannular abscess. a total of patients required some surgical intervention for the ie [ valvular replacement ( of them associated with aortic graft), pacemaker extraction and aortic graft]. the majority of the patients received outpatient monotherapy ( %). the most frequent antibiotic used was ceftriaxone ( % of the cases), followed by cloxacillin %, gentamycin %, vancomycin %, teicoplanin %, ampicillin % and other antibiotics in %. in % of the patients the vascular access was a perifericallyinserted venous central catheter and in % we used a portable infusion system. twelve patients ( %) had some complication during opat that require hospital readmission, of which could return to opat program. three patients had a fatal outcome (deaths) during admission, not related to ie complications. the mean duration of opat was . days per patient, and globally supposed . days of hospital admission savings. conclusion: opat for ie can be a good therapeutic option for ie stable patients. this procedure can represent a considerable amount of hospital admissions savings, improving also patients' well-being, and must be take into account for the treatment of this disease. objectives: botulism, a neuroparalytic illness, is caused by toxin produced by clostridium botulinum. food born botulism, a potentially lethal neuroparalytic disease, is caused by ingestion of preformed toxin. clinical illness is characterised by cranial nerve paralysis, followed by descending flaccid muscle paralysis. in this article we report a case series including a family group of type e botulism after ingestion of an iranian traditional soup. methods: in january , patients of a family group developed clinical manifestations of botulism - hours following ingestion of a traditional soup. their main clinical presentations were severe weakness ( . %- case) and lethargy ( . %- case). other signs and symptoms were blurred vision, fixed and dilated pupils, diplopia, dry mouth and decreased gag reflex. based on clinical finding, all patients received monovalent antitoxins (a, b, c). stool, gastric fluid and serum samples were sent for toxicological evaluation using the standard mouse bioassay. results: type e toxin was detected in the stool and serum sample of only one patient. all patients recovered and discharged one week after admission. conclusion: this study confirmed that prompt administration of antitoxin can prevent progression of disease based on clinical judgment and also may be life saving. in this case series study, we observed a short incubation period of - hours only in type e botulism. an outbreak of group g streptococcal pharyngitis among hospital personnel considered to be foodborne n. karabiber, a. gurbuz ertas, m. karahan, e. aykut arca, z.c. karahan, a. tekeli (ankara, tr) introduction: food-born outbreaks of streptococcal pharyngitis are relatively rarely reported,and while group a streptococci are the main causative agents, only a few epidemics caused by group g streptococci have been published. we describe here an outbreak of group g streptococcal pharyngitis occurred among the staff of a teaching hospital in ankara.the outbreak: an explosive outbreak of pharyngitis occured mainly among the staff working in certain departments (i.e. intensive care units, operation rooms) of tü rkiye yü ksek ihtisas teaching hospital, in january . methods: a total of ( and ; and from catering firm personel) throat cultures were evaluated in days,and bhs strains were isolated, and on the first and the second days, respectively. presumptive identification by nbacitracin and trimethoprim/sulfametoxazole disk diffusion test showed that strains were non-group a, strains were group a streptococci. in definite grouping by streptococcus grouping kit (avipath-strep,omega), strains were found to be lancefield group g, strains were found to be group a streptococci (gas). one of the gas strains was isolated from a catering staff on the first day, the other two were isolated from two health care personnels on the second day. during the outbreak, of catering firm personel ( %) were found to positive for group g streptococci. all the bhs tested were found sensitive to penicillin g and erythromycin by agar disc diffusion method. conclusions: the configuration of the epidemic curve suggested a common source of exposure. since respiratory spread of streptococci in such a rapid fashion would be highly unlikely and that of positive throat culture were from the staff of the catering firm that provide all the food services for the hospital, and that most of them were working at the departments in which the outbreak occurred, we considered that the outbreak might be food-borne. prompt treatment with penicillin all the ill personnel and -day holiday coming consequently january, terminated the outbreak. all the strains were cryopreserved for further typing studies.we are now typing these strains by pulsed field gel electophoresis (pfge) after digestion with smai restriction endonuclease. our initial results show that these strains are of the same origin. outbreak of acute gastroenteritis in an air force base in western greece e. jelastopulu, t. constantinidis, t. kolokotronis, d. venieri, g. komninou, c. bantias (patras, andravida, gr) objectives: on september , an operative training day at the air force base in western greece, soldiers and staff experienced an outbreak of acute gastroenteritis. the purpose of this study was to determine the causes of the outbreak and develop control measures. methods: following the assessment of descriptive epidemiology, a case-control analytic approach was utilized with randomly selected cases and controls. patients completed a questionnaire pertaining to the presence and severity of gastrointestinal disturbances, date and time of symptoms onset and consumption of food items served in the base on the implied training day. adequate questionnaire was administered to the controls. odds ratios were calculated and statistical significance was determined using x test. samples of food items were collected for bacteriological examination. results: the overall attack rate was at least % among the approximately attendees. the outbreak started abruptly in the late afternoon on september, peaked at midnight and ended about hours later. from the interviews and the analysis it was established that the lunch (beef, macaroni, tomato sauce and grated cheese) consumed several hours prior to onset of symptoms by affected military personnel was the likely source of the outbreak with a strong statistical association. there was only one subject who did not eat lunch. among the symptoms the most prominent were watery diarrhoea ( %) and abdominal pain ( %). relatively few indicated vomiting ( %) and nausea ( %). the mean incubation period was h. in the bacteriological examination, staphylococcus aureus was detected in a sample of raw beef and in two samples of grated cheese (rest-cheese from lunch and an unopened package). conclusion: the short incubation period with abrupt onset, the symptomatology and the short, self-limiting nature of the illness, are suggestive of gastroenteritis caused by an enterotoxin-producing bacterium. s. aureus is considered to be the most likely cause. although mortality and longer-term morbidity are uncommon with food poisoning caused by enterotoxin-producing bacteria, this outbreak highlights its capacity to cause short term, moderately-severe illness in a young and healthy population. it underscores the need for proper food handling practices and reinforces the importance of appropriate microbiological specimen collection from cases, as well as the public health importance of timely notification of such outbreaks. occurrence, characterisation and antimicrobial resistance pattern of staphylococcus aureus strains isolated from dairy products in southern italy g. la salandra, e. goffredo, c. pedarra, m.c. nardella, a. parisi, a. dambrosio, n.c. quaglia, g.v. celano, g. normanno (foggia, valenzano, it) objectives: the ingestion of food contaminated by enterotoxins (ses) synthesized by staphylococcus aureus is responsible of one of the most common foodborne diseases (staphylococcal food poisoning-sfp). since s. aureus is often involved in cases of subclinical mastitis of ruminants, milk may results contaminated. infact, the dairy products are frequently related to cases of sfp, expecially in areas characterized by a high level of consumption of these products. consequently an active microbiological surveillance is needed in order to control the risk of sfp and to allow the improvement of the public health standards. s. aureus also show a large antimicrobial resistance pattern. in this work are reported the results of a survey conducted on the occurrence of s. aureus in dairy products from apulia region (southern italy). furthermore, the isolated strains were characterized in order to determine their ability in synthesizing ses and to evaluate their antimicrobial resistance pattern. methods: samples of dairy products (milk, cheese, mozzarella cheese, ricotta cheeses) were analysed for the detection of s. aureus. the isolated strains were tested for the detection of ses, using the reverse passive latex agglutination test (sea to sed) and submitted to pcr to detect enta, entb, entc, entd and ente genes. furthermore, the strains were tested for susceptibility to ampicillin, tetracycline, gentamicin, eritromycin, enrofloxacin, co-trimoxazole, teicoplanin and vancomycin, by the agar diffusion method. results: out of samples analysed, ( . %) resulted contaminated with s. aureus and, among these, ( . %) have been recognized as enterotoxigenic strains ( samples of milk, samples of mozzarella cheese, samples of cheese from ovine milk and sample of cheese). all the strains tested (one per each positive sample) showed antimicrobial resistance properties but none of these was resistant to teicoplanin and vancomicin. conclusions: the results obtained from this survey show that milk and dairy products from southern italy are frequently contaminated by enterotoxigenic strains of s. aureus and highlighted the need to implement strict hygienic control measures along the food chain in order to decrease the risk of spf. furthermore, the presence of antimicrobial-resistant strains of s. aureus in food may be considered a source of communityacquired infections, with the direct risk of transfer of the antimicrobial-resistance to intestinal human microflora. objectives: infection accounts for about one-third of cases of fever of unknown origin (fuo), which remains a major diagnostic challenge. recently, f- -fluorodeoxyglucose (fdg) positron emission tomography (pet) has entered the field of clinical infectious diseases. fdg accumulates in tissues with a high rate of glycolysis, which is present in malignant cells and in all activated leukocytes. the aim of this prospective multi-centre study was to validate the use of fdg-pet as part of a structured diagnostic protocol in the general patient population with fuo. methods: from december to july , patients with fuo, defined according to the revised petersdorf criteria, were recruited from one university hospital and five community hospitals. a structured diagnostic protocol was used. fdg-pet was performed after certain obligatory laboratory tests, chest xray and abdominal ultrasound. the final clinical diagnosis was used for comparison with the fdg-pet results. results: a final diagnosis was established in patients ( %): infections, malignancies, non-infectious inflammatory disorders and miscellaneous causes. of the total number of fdg-pet-scans, % were helpful. positive predictive value of fdg-pet was % and negative predictive value was %. fdg-pet was helpful in all patients diagnosed with an infection except for one case of pyelonephritis. contribution of fdg-pet to the final diagnosis did not differ significantly between the university hospital and the community hospitals. fdg-pet was not helpful in any of the patients with normal erythrocyte sedimentation rate (esr) and c-reactive protein (crp). conclusion: in addition to the apparent value of fdg-pet in diagnosing different infectious diseases as described in several case series, fdg-pet is a valuable imaging technique as part of a diagnostic protocol in the general patient population with fuo and a raised esr or crp. based on previous studies comparing gallium- -citrate or labelled leukocyte scintigraphy and fdg-pet in patients with fuo and resulting from favourable characteristics of fdg-pet, conventional scintigraphic techniques may be replaced by fdg-pet in institutions where pet is available. emergence of clindamycin-resistant streptococcus pyogenes causing cellulitis epidemiology of viral respiratory infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus as a cause of community-acquired respiratory illness seroprevalence of human metapneumovirus in japan - . carriers into account when studying the dynamics of pneumococcal transmission and modelling the effect of pneumococcal vaccination in young children erythromycin-resistant streptococcus pneumoniae isolated in spain: serotypes, clones and mechanisms of resistance ( %) and f ( %) that accounted for % of eryr strains. among eryr strains ( %) had mlsb phenotype ( % constitutive and % inducible) and ( %) had m phenotype. the genes detected in mlsb isolates were: ermb in isolates, and ermb and mefe genes in isolates. all ( %) mlsb isolates with resistance to tet had tetm gene and of them had int gene (related to tn -like). seven positive ermb strains susceptible to tet had int gene spain f- , spain b- , sweden a- and st - f) accounted for % of these strains. capsular switching was observed in two clones, spain f- (serotypes f and a) and sweden a- (serotypes a, f suggesting the spread of tn -like elements. the ermb positive strains, related to spain f- , spain b- , sweden a- and st - f clones, were more frequently isolated in adults us) objective: to characterize changes in the frequency of occurrence of bacterial pathogens responsible for pneumonia in hospitalized patients in europe for the years - and examine select antimicrobial susceptibilities (s) for predominant pathogens. the emergence of resistance (r) among pathogens responsible for pneumonia has resulted in changes to empiric therapy, with increasing reliance upon third-and fourthgeneration cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, carbapenems and fluoroquinolones. methods: participating european medical centres ( - /year) referred consecutive, non-duplicate pathogens ( isolates) from lower respiratory tract sites determined to be significant by local criteria as the probable cause of pneumonia. all identified isolates were tested for s by the broth microdilution method and . %, respectively), increased significantly in ( . % s), and returned to near- levels in . esblphenotypes (cro or caz or aztreonam mic ‡ mg/l) remained essentially unchanged among ec between and ( . % and . %, respectively), whereas among ksp increases were more substantial ( . % and . %). metallobeta-lactamase-producing pa were identified during the study from italy vim- ) methods: four-hundred consecutive mrsa isolates were collected at centres (max. isolates per centre) as part of a multicentre study conducted throughout germany in . isolates were collected from various sources, including colonization sites as well as infectious foci. only one isolate per patient was included and all isolates were spa-genotyped. cps were determined by slide agglutination with cp-specific antibodies (anti-t -dt, anti-t -conjugate, anti- -repa). the serotypes were confirmed by immunodiffusion using lysostaphin-digested cell lysates. results: in the present study, we serotyped mrsa isolates collected most recently in a german multicentre study. all mrsa isolates evaluated were one of the serotypes tested invasive pneumococcal disease in adults in north-rhine westphalia methods: surveillance for our current study focused on north-rhine westphalia, the largest federal state in germany ( million inhabitants). ( . %) acute care hospitals microbiological laboratories serving these hospitals agreed to participate. we studied hospitalized patients older than years of age. a case of ipd was identified by the isolation of s. pneumoniae from an otherwise normally sterile site. isolates were verified for species diagnosis by optochin testing and bile solubility, and for serotyping by the neufeld quellung reaction. mics of penicillin g, amoxicillin, cefotaxime, cefpodoxime, cefuroxime, clarithromycin us) objectives: carbapenems are the most reliably active betalactam antibiotics against enterobacteriaceae and are often the treatment of choice for infections caused by multi-drug resistant isolates. while carbapenem resistance has occasionally been reported in enterobacteriaceae, there are limited data on its frequency and distribution. methods: two large ongoing surveillance databases were searched for imipenem (imp) and ertapenem (etp) resistance in enterobacteriaceae: smart (study for monitoring antimicrobial resistance trends), a worldwide program focusing on community-and hospital-acquired intra-abdominal pathogens, and iss (icu surveillance survey), a us program focusing on icu isolates from any sterile body site. results: the overall frequencies of carbapenem-resistant enterobacteriaceae remained < % in smart and < % in iss throughout the periods of observation (see table). for % of esbl producing k. pneumoniae and e. coli were resistant to etp or imp and rates varied by geographic region. all isolates studied to date have exhibited multiple resistance mechanisms. conclusion: carbapenem resistance was uncommon among clinical isolates of enterobacteriaceae in these surveillance studies. its observed frequency varied by species and geographic region no significant seasonal variability in the prevalence of emergence of streptococcus b-haemolyticus strains in swabs was observed. conclusions: . seasonal fluctuations of pharyngeal discussion: with regard to high prevalences of giardiasis and enterobiasis it increase the prevalence that intestinal parasitic infections. it is suggested to decrease the rate of these parasitic infections in the region by strict programes that help to increase the knowledge of students, their parents and teachers about hygen. results: the study included patients, with a mean age of . ± . . ( . %) were women. the predisposing factors were: renal lithiasis patients ( . %), prostatic adenoma ( . %), vesical structure disease ( . %), vesical functional disorder ( . %), chronic kidney failure ( . %). the underlying diseases included: diabetes mellitus ( . %), immunosuppression ( . %), previous urinary tract instrumentation ( . %), permanent catheter ( . %). the mean hospital stay was . ± . days. the mean duration of symptoms was . ± . days the absence of leukocyturia or mictional syndrome does not exclude the presence of complicated upper uti. ) the high percentage of bacteriemia necessitates blood cultures, with e. coli being the most common pathogen. ) the associated morbidity and mortality are important in association with sepsis or septic shock gr) objectives: to estimate the incidence of streptococci in community acquired urinary tract infections (uti) and also to carry out the in vitro antibiotic resistance of streptococci in urinary tract infections %) were enterococcus avium, ( . %) were enterococcus gallinarum and ( . %) were streptococci group b. the in vitro antibiotic resistance of enterococcus faecalis was: penicillin g . %, ampicillin . %, gentamicin %, streptomycin . %, nitrofurantoin %, ciprofloxacin . %, tetracyclines . %, vancomycin . %, linezolid %. the in vitro antibiotic resistance of enterococcus faecium was: penicillin g %, ampicillin . % tremolieres for the french aup study group background: short-course therapy for acute uncomplicated pyelonephritis (aup) is the newly suggested standard. talan et al. have demonstrated that oral ciprofloxacin (cip) for days, was associated with greater cure rates than a -day trimethoprim-sulfamethxazole regimen. we assessed efficacy and tolerance of a -d. regimen of cip (study i), then of levofloxacin (lvx) in study ii results: of (i) and (ii) enrolled pts. aged . ± . y., and , aged . ± . y. were retained for itt analysis; . % and . % had positive blood cultures. escherichia coli was the uropathogen in . % and % of cases. finally (i) and (ii) were retained for per protocol (pp) analysis. at v bacterial eradication rates were . % and . %. global cure rates were . and . % at v and . % and . % at v with only less than % of lost to follow-up between v and v in both cases. side effects were observed in . % and . % of pts. who received or more fq doses. conclusions: aup treatment with lvx mg hiv-infected patients and drug addicts were excluded. antimicrobial susceptibilities of all s. pyogenes isolates were studied by microdilution method, and macrolide resistance phenotype by double disc test. macrolide resistance genes were detected by pcr. results: over the -year study period, there were episodes of cellulitis. the infection was microbiologically ). of note, all cases of cellulitis due to clindamycin-resistant strains occurred during the last years of the study. five ( %) patients presented with stss and died ( due to an erythromycin-resistant strain). overall mortality (< days) was % this resistance might become a problem when treating s. pyogenes infections, especially stss cases. p risk factors for community-acquired bacteraemic gram-negative cellulitis an administrative database was used. then we selected the patients with blood cultures obtained at the time of the cellulitis episode using the microbiology laboratory database. nosocomial cellulitis were excluded. a standardized data collection form was used to review the hospital records. in statistical analyses, student's t test was used for the comparison of mean values and chi square test and fisher's exact test for the comparison of categorical data (two tailed). results: of the patients with limb cellulitis identified in the study period, patients had blood cultures and were selected for the analysis. bacteremia was detected in of the patients ( . %), of them due to gram-negative microorganisms hemorrhagic rash was present in . % cases. koplick spot was found in . % cases. measles was associated with streptococcal tonsillitis in . % cases, with oral candidiasis in . % cases and with pulmonary tuberculosis in . % cases. severe forms of evolution were observed in complicated cases with: encephalitis ( . %) or bronchopneumonia ( . %), which required intensive care unit survey. we registered only one deceased, in a case of measles encephalitis in gipsy collectivities even it's very difficult, it's necessary to was performed. respiratory samples were tested routinely for twelve common respiratory pathogens. results: over the study period, of samples processed, -six cases were community-acquired and ( %) patients had significant co-morbidities. cough was the predominant reported symptom. chest x-ray was performed in cases, of which showed abnormalities. bronchiolitis ( / ) was the commonest initial clinical diagnosis. the majority ( %) of patients received antibiotic therapy, but a convincing bacterial pathogen was isolated in less than half of these cases. thirty patients were admitted for management. more than one virus was identified from patients, with rhinovirus being the predominant co-infection. overall, the average length of stay was . days. however, where hmpv was the sole pathogen identified, average length of stay was . days. conclusion: our data suggests that hmpv infections are more common in children with underlying co-morbidities. the rate of radiological imaging was higher than expected and perhaps is a reflection of the patient population or the degree of severity of illness. nosocomial acquisition occurred in cases, which has implications for patient cohorting acknowledgements: the financial support for this study was provided by kuwait university research grant / . evaluation of infection control practices in haematopoietic stem-cell transplant facilities in german-speaking countries: variation of measures reflects lacking evidence s. wenzler-rö ttele, a. conrad, w.v. kern, h. bertz, f.d. daschner, m. dettenkofer (freiburg, de) objective: haematopoietic stem cell transplant (hsct) recipients are highly immunocompromised during pre-and postengraftment. thus, they are cared for in specialised facilities and versatile precautions are practised in order to prevent nosocomial infections. however, there is a lack of evidence whether these interventions are effective. furthermore, most of the measures are cost-intensive and restrict the patients' comfort. for evaluation of precautions, a survey was performed to assess the spectrum of measures commonly practised. methods: a questionnaire was compiled asking in detail for infection control measures differing according to allogeneic and autologous hsct recipients. the questionnaire was sent to hsct facilities in germany, austria and switzerland. results: questionnaires ( %) were filled in and sent back. among the centres, were university hospitals, and teaching hospitals. the overall number of transplantations that were performed by the facilities varied considerably and ranged from to /y for auto hscts and from to /y for allo hscts. % of the institutions performing allo and auto hsct have implemented different precaution standards for each group. some measures regarding allo hsct were routinely adhered to in practically all institutions: accommodation in single rooms ( %), interdiction of plants and opening of windows ( % each) and protection from waterborne bacteria by use of terminal tap water filters ( %). % of hsct facilities perform their allo transplantations in hepa-filtered rooms and % are providing laminar air-flow for this population. there was a broad spectrum of different measures regarding barrier precautions: gowns when entering the room (required in % of centres for allo and % for auto hsct) and face masks ( % allo and % auto hsct). precautions to be followed by the patient varied among centres, e.g. specification of the face mask/respirator to be worn outside the isolation room (for allo hsct: % surgical mask, % ffp , % ffp and % ffp ). conclusion: the broad variety of different preventive measures performed by the different facilities reflects lacking evidence for many infection control precautions that are commonly practised in the care of hsct recipients. this survey provides the basis for further studies within the onko-kiss project (hospital infection surveillance system for patients with haematologic/ oncologic malignancies). objectives: in this study it was our aim to evaluate the microbiological contamination of physiological serum flasks in use in medical day center for wound cleaning and to identify the isolated microorganisms. methods: we have collected saline solutions from health care centres localized in the health sub-region of coimbra. from each centre we have recovered aleatory flasks in current use.the samples were transported at ordm;c and maintained at this temperature until its processing. saline solutions were seeded by the pour-plate technique in plate count agar and plates incubated at and ordm;c for h. the saline solutions were evenly spread over the surface of blood agar and sabouraud chloramphenicol agar (sab chl-d). the transfers of saline solutions flasks were also tested for microbiological contamination with a sterile cotton swab that was rubbed vigorously, over the transfer surface and directly applied on blood agar media. blood agar plates were incubated at °c for h and sab chl-d plates were incubated at °c and °c and examined daily for a period of days before declared as culture negative. microbial identification was firstly accomplished by employing conventional morphological and biochemical tests. when identification was not possible by these methods, s rrna gene sequence determination and phylogenetic analysis were used for bacterial strains and in the case of moulds we performed the amplification and sequencing of internal transcriber spacers region of . s gene. results: from the saline solutions analysed, . % were contaminated. a total of strains were isolated, % could be identified to species level using morphological and biochemical tests, the remaining % were identified by gene amplification and sequencing. about . % of the identified strains were gram-positive cocci, the second dominant type of strain were gram-positive bacilli ( %), and the third dominant type of strains were gram-negative bacilli and moulds, both with . %. the most frequent contaminants belong to human normal flora ( %), supporting the idea that the source of contamination of saline solutions analysed was human, in contrast with % of contamination due to the environment. conclusions: the contamination of the saline solutions is due to inadequate clinical practices. these results claim for more strict hygienic measures and for the replacement of big flasks by single use flasks with an incorporated overture used for wound irrigation. frequencies of cmv-ie specific memory t cells are inversely correlated with alloimmune memory and serum creatinine in kidney transplant patients p. nickel, g. bold, f. presber, c. schö nemann, j. pratschke, d. bitti, f. kern, h.-d. volk, p. reinke (berlin, de) background/aims: cytomegalovirus infection is a significant cause of morbidity in transplant patients and has been associated with allograft rejection. in this study frequencies of ifng-producing t cells following ex-vivo stimulation with protein-spanning peptide pools for cmv proteins pp and ie as well as donor-reactive t cell frequencies were serially determined during the first months after renal transplantation (tx) to analyse the relation of cmv specific t cells, virus control and alloimmunity. patients: kidney transplant recipients were included. immunosuppression generally consisted of anti-il- r mab, calcineurin inhibitor, mmf and steroids. presensitized patients received an induction by x low dose okt- , anti-tnf mab, anti-cd mab and x plasmapheresis. patients received fty- , cyclosporine and steroids. methods: pbmcs from renal transplant recipients were analysed in a computer-assisted elispot assay before and at multiple times (mean ) post-transplantationfor ifn-yproducing t cells following in-vitro stimulation for hrs by irradiated donor cells and pools of overlapping peptides conclusions: although temporary r declines were seen among some european pneumonia pathogens, all showed increasing r to most class agents during the study period. the increase in esbl among enterobacteriaceae, and r among pa to most agents except polymyxin b, are especially worrisome. continued longitudinal comparisons of emerging pathogens and changing susceptibility profiles are critical elements in guiding empiric therapies and epidemiologic interventions. week, all participating hospital inpatients were swabbed on three anatomical sites: throat, nose and groin. we investigated the molecular epidemiology of the mrsa isolates collected from patients in hospitals using the pfge method with the smai restriction enzyme. cluster analysis was carried out using bionumerics software. band-based similarity dice coefficients were used for dendrogram construction, which provides a quantitative assessment of strain similarity. samples were defined to belong to a cluster using a similarity coefficient of % or higher. pfge profiles were compared with the most similar strains from the harmony iums global mrsa database.results: different restriction profiles were observed among the mrsa isolates and patients. isolates from the same patient but from different anatomical sites had similar pfge profiles. clusters of mrsa strains could be identified with the two largest clusters containing ( %) and ( %) patients, respectively. strains from these major clonal clusters occurred in and out of the hospitals, respectively. isolates from the cluster with patients were most similar to the well-known iberian clone: france a ( strains), belgium e ( strains), france b, france c and northern germany i ( strain each). isolates from the next largest cluster of patients correlated with a group of strains previously found in finland and belgium: belgium e ( strains), finland e ( strains) and finland e ( strain). the remaining strains were most closely related to belgium ec ( strains), berlin iv ( strain), southern germany ii ( strains) and uk e ( strains). conclusion: two major clonal clusters of mrsa strains were found to be dominant among hospitals inpatients in luxembourg. the molecular diversity of circulating strains was fairly diverse and profiles were very similar to previously described patterns in neighbouring countries and europe. further sequence-based genotyping is warranted to gain a better understanding of the clonal structure and elucidate transmission patterns. enterococci were identified by basic tests and by pcr amplification of ddl genes. susceptibility testing was performed using the icls broth microdilution method. resistance genes were detected by pcr, selected vana, vanb and vanc amplicons were sequenced. macrorestriction analysis (smai) resolved by pulsed-field gel electrophoresis (pfge) was performed. results: during the study vre isolates with different phenotypes of resistance to glycopeptides were obtained from specimens. the prevalence of vre in the gastrointestinal tract was . %. one e. faecalis (isolated from patient arrived from us) and e. faecium isolates, harbouring vana genes, demonstrated mic's of vancomycin (van) and teicoplanin (tec) - and - mg/l respectively. three e. faecium and four e. gallinarum isolates were vanb-positive, with van and tec mic's > and . - mg/l respectively. all stains were susceptible to linezolide. among e. faecium isolates with vana genes one predominant pegf type was observed, differentiated in nine pegf sub-types. each of three other pegf types detected seemed to be unique. among six vana genes sequenced, four demonstrated similarity to vana gene from e. faecium (genebank af ) and two to -vana gene from e. faecalis (genebank ay ). in two sequenced vanb genes from in e. gallinarum nucleotide substitutions, resulting in seven new amino acid substitutions, were detected. conclusions: heterogeneity of glycopeptide-resistance genes, circulating in haematological centre, leads to the conclusion that their spread is not a local phenomenon. spread of vre is an emerging and, possibly, underestimated problem for russia. study of resistance and clonal relatedness of clinical isolates of stenotrophomonas maltophilia from a hospital in northern spain c. valderrey, e. sevillano, f. calvo, l. gallego (bilbao, es) objectives: the aim of this work was to study the antibiotic resistance and genetic relatedness among clinical isolates of s. maltophilia isolated from patients with tract respiratory infections. methods: the study included s. maltophilia isolates obtained in a hospital from bilbao (northern spain) during (from january to october). susceptibility to antimicrobial agents was determined by the disk diffusion method following the nccls recommendations. the antibiotics tested were imipenem, meropenem, cefotaxime, ceftazidime, cefepime, aztreonam, amikacin, tobramicin, ciprofloxacin, ofloxacin and trimethroprim/ sulfamethoxazole. total dna was used as target for pcrfingerprinting experiments with primers rd , eric , ap , m and rnar and . to detect class integrons, primers cs and cs were used in amplification experiments.results: resistance to antibiotics tested was the following: imipenem ( %), meropenem ( %), cefotaxime ( %), ceftazidime ( %), cefepime ( %), aztreonam ( %), amikacin ( %), tobramicin ( %), ciprofloxacin ( %), ofloxacin ( %) and trimethroprim/sulfamethoxazole ( %). pcr-fingerprinting technique was only useful when eric primer was used identifying distinct genotypes. the other primers were not able to produce reliable band patterns. patients with several isolates maintained the same clone along time, although there are two patients from which two different genotypes have been isolated, and two clones that have been isolated from more than one patient. class integrons were detected in % of isolates ranging in size from of to bp ( isolates bored combinations of two structures). conclusions: trimethroprim/sulfamethoxazole and amikacin showed the best activity against the isolates tested. for pcrfingerprinting experiments the best primer was eric which produced reliable and reproducible band patterns. there was a high clonal diversity since different genotypes were identified among the patients included in the study. many isolates bored class integrons with sizes similar to those detected in other nonfermenters bacilli from the same environment.methods: -identification: the strains were identified by colonial morphology, haemolysis on blood agar plates, biochemistral and antigenic identification; antibiotic susceptibility testing: all the strains were tested by disk diffusion according to the national committee for clinical laboratory standard methods. mics were determinated by screening test or mic evaluation in solid media. results: our study concerns bacterial strains isolated from january to june . among the strains isolated, neisseria meningitidis represented the most number of cases with . %.these were distributed among all different age groups. serogroup a was the most predominant and represented . % of total strains while groups b and c represented . % and . % respectively. streptococcus. pneumoniae represents the second causes of purulent meningitis with . % while haemophilus. influenzae b is the third causative bacterial agent with . %.this last agent is most predominant among infants less than years of age in % of cases. neisseria. meningitis is susceptible to all types of antibiotics tested. however, haemophilus. influenzae b produced an inactivating enzyme (penicillinase) in . % of cases. the resistance was associated to cotrimoxazole in . % of cases. the results of mic done on streptococcus. pneumoniae show that . % of strains has an intermediate resistance to penicillin and high level of resistance in . %. the amoxycillin is active in . % of the strains,in the opposite cefotaxim has an intermediate resistance in . % and a high level of resistance in . % of the strains. the resistance to penicillin was associated with resistance to erythromycin, cotrimoxazole or to both in some cases. conclusion: streptococcus. pneumoniae represents the second causative bacterial agent responsible of purulent meningitis and showed an increasing prevalence of resistance profiles to penicillin and cefotaxim in our hospital. this implicates an effective microbiological and epidemiological control.conclusion: as expected in a referral hospital with a cardiac surgery department, the prevalence of s. aureus ie was elevated as well as the attributable mortality rate. the high global mortality rate may be explained by the high frequency of severe co-morbidities and by the late referral of patients to hospital. our data suggest that there is room for improvement in the diagnosis and management of ie in a multidisciplinary collaborative approach. objective: to determine the clinical, epidemiological, diagnostic, and therapeutic characteristics of a series of cases of prosthetic valve endocarditis. methods: we undertook a retrospective, descriptive study of cases of prosthetic valve endocarditis obtained from a series of definite or probable left sided infectious endocarditis from six second-or third-level andalusian hospitals from to . results: of the cases of prosthetic valve endocarditis, ( . %) were definite and ( . %) possible. the mean age was ± years, and they were more common in men ( %). late infection was more common than early involvement ( vs. cases). the aortic valve was involved in cases ( %) and the mitral valve in cases ( %. most ( %) of the valves were made of metal and prior handling had taken place in cases ( %). clinical characteristics were fever %, constitutional syndrome %, murmur %, vascular events %, and immune phenomena %. complications included left ventricular failure %, kidney failure %, peripheral embolism %, cns embolisms % and heart block %. the etiology was as follows: in early prosthetic valve endocarditis the three most common pathogens were s. coagulase-negative ( %), s. aureus ( %) and enterococcus ( %). late prosthetic valve endocarditis involved s. viridans ( %), s. coagulase-negative ( %) and s. aureus ( %). transesophageal echocardiography alone in cases ( %), and transthoracic followed by transesophageal echocardiography in cases ( %). medical therapy was applied in cases ( . %) and surgery in ( . %). a cure was achieved in cases ( %), the other ( %) dying. of those who underwent surgery, . % died and . % of those who were treated medically died. the death rate from early prosthetic valve endocarditis was greater than that for late prosthetic valve endocarditis ( % vs. %). conclusions: ) prosthetic valve endocarditis is a very serious infection which is still associated with an excessively high mortality, despite advances in diagnosis and treatment. ) early prosthetic valve endocarditis has a worse prognosis than late prosthetic valve endocarditis, due to its distinguishing pathophysiological features. ) the greater mortality seen in patients who underwent surgery is probably associated with the fact that they had more complications, such as perivalvular abscesses or persistent infection. outcome of infective endocarditis e.e. hill, s. vanderschueren, p. herijgers, m-c. herregods, p. claus, w.e. peetermans (leuven, be)objectives: despite progress in diagnosis and therapy, almost half of patients with infective endocarditis (ie) has at least one complication and overall mortality remains high. the aim of the present -year prospective observational study was to define predictors of outcome in patients with ie. methods: from june through december , all first episodes of definite ie by the modified duke criteria, encountered in a single tertiary-care medical center, were registered and followed-up for months. results: overall, patients suffered ie episodes. sixtyone percentage were males. the median age was years (range - ). fifty-five percentage of episodes were referred from another hospital. at least one complication occurred in %. surgical intervention was performed in % and was mainly indicated because of congestive heart failure. the median time from diagnosis to surgery was days (range - ). six-months mortality was % (n = ). in bivariable analyses, factors associated with -months mortality were: age, female gender, causative microorganism, nidus of infection and therapeutic policy. six-months mortality was % for native valve ie and % for prosthetic valve ie; twenty-five% for nosocomial ie and % for community-acquired ie. six-months mortality rates for microorganisms were: staphylococci % (n = ) [s. aureus % (n = ) and cons % (n = )], enterococci % (n = ), streptococci % (n = ) and other microorganisms % (n = ). the -months mortality for patients with a contraindication to surgery was % (n = ), for patients conservatively treated without a contraindication % (n = ) and for combined surgical-medical treatment % (n = ). in multivariable logistic regression predictors of -months mortality were age (or, . ; % ci, . - . ; p = . ), causative microorganism (or, . ; % ci, . - ; p = . ) and a contraindication to surgery (or, . ; % ci, . - ; p < . ). conclusion: in the present prospective single centre study of patients with definite ie, -months mortality rate was . , and was especially high in patients with preestablished contraindications to surgery, in the elderly and in patients with staphylococcal ie. six-months mortality in patients with combined surgical-medical treatment versus exclusively medical therapy in patients without a contraindication to surgery was not statistically significant. staphylococcal and enterococcal ie had a worse prognosis compared to streptococcal ie. epidemiology and aetiology of infective endocarditis e.e. hill, p. claus, m-c. herregods, p. herijgers, s. vanderschueren, w.e. peetermans (leuven, be)objectives: the epidemiological features of infective endocarditis (ie) have changed. we report the results of a -year prospective observational study investigating trends in the epidemiology and etiology of ie. methods: from june through december , we registered definite ie episodes according to the modified duke criteria in patients older than years, hospitalized in a single tertiary-care center. results: sixty-one% of episodes involved males. the median age was years (range - ).fifty-five percentage (n = ) were referred from another hospital. forty-four percentage (n = ) were nosocomial. thirty-four percentage (n = ) involved prosthetic valves and % (n = ) thereof were of early postoperative onset. the mitral valve was most frequently involved. exposure to ie risk factors during the previous months was recorded in % (n = ) of the episodes. twenty-four percentage (n = ) were intravascular catheter- objective: to determine the eco-epidemiology of cryptosporidiosis in the health services executive -western area (formerly the western health board).concerns about the incidence of cryptosporidiosis in the western area prompted the department of public health to undertake further investigation of potential links between cryptosporidiosis and environment by focusing on farming activity and water supplies in the first instance. background: cryptosporidiosis was not notifiable in the republic of ireland prior to , unless cited as a cause of gastroenteritis in a child less than two years old. as a result the incidence of cryptosporidiosis in the republic of ireland at the time was unknown. nationally it was estimated that up to % of cases of gastroenteritis in children less than two years old could be attributed to cryptosporidium. in the western area from to the proportion of cases of gastroenteritis in children less than two years old attributable to cryptosporidium ranged from . % to . %. this was cause for concern.many rural locations in the western area are served by voluntarily-operated water schemes. water quality from these schemes is often microbiologically unsatisfactory. the department of public health methods: initial research involved analysis of notification records for cases of cryptosporidiosis received from to inclusive. crude incidence rates for cryptosporidiosis in the western area were compared with crude incidence rates in england & wales, northern ireland, and scotland for the same time period. cases of cryptosporidiosis from the western area were geo-coded and mapped to visualize the geographic spread of cases, and are being contrasted with geographic data for farming activity, and also with available data on water supplies. the results of the initial phase of this research indicated the incidence of cryptosporidiosis in the western area may be cause for concern. the geographic spread of cases and potential links to farming practices and water supplies will be presented. objective: the evaluation of epidemiology and seasonal fluctactions of bacterial flora in pharyngeal swabs taken from family doctors' patients. material and methods: a total of of positive pharyngeal swabs ordered by primary care physicians from silesia were examined during the - period. the microbiological analysis was performed in silesian analytic laboratories. the intake of material, its transport and final identification complied with laboratory standards. results: the most common pathogens were, in order of prevalence: streptococcus viridans ( . %), moraxella catarrhalis ( . %), staphylococcus aureus along with mrsa ( . %) and mrsa alone ( . %), e. coli ( . %), klebsiella pneumoniae ( . %), streptococcus b. haemoliticus ( . %). candida albicans was identified in . % of positive specimens. considering seasonal fluctuation, the number of positive swabs in each month tended to gradually increase in spring with its culmination in may ( . %). as for the most common pathogens streptococcus viridans and moraxella catarrhalis mirrored the general tendency and dominating in spring season (up to . and . %, respectively) and having less stronger impact in automn (up to . and . %). the frequency of isolation of the other pathogens revealed seasonal fluctuations confined to either spring, as in the case of klebsiella pneumoniae, escherichia coli and staphylococcus aureus strains (up to . , . clinical microbiology and infection, volume , supplement , aim: the aim of this study was to identify the microorganisms isolated from corneal and conjuntival samples, isolated from patients attending the ophtalmology department of a spanish hospital. material and methods: a total of corneal scrapes and conjunctival swabs were obtained since october of to october of in an university hospital of madrid. samples were cultured into blood and chocolate agar plates and incubated at ordm;c in o and co atmospheres, respectively, for two days (conjunctival swabs) and fifteen days (corneal scrapes). identification and susceptibility tests were performed following standard methodology. results: thirty four ( . %) out of corneal samples and ( . %) out of conjunctival swabs yielded positive cultures, respectively. results are summarized in the following table:conclusions : corneal scrapes yielded a higher number of positive cultures than conjunctival swabs. gram-positive microorganisms were more prevalent both from corneal scrapes and conjuntival swabs although the difference was more evident in corneal scrapes. s. aureus was the specie most prevalent in conjunctival samples meanwhile cns were the most prevalent in corneal scrapes. methods: vitreous fluid samples (n = ) were obtained from patients ( male, female) undergoing vitrectomy for endophthalmitis between january and october . specimens of undiluted aqueous and vitreous fluid were cultured for aerobic, anaerobic bacteria and fungi by conventional methods. identification and antibiotic susceptibility were performed by the api system, vitek ii system (biomerieux) and the agar disk diffusion methods according to clsi recommendations. results: ninety one isolates were recovered from the samples. gram stain was positive in / ( . %), while cultures were positive in / ( . %) samples. gram-positive bacteria were the most common isolates ( / , %), followed by gramnegative bacteria ( / , %) and fungi ( / , %). staphylococci coagulase-negative were isolated in / ( %). the next most common species isolated among gram-positive bacteria were s. aureus ( . %), streptococcus spp ( . %), propionibacterium acnes ( . %), bacillus spp ( . %), streptococcus. pneumoniae ( %) and enterococcus faecalis ( %). among gramnegative bacteria eight isolates were enterobacteriaceae, two were non fermenters and one was haemophilus inlfuenzae. two of the fungal isolates were candida albicans, one acremonium spp and six aspergillus fumigatus. polymicrobial growth was observed in six patients with two at least isolates. of staphylococci coagulase-negative / ( %) were resistant to methicillin. only one strain of staphylococcus aureus was methicillin resistant. all gram positive isolates were susceptible to vancomycin. all isolates were sensitive to amikacine and ceftazidime while resistance was observed in / ( %) isolates to fluoroquinolones. conclusion: a variety of microorganisms was isolated from the vitreous fluid of patients. the predominant isolates were grampositive bacteria, especially staphylococci coagulase-negative with low resistance rate to methicillin. so, therapy should be based on the isolation and identification of the infecting agent and the in vitro antibiotic susceptibility to the appropriate antibiotics. the prevalence of intestinal parasitic infection in the students of primary schools in nazloo region in urmia during [ ] [ ] k. hazrati tappeh (urmia, ir)background: intestinal parasitic infections are of the most important hygienic and economical problems of millions of people in all over the world, mostly from developing countries. understanding their epidemiological situation and relation to environmental and social factors is necessary for struggling with them in every society. this investigation was designed to study the prevalence of parasitic intestinal infections among primary school attending students in nazloo region of urmia district in . materials and methods: students were chosen randomly from schools upon their population. having their questionnaires filled, two faecal samples were taken from each student and examined with direct wet mount and formalinether sedimentation technique. scotch tape was also applied in order to detect the enterobiasis and taeniasis. students completed the test. all infected persons by e. vemicularis, h. nana were treated by mebandazole and giardia lamblia were treated by metronidazole. results: overall prevalence of parasitic protozoan infections was . %. giardia lamblia was found in cases ( . %), entamoeba coli in cases ( %) and blastocystis hominis in cases legionella pneumophila as an occupational risk factor for inter-city bus drivers y. polat, Ç . ergin, i. kaleli, a. pinar (denizli, ankara, tr)objectives: legionellaceae are ubiquitous aquatic microorganisms that usually isolated from evaporative condensers. various man-made sources such as cooling towers, whirlpools and spas are sources for legionella pneumophila. in hot climate, bus air-conditioning and aircirculating systems are possible sources for the organism. in this study, serologic status of bus drivers and their assistants for legionella infections as well as bus air-conditoner moisture exit samples for legionella species were investigated.methods: serum samples were collected from bus drivers (n = ) and their assistants (n = ). samples were tested for anti-legionella antibodies by indirect immunofluorescence technique. / dilution was accepted as a positive result for anti-legionella pneumophila antibodies. results were analysed according to risk factors based on hot/cold climate route (aegean and mediterranean parts of the turkey were accepted as hot climate region), immundeficiency, chronic diseases and work hours. according to serologic test results, air-conditioners of buses which has been driven by / dilution seropositive persons, were investigated. air-conditioner moisture exit samples were cultured on bcye-alpha agar supplied with bmpa. same samples were tested by pcr targeting a -bp fragment of the s rrna gene of legionella. results: anti-legionella pneumophila antibodies were positive in ( . %) bus-persons. bus drivers' seropositivity was higher than assistants (p < . ). in hot climate route, seropositivity was higher than cold climate route (p < . ). no positive pcr result was detected. coclusion: in conclusion, higher seropositivity rates in bus drivers were pointed out a newer occupational risk factor for legionellosis. although pcr positivity was not detected for bus air-conditioners, high seropositivity rates show that bus drivers have been somehow exposed to legionella. further legionellosis surveillance studies for bus drivers may help to understand legionella exposure during travel. objective: asymptomatic bacteriuria is an important risk factor contributing to pyelonephritis and renal disfunction in diabetic patients. in this study, the relationship between microalbuminuria and age, body mass index, duration of the disease, the level of glycohemoglobin, glycosuria and glomerular filtration rate is studied prospectively in diabetic patients who have asymptomatic bacteriuria. methods: a hundred and twenty-three type diabetic outpatients who were admitted to baskent university konya medical and research center between january-october were included in the study. ages of the patients were within the range of - years. the diagnosis of asymptomatic bacteriuria was established according to the cdc criteria. concurrent samples for urinary culture, glomerular filtration rate, microalbuminuria and glycohemoglobin were obtained. results: twenty-two of ( . %) patients had significant bacteriuria. of these patients % were female. although age, body mass index, creatinine clearence and presence of microalbuminuria were similar, there was a significant difference in glycohemoglobin levels, duration of diabetes and glycosuria between the two groups (p < . ). e. coli was the most common microorganism obtained from urinary samples. risk factors for asymptomatic bacteriuria were shown in the table. conclusion: the frequency of asymtomatic bacteriuria was found to be similar with the previous studies. high glycohemoglobin levels and long duration of diabetes were found to be the risk factors contributing to asymptomatic bacteriuria in type diabetic patients. descriptive study of complicated pyelonephritis objective: the evaluation of prevalence and contributory factors associated with the development of urinary tract diseases among women with urinary incontinence. material and methods: women aged from to years had their urine culture examination performed. the material was taken from the central stream of first catch urine and transported on uromedium. antibiogram was carried out with the use of becton-dicinson's discs. results: in cases the urine culture tested positively which accounted for . % of subject women. the most common pathogens of urinary tract were, in order of prevalence:e. coli- . %, staphylococcus aureus - . %,citrobacter diversus- . % and klebsiella pneumoniae- . %. candida albicans strains were isolated in one patient. e. coli had the highest sensitivity to norfloxacin - % and cefuroxim - %, amoxicillin with clavulonian acid - . %, ampicillin nitrofurantoin and trimethiprim -sulfamethoxazole - . % in each case, cefalothin - . %, tetracycline - . %, and amikacin - . % but only in . % to amoxacillin. staphylococcus aureus proved sensitivity only to gentamicin ( %) and nitrofurantoin ( %). in the case of citrobacter diversus % sensivity to norfloxacin, nitrofurantoin, tetracycline, trimethoprim / sulfamethoxazole, ceftazidim and cefotaksym was confirmed.klebsiella pneumoniae also proved sensitivity to amoxicillin with clavulonian acid, cefuroksime, nitrofurantoin, norfloxacine, tetracyclin and trimethoprim / sulfamethoxazole. when considering the sensitivity of pathogens to antibiotics in the family practise setting of higher reliabilty are nitrofurantoina, norfloksacyna.after the administration of guided therapy complete release from symptoms was observed in women ( %).conclusions: women with urinary incontinence relatively seldom suffer from urinary tract infections. the most common pathogen among women with urinary incontinence was e. coli sensitive to floxacins and cephalosporins but with impaired reaction to amoxycillin. incidence and in vitro antibiotic resistance of streptococci in community-acquired urinary tract infections uncomplicated community-acquired urinary tract infections (ca-utis) and non-pregnant women in london hospital in kuwait over a period of two years. methods: eighty-six pregnant and non-pregnant women with signs of ca-utis were enrolled in the study. the strains isolated from the patients who had significant bacteriuria were included in the microbiological analyses. the identification of the strains was performed using the api e system (biomerieux), while their susceptibility was determined by disk diffusion method. the interpretation of the results was realized according to nccls guidelines. quality control was performed using reference strain e. coli atcc . oserotyping was carried out with polyvalent and monovalent antisera. hemolysin production was tested on human blood agar plates. possession of k antigen by e. coli was tested with agglutination by murine monoclonal antibodies to the group b meningococcal capsule. results: we found o serogroups o , o , o , o , o , o and o among strains isolated from pregnant and non-pregnant women. hemolysin was presented in % and % respective. k antigen was presented in % of strains in studied groups.there are some statistically significant differences in antimicrobial resistance between both groups. amoxicillinclavulanate (amx-clv) resistance was higher among uti haemolytic isolates of e. coli in pregnant women ( %) then in non-pregnant women ( %). similar distinction in cefuroxime resistance was found - % and . amikacin resistance was higher among uti isolates of e. coli in non-pregnant women ( %) then in pregnant women ( %).conclusions: there are no significant differences in expression of virulence factors of e. coli from pregnant and non-pregnant women with ca-utis in london hospital, kuwait. the resistance rates of e. coli from pregnant women to amx-clv and cefuroxime are significantly higher than in non-pregnant women. the penetration of telithromycin in gynaecological tissues and activity in cervicitis patients h. mikamo (gifu, jp)objectives: chlamydia trachomatis and neisseria gonorrhoeae are major causative organisms for sexual transmitted infections in japan. although several oral antimicrobial agents are active against c. trachomatis, few effective oral antimicrobial agents against n. gonorrhoeae exist in japan. two studies were conducted: a clinical pharmacology study examining penetration of telithromycin (tel), an oral ketolide antibiotic, in female genital organ tissues and a clinical study examining tel mg once daily (qd) in cervicitis patients (pts chronic prostatitis (cp) is believed to be an infectious disease in most cases. both aerobic and anaerobic bacteria are involved in the polymicrobial microbiocenosis found in prostate specific specimens. coryneform bacteria form a remarkable part of this community, yet scarce knowledge exists about their clinical significance, species composition and antibiotic susceptibility.our aim was to compare the corynebacteria of the seminal fluid of cp patients and controls and to evaluate their antibiotic susceptibility.material and methods: semen samples from controls and cp patients (nih iiia or iv category) were analysed. corynebacterium seminale was identified by beta-glucuronidase activity, the rest of coryneforms using api coryne (biomerieux). e-test method was used for susceptibility testing.results: coryneforms were found from % cp patients and % controls (p > . ). twelve species and genera were found among strains identified, the most frequent being c. seminale (in % cp patients and % controls). cp patients harboured significantly more arthrobacter sp. ( % vs %, p = . ) andcorynebacterium group g ( % vs %, p = . ), the latter association was especially eminent in case of patients with serious inflammation (> wbc/ml): % vs %, p = . . all tested strains were susceptible to ampicillin-sulbactam, single strains were resistant to doxycycline ( %) and tmp/smx ( %), however, moderate resistance was common to doxycycline ( %). resistance to clindamycin ( %), benzylpenicillin ( %), nitrofurantoin ( %), erythromycin ( %) and norfloxacin ( %) was observed as well. half of cp-related corynebacterium group g strains showed resistance to nitrofurantoin and benzylpenicillin. in addition, they were often moderately resistant to clindamycin, erythromycin and, finally, norfloxacin frequently used to treat cp. conclusions: most of men have coryneforms in their semen, more than half harbour c. seminale. corynebacterium group g and arthrobacter sp are more frequently found in cp patients than the controls. in the treatment of cp of unknown etiology it is useful to take into consideration the susceptibility profile of corynebacterium group g. objective: to evaluate the role of cmv and listeria monocytogenes in abortion.methods: this descriptive prospective study was done on women, women with spontaneous abortion before th weeks of pregnancy as a case group and healthy woman with full term delivery as a control group. serum samples were taken from all patients. elisa test was done for evaluation of cmv (igg and igm) and listeria antibodies in both groups. prevalence of seropositivity was determined. data were analysed by x and chi-square test.results: seologic tests were done on samples. average age in case group was . ± . and in control group was . ± . years. in cases with abortion ( . %) and in control group ( . %) were seropositive for listeria monocytogenes. difference in seropositivity between groups is statistically significant (p = . ). cmv igg antibodies were positive in ( %) of case group and in ( %) of control group; the difference is significant statistically (p = . ). cmv igm antibody was positive in ( . %) of case group and none in control group. difference is significant (p < . ) there was no correlation number of previous abortion and seropositivity for listeria and cmv. conclusion: the present study showed an important role of listeria monocytogenes and cmv infection in abortion. serum and prostatic tissue concentrations of moxifloxacin ( mg) after a single intravenous infusion in patients with benign prostatic hyperplasia undergoing transurethral resection of the prostate background: the spectrum of bacterial prostatitis comprises gram-negative, gram-positive and atypical pathogens. because of its broad spectrum of activity, moxifloxacin might be a suitable antibiotic for the treatment of bacterial prostatitis. aim: in this study the penetration of moxifloxacin into prostatic tissue after intravenous application of mg as single dose was investigated.methods: in a prospective, multicentric study patients with benign prostatic hyperplasia received a single dose of moxifloxacin mg in an hour lasting infusion ( ml) for perioperative prophylaxis before undergoing transurethral resection of the prostate (tur-p). serum concentrations were determined in all patients before infusion, at the end of infusion (time point ), . , and h after the end of infusion. patients were randomized for tissue sampling either , . , or h after the end of infusion. at the beginning of tur-p approximately g of tissue was sampled for analysis. concentrations of moxifloxacin in serum and tissue were determined by hplc. results: patients were evaluated in the study. the concentrations (mean, sd, median, / % quantile) are shown in the table. the prostatic tissue concentrations of moxifloxacin were approximately twice as high as in serum. at the end of infusion the tissue and serum concentrations were already equilibrated, because the tissue-serum ratios did not differ significantly from the end of infusion until h after the end of infusion. after an intravenous infusion of mg the serum and prostatic tissue concentrations of moxifloxacin were well above the mic values of the most important prostatic pathogens until h after the end of infusion. therefore, moxifloxacin might be a good alternative for the treatment of bacterial prostatitis and/ or perioperative prophylaxis for tur-p. statistical significant differences were detected between patients with and without bgnc in the proportion of patients older than years ( . % vs . %), the antecedent of recent animal bite ( . % vs . %), the presence of immunosuppression ( . % vs . %), the presence of haematological illness ( . % vs . %), and the degree of leukocytosis at admission ( ± vs ± cel/ll). conclusions: bgnc is frequently detected in our patients. age older than years, the existence of immunosuppression, the existence of haematological illness, and the antecedent of animal bite are more frequent among patients with bgnc. patients with bgnc had a lower degree of leukocytosis at admission. these factors should be borne in mind to select empiric therapy for patients with cellulitis. is erysipelas-associated tinea pedis a site of streptococcal colonisation? objectives: tinea pedis is considered the most frequent portal of entry of erysipelas of legs (sel) but whether it is the site of streptococcal colonisation is unknown. methods: from june to october we prospectively searched for clinical tinea pedis in patients hospitalised in our infectious diseases ward for sel (acute and unilateral feature with fever were only retained). all patients had bacteriological samples on inter-digital spaces of both feet (sel side and contra lateral side).results: fifteen patients were included. all but one were treated by intra-venous penicillin-g followed by oral amoxicillin. on sel side: tinea pedis was found in / ( %) and, when present, streptococcal colonisation (c or g streptococcal groups) was found in / ( %), although streptococcal colonisation was never found ( / ) in its absence. on contra lateral sides : no streptococcal colonisation was found without tinea pedis, which was observed in / , with streptococcal colonisation in / . then there is a strength statistical association between streptococcal colonisation and tinea pedis, on sel side (p = . ) as well as on contra lateral side (p = . ). in one patient blood-cultures yielded with the same streptococcus than found in foot samples. discussion: streptococcal colonisation of tinea pedis is a common finding on both feet of patients hospitalised for sel. whether inter-digital colonisation is a primary stage of invasive disease remains unproved. in our experience, a strain of streptococcus that colonised inter-digital space was isolated in patient's blood, suggesting this hypothesis may be true in some cases. if confirmed, this concept could lead to a new strategy for secondary prophylaxis of recurrent sel by decontaminating streptococcal colonisation of tinea pedis. among ggs, different emm types were found; stg , stg and stg predominated. among gas, types were found, emm predominated. one patient had the same ggs isolate in throat and skin. six patients had recurrent infections during the study; two of them with disease episodes. of the culture positive skin samples, were taken from the erysipelas infection focus ( % positive for ggs) and from another site ( % positive for ggs), e.g. wound, intertrigo, between toes or an unknown site.conclusion: a predominance of ggs was seen in the throat of erysipelas patients and their families whereas ggs was not present in control subjects. ggs, instead of gas, also seems to predominate in erysipelas skin lesions. several emm types were present in both groups and there was no clear predominance of a distinct type. the recurrent nature of erysipelas became evident also during this study. the evaluation of fournier's gangrene severity index score in patients m. ulug, m.k. celen, m.f. geyik, c. ayaz, s. girgin (diyarbakir, tr)objectives: fournier's gangrene is synergistic necrotizing fasciitis of the perineum and abdominal wall along with the scrotum and penis in men and the vulva in women. it is rare but life-threatening process. in this study we identify effective factors in the survival of patients with fournier's gangrene and to determine the accuracy of the fournier's gangrene severity index score (fgsis). methods: we evaluate patients with fournier's gangrene who were threated and follewed up from us between january and september in the department of general surgery prospectively.results: the results were evaluated in two groups: those who died (n: ) and those who survived (n: ). no statiscally significant difference was found between the age of the survivors and those who died. the admission and final laboratory parameters that correlated statiscally signinificant with outcome includes leucocyte count, hematocrit, urea, creatinine, lactate dehydrogenase, bicarbonate and albumin. sites of culture were skin/soft tissue ( , and %), respiratory tract ( , , and %) , blood ( , and %), urine ( , and %) , and other ( , and %) . -day mortality was % in this population. % of patients received antibiotic therapy alone, % surgery alone, % antibiotics + surgery, % other therapy, and % no treatment. the most common antimicrobial classes received were vancomycin ( %), beta-lactams ( ), fluoroquinolones ( ), and cotrimoxazole ( ) with % of patients receiving multiple agents. median duration of antibiotic therapy was , , and days, in the ca-mrsa, ha-mrsa and ca-mssa groups respectively. , , and % received adequate antimicrobial therapy (p < . ). hospital admission was required in , , and % of patients (p < . ). clinical success rates of initial therapy were , , and % (p < ), and recurrences were more common in the ca-mrsa group, ( , , and %, p < ). characteristics associated with outcome are listed in table . in multivariate analysis, presence of mrsa and diabetes were predictive of clinical failure.conclusion: in the community setting, mrsa infections are associated with an adverse impact on outcome compared to mssa infections and patients with ca-mrsa are significantly less likely to receive adequate antibiotic therapy. microbiological analysis of root canals associated with periapical abscesses and the antimicrobial susceptibility of isolated bacteria s. ozbek, a. ozbek, m. koseoglu, s. evcil, a. erdogan, a. ayyildiz (erzurum, tr)objective: the periapical abscess is a collection of pus in the pulp or around the root of teeth. many odontogenic infections can be managed without antimicrobial therapy or bacteriologic investigation. however, when an acute bacterial infection has progressed or antimicrobial therapy might be of benefit to patients, antibiotics are prescribed. we aimed to identify microorganisms in root canals with periapical abscess and the antimicrobial susceptibility profile of them and to revise antimicrobial treatment protocols when antimicrobials is used empirically. methods: patients with odontogenic infections included in this study. the microbiologic investigation was performed under strict aseptic conditions. a standardize routine of root canal therapy was instituted, and in each case a single root canal was sampled. in multirooted teeth only the largest canal was sampled to preserve the identity of a single endodontic/ microbiologic ecosystem. for microbial sampling, two sequential paper points were introduced into the full length of the canal, and kept in place for min. one of the paper points was used for aerobic culture and the other one for anaerobic culture. to identify isolated bacteria, whole bacterial fatty acid profiles were evaluated by using microbial identification system. antimicrobial susceptibility results were obtained by disc diffusion test for aerobics, and e-test for anaerobics. results: totally bacterial strains were isolated. of them were aerobic and of them were anaerobic. or % of cultured specimens yielded mixed (aerobic and anaerobic) species. the most prevalent bacteria were staphylococcus spp. as aerobic, peptostreptococcus prevotii and streptococcus morbillorum as anaerobic. conclusion: beta-lactam antibiotics combined with beta-lactam inhibitor (amoxicillin-clavulanic acid) had a quite effect on gram (+) and (-) aerobics. when we take into consideration that beta-lactam antibiotics stimulate production of beta-lactamase, amoxicillin-clavulanic acid combination appears a good first step antimicrobial. clindamycin may be second alternative for that purpose. for anaerobics, cefoxitin and metronidazol had well effect. although imipenem and piperasilin-tazobactam are perfect, they should not be first step of therapy. due to the frequency of mixed infections, a combination of amoxicillinclavulanic acid and metronidazol or a combination of clindamycin and metronidazol considered to have well effect for mixed infections. clinical microbiology and infection, volume , supplement , study is to review the spectrum of p. multocida infections in our centre. methods: we studied the medical records of all patients who had positive cultures for p. multocida between and . demographic, epidemiological, clinical and microbiological data including age, sex, animal exposure, site of infection, underlying diseases, type of therapy and outcome were evaluated. all isolates were identified by standard conventional microbiological methods. antibiotic susceptibility testing was performed by the disk diffusion method onto muller-hinton agar supplemented with % sheep blood and the mics of the antibiotics tested were determined by the e-test method. results: thirteen cases of p. multocida infections were diagnosed during this period. the male to female ratio was : and most patients ( %) were > years of age. respiratory tract infections were most commonly encountered ( . %), followed by soft-tissue infections ( . %) and septicemia ( . %). underlying disease was present in ( . %) patients. among them, presented a kind of malignancy. bullous pemphigoid, mitral valve stenosis, coronary disease, chronic obstructive pulmonary disease, and intracranial haemorrhage served also as predisponding factors. a traumatic animal exposure was reported in only patients and non-traumatic in cases. all isolates were susceptible to beta-lactams (penicillin, amoxicillin, amoxicillin/clavulanic acid, cefepime, cefuroxime, ceftriaxone, imipenem, and meropenem), quinolones (ciprofloxacin, norfloxacin, levofloxacin, and sparfloxacin), chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and % were intermediately resistant to aminoglycosides (gentamicin). appropriate antibiotic therapy was administered to all patients and a clinical response was observed in ( %) of them. mortality rate was %. conclusions: pasteurella multocida must be considered as a possible etiology for a variety of infections, even without an obvious animal exposure. although this organism is susceptible to a large spectrum of antibiotics, a failure to treatment may be recorded especially in severe infections and in compromised patients. infections caused by nocardia cyriacigeorgici in zaragoza, spain: identification and antibiotic susceptibility c. villuendas, b. moles, v. rodriguez-nava, a. couble, f. laurent, m. revillo, p. boiron (zaragoza, es; lyon, fr)objectives: nocardia species known to date differ in their clinical presentation, antibiotic resistance patterns and geographic distribution. nocardia cyriacigeorgici is a recently described species.the aim of this study is to analyse the identification results, antimicrobial susceptibility together with the clinical data, of n. cyriacigeorgici clinical isolates, recovered from to in our laboratory. methods: identification of nocardia spp. isolates was achieved in our laboratory on the basis of the following: visualization of the colony, gram stain and parcial acid-fast positivity by modified acid-fast staining, casein, xanthine and tyrosine hydrolysis, opacification of middlebrook h agar, production of arylsulphatase after days incubation and antimicrobial susceptibility pattern.identification at species level was achieved by s rdna gene sequencing (laboratoire de mycologie. faculté de pharmacie. lyon. france)antimicrobial susceptibility tests included commercial broth microdilution (emiza ef sensititre Ò ) and gradient strip agar dilution (e-test ab biodisk Ò ). interpretation of results was done according to nccls standard guidelines. in the six years of study, isolates of nocardia spp. were recovered, of them belonging to n. cyriacigeorgici species ( %). n. cyriacigeorgici represents the third species in frecuency in our serie, after n. abscessus and n. farcinica. the strains were recovered from patients, from respiratory specimens and one from blood-culture.pneumonia was the most frequent clinical manifestation, being copd and previous corticosteroid therapy the most common predisposing conditions. all n. cyriacigeorgici isolates showed susceptibility to: amikacin, tobramycin, cefotaxime, imipenem, trimethoprimsulfamethoxazole and linezolid, and resistance to: amoxicillinclavulanic acid and ciprofloxacine. conclusion: n. cyriacigeorgici is not an infrequent cause of nocardiosis in our geographical area. the uniformity showed in the antimicrobial profile can be useful for its identification. in our hospital, patients with copd and receiving corticoid therapy is the most important group of risk for adquiring n. cyriacigeorgici infection. whit the technics available in our laboratory the isolates were identified as nocardia spp. and identification at species level was only possible by phylogenetic analysis using rdna sequencing. high frequency of single-step resistance mutations in nocardia farcinica exposed to quinolones u.s. jensen, j.d. knudsen, k. schønning (hvidovre, dk)objectives: nocardia farcinica infections often require prolonged antibiotic therapy and perorally administered agents are desirable. isolates commonly display in vitro susceptibility to quinolones when tested by disc diffusion methodology. in the present study, we investigated the activity of three different quinolones (ciprofloxacin, levofloxacin and moxifloxacin) against n. farcinica and assessed the robustness of their activity by determining the frequency of single step resistant mutants when exposed to inhibitory concentrations of quinolones. methods: isolates of n. farcinica were used in the study; correct identification to the species level was verified by s rdna sequencing. mics of ciprofloxacin, levofloxacin and moxifloxacin against n. farcinica as well as s. aureus atcc and e. coli ccug were determined by the agar dilution method using inocula of approximately . cfu and h of incubation. single step mutation frequencies were determined by heavily inoculating selective agar plates containing quinolone at a concentration of x mic and counting resistant colonies after days incubation. inoculum was quantified by seeding a dilution series of the inoculum employed on unselective plates and counting colonies after h of incubation and frequencies were calculated by dividing the number of resistant colonies by the number of cfu present in inoculum. results: when mics were determined by agar dilution method all quinolones displayed roughly the same potency against n. farcinica isolates (mics between . and ). as expected moxifloxacin were the most potent quinolone against s. aureus. however, all three quinolones selected for single step resistant mutants, the frequency of which was higher for ciprofloxacin (~ ) ) than for levofloxacin ( ) - ) ), which again was higher than for moxifloxacin ( ) - ) ). however, even for moxifloxacin the frequency against n. farcinica was comparable to the single step mutation frequency of ciprofloxacin against s. aureus ( - ).conclusions: although quinolones may exhibit activity against n. farcinica, n. farcinica is capable of rapid development of resistance. therefore, quinolones should probably be avoided, at least as single agents, in the treatment of nocardia infections. correlation between clinico-laboratory findings and a positive igm elisa test for leptospira: a retrospective study e. mendrinou, p. goudas, a. regli (patra, gr) objective: to correlate a positive elisa test for igm antibodies against leptospira with the clinical and laboratory findings in patients with suspected leptospirosis. method: we retrospectively analysed the history, clinical course and laboratory findings in a total of patients, with suspected leptospirosis. all patients fulfilled the criteria for clinical diagnosis of leptospirosis. from the patients, had to be transferred to the dialysis unit for haemodialysis and patients had to be admitted to the intensive care unit (icu) due to severe pulmonary haemorrhage. serum samples from all patients were tested for igm antibodies against leptospira. results: from the total of patients death occurred to only four, due to respiratory failure from severe pulmonary haemorrhage. the rest of the patients recovered completely. from the total of patients had a positive elisa igm test for leptospirosis ( . %). however, from the patients that were transferred to the dialysis unit, had a positive leptospirosis test ( %) and from the six patients admitted to the icu, three had a positive test ( %). among other laboratory findings there was a stronger correlation between very low platelet levels (< . mm ) and very high blood bilirubin levels (> mg/dl) with a positive test for leptospirosis. all patients with a positive test had less than . platelets per mm and had blood bilirubin over mg/dl. the differential diagnosis of icterohemorrhagic fevers includes a vast number of pathogens, some of which are untraceable with the common laboratory methods. in our study, from the total of patients, only . % had a positive test for leptospirosis. in of the rest patients, many different pathogens were traced, most of them being several kinds of viruses (cmv, ebv), brucella and coxiella. in of the patients no pathogen was traced. conclusions: taking into consideration the high sensitivity of the elisa test we conclude that: . icterohemorrhagic leptospirosis comprises only a small subtotal of icterohemorrhagic fevers; . there is a correlation between higher levels of bilirubin and/or very low platelet levels with leptospira infection; . there seems to be a correlation between leptospira infection and severity of icterohemorrhagic fevers. evaluation of continuous ambulatory peritoneal dialysis-related peritonitis attacks in ankara s. tekin koruk, m.a. yetkin, i. koruk, f.s. erdinc, s. sahan, n. tulek, m. duranay, a.p. demirö z (ankara, konya, samsun, tr) objectives: peritonitis is a common clinical problem that occurs in patients with end stage renal disease treated by peritoneal dialysis. the aims of this study were to assess demographic aspects, rates of peritonitis, causative organisms, clinical outcomes and treatment approach for continuous ambulatory peritoneal dialysis (capd) -related peritonitis cases. methods: seventy cases of peritonitis occurred in patients treated in infectious diseases and clinical microbiology department between may and april were enrolled into this study. the mean age of the patients was . years (range - years). cloudiness of the peritoneal dialysis fluid and/or abdominal pain were considered suggestive of peritonitis and were confirmed by cell count and culture. baseline cell count, gram stain, and cultures were obtained, and repeated with periodic follow-up. results: the overall incidence of peritonitis was . ± . episodes/patient-year. in . % of patients there were only one peritonitis attack, where as in . % of them had two or more attacks. age, gender, education and profession of the patients have not been found as a risk factor in peritonitis attacks.the most common presenting symptoms of the patients were abdominal pain, cloudiness of the peritoneal dialysis fluid, nausea and vomiting. peritoneal dialysate fluid white blood cell count was ± /mm in episodes. cultures were positive in ( . %) peritonitis episodes; coagulase-negative staphylococci was the most common organism (% . ), followed by staphylococcus aureus (% . ), episodes (% . ) had negative culture results. there was a statistically significant decrease in serum crp and esr levels and at the end of the treatment when compared with the levels on admission.at the end of the study, episodes of peritonitis cases were treated with ip cefazolin and gentamicin protocol. seven of the patients did not respond initiate therapy and the therapy was converted to iv protocol. seven episodes were treated with iv antibiotics on admission for medical reasons (systemic infection and/or concurrent exit-side or tunnel infection). there were two deaths. two catheters were removed and the patients were transferred to haemodialysis programme. conclusion: despite all technical improvements during recent decades peritonitis is still the major complication of capd. for the accurate treatment of complications, causative organisms and their antimicrobial susceptibilities must be known. objective: viruses are a frequent cause of upper respiratory tract infections in children. among the respiratory viruses, influenza viruses are known to cause outbreaks globally. the present study was carried out to identify the influenza virus serotypes causing acute respiratory infection in children attending univesity hospital in konya in turkey. methods: thorat swabs were collected from acute viral upper respiratory infection suspected children attending the out patient clinic of meram medical faculty hospital. two swabs were taken fron each chidren and one of the swabs was used for bacteriological cultures and if these were negative the other one was used for viral diagnosis. totally bacteriological cultures negative swabs were investigated by real-time pcr for the presence of parainfluenza , and , influenza a and b. results: one or more viral pathogens were detected in children, with parainfluenza % being the most commonly identified virus. parainfluenza in % and parainfluenza in %, influenza a were identified in % and influenza b in %. from the specimens of children more than one virus detected. conclusion: the influenza viruses cause morbidity and mortality among children and elderly. this study analysed the occurrence of influenza and paranfluenza respiratory ifections due to influenza and paranfluenza viruses. molecular methods used directly on clinical material have an important role in the rapid diagnosis and surveillance of influenza viruses and can be applied in clinical practice for correct diagnosis and administration of effective treatment. , , , , , and . the demographics, clinical presentations and laboratory findings of the patients with serotype were presented. results: the mean age was y m, ranging from months to y m. seventy percents of children with serotype infection clustered between october and january . the mean duration of a positive culture result was . days. the mean duration of fever was . days, with days before admission. forty ( %) children were treated as outpatients. the mean length of hospital stay was . days. the most common diagnoses were exudative tonsillitis ( %), pneumonia or bronchopneumonia ( %) and pharyngoconjunctival fever ( %). the most common symptoms and signs were fever ( %), cough ( %) and coryza ( %). neurologic complications were noted in children. eighteen children had documented coinfection (including virus, bacteria and mycoplasma pneumoniae). leukopenia (wbc < /microliter) was noted in two of cases while leukocytosis (wbc > /microliter) in ( %). six ( . %) of cases had a normal serum c-reactive protein (crp) level (< mg/l), while % of children had a serum crp greater than mg/l. seventy ( . %) of children ever received antibiotics therapy. the outcomes were excellent in these cases. conclusion: recognizing that children with adenoviral serotype infection may present with prolonged high fever, leukocytosis and elevated crp, which mimics bacterial infection, the clinician may not prescribe unnecessary antibiotics for these children. the infectious mononucleosis like syndrome (im) is an acute febrile disease of older children and young adults, and is characterized by lymphadenopathy, tonsillitis, splenomegaly, liver dysfunction and by the presence of peripheral lymphocytosis with > % atypical lymphocytes. epstein -barr virus (ebv) is responsible for over % of the cases, cytomegalovirus (cmv) for %- % and toxoplasma gondii < %.herpes simplex, rubella and adenovirus are rare. the infection is usually characterized by mild symptoms. however in some cases the clinical manifestations may be rather atypical and severe. objective: to determine the prevalence of im like syndrome among patients in a children's hospital and its possible association with etiologic factors, age, major symptoms and atypical manifestations. material and methods: during a one-year period (january to december ) a total of samples were examined in our laboratory. the study population was children between - years old, which either examined in the outpatient's clinic or hospitalized. all serum specimens were examined by . indirect immunofluorescence for the presence of igg and igm antibodies against the viral capsid antigen (vca) ebv, .immuno chemistry luminescence for the detection of igg and igm antibodies to cmv and .eia for the detection of igg,igm abs of herpes simplex i and ii and toxoplasma gondii. results: of the children examined ( . %) were found positive for igg and igm vca antibodies and ( %) showed positive specific igg and igm antibodies for cmv. these patients had one or more of the primary following symptoms: fever ( %), lymphadenopathy ( %), pharyngalgia ( %), cough ( %), skin eruption ( %). atypical manifestations as meningoencephalitis were found in two children one aged months (caused by ebv) and the other of years old (caused by hsv i) confirmed by pcr. the laboratory data showed positive serology for ebv and cmv infection, the existence of atypical lymphocyte ( %), ldh, asat and alat were moderately elevated ( %) and crp increased ( %). conclusion: the frequency of im like syndrome in greece, though it's relatively low, it's not rare. the above results suggested that ebv, cmv, hsv should be considered in any young patient with im and acute neurological illness of uncertain etiology. objectives: enterovirus, parvovirus b and human herpes virus type (hhv- ) are a common cause of infection in young infants. the objective of this study was to determine what portion of the infants who received a clinical diagnosis of febrile syndrome have a viral etiology by these three genera of viruses. methods: ninety-six patients were included in the study, all of them were admitted to the pediatric casualty of a tertiary care hospital, and all of them presented a febrile syndrome without a clear focus of infection (urinary tract, lung and meningeal infections were discarded). the assay was carried out in blood samples by real-time pcr. dna was isolated from ll of blood by semi-automated system magna pure lc total nucleic acid isolation kit (roche diagnostics, nederland bv). pcr was performed in a lightcycler instrument (roche molecular biochemicals) by a uniform cycling parameters: min at °c for polymerase activation, and cycles of s at °c and s at °c for amplification of the specific target sequence ( utr gene for enterovirus, vp gene for parvovirus b and dna polymerase gene for hhv- ). pcr product formation was detected continuously by the use of taqman probes. results: a viral amplification was detected in ( %) of the patients included in this study. enterovirus was detected in ( . %) of the patients, parvovirus b in ( . %) and hhv- in ( . %). in five cases two viral amplifications were detected at the same time: parvovirus b /hhv- and enterovirus/hhv- . the mean age of the patients was years old (range from days to years). in group of infants < months old (n = ) there were enterovirus and hhv- . in the infants from months to years old (n = ) there were enterovirus, parvovirus and hhv- . in the last group of infants > years old (n = ) there were enterovirus and parvovirus b . conclusions: viral infections are an important cause of sepsis in infants admitted to hospital. enterovirus was the most frequent virus detected in infants < months, parvovirus b the most frequent in children > years old, and the hhv- was detected in all age groups. qualitative real-time pcr in blood is a rapid and sensitive method for diagnosis of enterovirus and parvovirus. however, is not the better method for diagnosis of hhv- , a latent virus, in which this technique is not capable of distinguish between recent and acute infection. objectives: group a rotaviruses are a major cause of acute gastroenteritis in infant and young children worldwide. in this study, the molecular epidemiology and clinical features of rotavirus infection in iranian children was investigated. methods: between february to january , thirty hundred and seventy two diarrhoea stools from children under -years-old with acute diarrhoea that attended the biggest paediatric hospital in tehran (iran), were analysed using elisa, electropherotyping and reverse transcriptionpolymerase chain reaction (rt-pcr). results: ninety-four samples ( . %) were positive for the presence of rotavirus either by page, elisa, or both. according to page, the predominant electrophoretic pattern detected was the long profile of ( . %) followed by the short electropherotype five of ( . %). out of the positive samples, were further characterized by rt-pcr typing assay for identification of g types, resulting in strains of g genotype while samples could not be assigned a g type. all of g genotypes had a long rna electropherotype. among the patients with rotavirus infection, ( . %) required hospitalization. watery diarrhoea ( . %), vomiting ( . %) and fever ( . %) were significantly more frequent in children suffering from rotavirus gastroenteritis. seven out of rotavirus-positive patients had severe dehydration (p < . ). rotavirus infection mostly affected children under years of age with a peak incidence of % in children - years of age and it occurs year round with a seasonal pattern: more frequently during winter ( . %). conclusion: this study revealed that rotavirus is an important etiological agent of acute gastroenteritis in tehran. we found that a major proportion of the specimens were untypeable. improved detection and characterization of incompletely typed strains will help to develop comprehensive strain information that may be required for tailoring effective rotavirus vaccines. serological study of prevalent rotaviruses in tehran e. habibi, s. ghorbani, a. jarollahei, z. habibi (tehran, zanjan, ir)objectives: rotaviruses are icosohedral and non-enveloped viruses that belong to reoviridae family which consist of three layers of protein surrounding segments of dsrna. rotavirus is one of the most important agents of acute gastroenteritis in children. in this survey, the most prevalent serotypes in tehran and seasonal distribution in a year were detected. methods: in this study, a total number of specimens of faecal samples of children and infants with acute gastroenteritis were collected from two children hospitals in tehran. the samples were tested by elisa procedure. serotyping investigation of iranian rotavirus isolates, using serotypes monoclonal antibodies (g -g -g -g -g -g -g ) in elisa tests and immunosorbent electron microscopical studies using trapping and decoration techniques were performed. results: rotavirus type a infection was identified in samples ( %). serotyping investigation in elisa tests proved that serotypes g and g were the most common serotypes circulating among infected children and infants in tehran. by electron microscopic studies the characteristic of rotavirus particles were observed in the faecal samples of infected children. the maximum incidence of infection was determined to occur among the cold months of the year. conclusion: it was approved that g and g serotypes are the main rotavirus serotypes present among children in tehran. it was detected that rotavirus diarrhoea was most prevalent among children of under years of age. results: from patients with varicella presented neurological manifestations (sex ratio m/f: / ). had acut cerebellar ataxia and one had encephalitis. we estabilished the diagnosis on the basis of clinical aspects (including neurological examination), cerebrospinal fluid examination and electroencephalogram. the age interval was between months and years. most cases were diagnosed in children and teenager ( ); one case toddlers, and cases in adults. neurological manifestations appeared in most cases among and days after the onset of rash ( cases). in the order of frequency: gait disorders ( ), cerebellar ataxia ( ), fever ( ), vomiting ( ), nistagmus ( ), seizures and coma ( ) . csf showed limphocytic pleiocytosis and elevated levels of protein ( cases); in cases csf had normal aspects. electroencephalogram had dominant theta wave with totally or partially suppression of alpha activity in all patients. all cases showed clinical and eeg improvement at the end of the treatment. conclusions: the most frequent neurological manifestation was cerebellar. the evolution was good under treatment, with no sequelae at month of follow up. key: cord- -bie veti authors: nan title: ecc- abstracts date: - - journal: int j antimicrob agents doi: . /s - ( ) -x sha: doc_id: cord_uid: bie veti nan f spain introduction: the prevalence of erythromycin resistance (er-r) in group a streptococci (gas) has increased in spain since early s with current rates exceeding % in some regions. this study determined the emm -types associated to erythromycin resistance in spain. material and methods: isolates belonged to the sauce* surveillance collection. rapid sequence analysis of specific pcr products was used to deduce emm -types corresponding to the majority of the known gas m serotypes. pcr primers used: gasm ( ?-tattgcgct-tagaaaattaa- ?) and gasm ( ?-gcaagttctt-cagcttgttt- ?). sequencing was done with the big dye terminator mix and autosequenator (applied biosystems). dna sequences were subjected to homology searches against the bacterial dna database. results: overall, gas isolates ( er-r) were analysed. three m-types (m , st and m ) accounted for . % of the er-r isolates, whereas they just represented a . % of the ery-s. for er-r isolates the strongest association was seen with m (or / ; % ci . Á/ . ), and m was second after m only in the last temporal period of the study ( Á/ ) . no homogeneous distribution of er-r m-types by centres was seen. conclusions: few m-types (leading by m ) are responsible for the er-r in spain. but for m , the remaining er-r m types (st , m and m ) did not show a temporally nor geographically homogeneous distribution. *sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en espana' (susceptibility to the antimicrobials commonly used in the community in spain ) and is the spanish word for the willow tree. significant increase in the prevalence of erythromycin-resistant, clindamycin and miocamycin-susceptible (m-phenotype) streptococcus pyogenes in spain ( ( Á/ purpose: a variety of methods is used for a molecular typing of enterococcus spp. and related gram-positive bacteria. these include dna-based methods such as macrorestriction analysis using pulsedfield gel electrophoresis (pfge), ribotyping, and amplification-based methods such as rapid amplification of polymorphic dna (rapd) and amplified fragment length polymorphism (aflp). we used a homogeneous strain collection of transconjugants resulting from filter-matings with different antibiotic-resistant e. faecium and a recipient isolate from our lab. the influence of transferred antibiotic-resistance determinants on the outcome of different typing methods was investigated. results: fragment patterns resulting from pfge indicated minor differences between the transconjugants and the recipient. in respect to different primers used for rapd, none or only a single fragment shift was detected in the resulting fragment patterns. aflp clusters all transconjugants into a group of major relatedness, but the result was strongly dependent on the mathematical method used for cluster analysis. fragment patterns of digested plasmids showed the possession of different or only widely related plasmids in the transconjugants. conclusions: the results of this study clearly show that under certain situations typing methods commonly used for enterococci and related gram-positive bacteria come to their limits. the sasss network aims to set up a national surveillance study to obtain standardized information on antimicrobial susceptibility to various bacterial pathogens. currently, hospitals are participating in the project from different geographical regions in saudi arabia. during the st year ( ), the sasss focused on setting up this network. overall, high frequencies of resistance to antibiotics to different bacterial pathogens in saudi arabia were seen. geographical variations of resistance were noticed, which could be related to different prescribing practices. approximately, and % of escherichia coli and k. pneumoniae , respectively, were extended spectrum â-lactamases (ebls) producers. resistance of enterobacteriacae group to carbapenem and pipracillin/tazobactam is low. resistance of pseudomonas aeurginosa to various anti-pseudomonal antibiotics including carbapenem is high and alarming. methicillin resistant staphylococcus aureus (mrsa) comprised % of s. aureus isolates. no vancomycin intermediate s. aureus (visa) was detected. high level resistance to gentamicin in enterococcus were seen in % of the isolates and only % of enterococcus facieum were glycopeptide resistant. resistance of streptococcus pneumoniae to penicillin ranged between % to almost %. surveillance of antibiotic resistance on a national level is necessarily to give guidance to practicing physicians on the best agents to use. the world-wide problem of betalactam resistance (r) in streptococcus pneumoniae (sp) has been complicated by increasing r to macrolides and some older fluoroquinolones (fq) (ciprofloxacin cip). aim of our study was to evaluate rate of acquisition of resistance to different fq: cip, sparfloxacin (spx) and levofloxacin (lev) of sp strains with different levels of susceptibility to penicillin (p). fifteen strains were serially and daily passaged in subinhibitory concentrations of these four antibiotics by a gradient plate method until acquisition of resistance. clinical strains isolated from children in day-care centers were used. five strains were susceptible (s) to penicillin (p) (one reference strain, four clinical isolates: p micsb/ . mg/l): five were intermediate (i) to p (one reference strain: p mic . mg/l, four clinical strains p mics: . Á/ mg/l), five were resistant (r) to p (p mics . mg/l). mean of number of passages (n ) necessary to reach i or r level with each fq as selecting agent are in the following table: spx and lev induced resistance but more slowly than cip. our results show that rate of acquisition of resistance to fq is strongly related to alteration of susceptibility to p, probably by modification of cell wall. these results are concordant with clinical results. clinical relevance of phase variation in pneumococcal opacity: nasopharyngeal (np) colonization in children from day care centers (dcc) soa . carsenti h, mancini g, bensoussan m, dunais b, pradier ch, dellamonica p. archet hospital, infectious disease, nice, france streptococcus pneumoniae (sp) adherence to nasopharyngeal (np) epithelium is a prerequisite for induction of otitis transparent sp (t) have been shown to colonize the np of infant rats better than opaque (o) sp. opaque sp has proven more virulent than the t form during systemic infection in a mouse model. aim of this study was to evaluate phase variation in the nasopharynx of children. sp strains were isolated during a winter epidemiology study of np samples in children from family dcc. mics determinations were performed by e -test for penicillin (p), amoxicilline (amx) and ceftriaxone (cro). serotypes were performed using the quellung reaction. upon oil immersion microscopic examination short chains of six to eight cocci were noted as , '/, '/'/, '/'/'/ for absence, , , !/ chains by field, respectively. phase variation was detected on catalase trypticase soja plates, amx and cro mics and bactericidal activity was determined for pairs of o and t variants with different serotypes and susceptibility to penicillin. seventy strains of sp were screened for phase variation. nine out of with chain length , '/ had o variants while out of strains with chain length '/'/ or '/'/'/ showed o variants. proportion of o variants was predominant when chain length increased. serotype f was prevalent. bactericidal activity of o variants showed a four-to eightfold increase of mbc. o variants may be present in np of children while t are predominant form for colonization. these virulent variants with lower level of autolysis showed less susceptibility to killing by antibiotics. they may persist in np and explain the absence of eradication by active molecules. antimicrobial resistance among clinical strains of s. pneumoniae isolated from patients with community-acquired respiratory tract infections (carti) in russia soa . kozlov rs a , bogdanovitch tm a , sivaya ov a , agapova ed b , ahmetova li b , furletova b , gudkova lv b , gugutsidge b , ilyina vn b , marusina b , multich ig b , ortenberg ea b , schetinin ev b , shturmina purpose: to determine the antimicrobial resistance of pneumococci causing carti in different russian cities. methods: a total of non-duplicate strains isolated in russian cities in were studied. antimicrobials tested included penicillin (pen), amoxicillin (amo), erythromycin (ery), azithromycin (azi), clarithromycin (cla), midecamycin (mid), spiramycin (spi), clindamycin (cli), levofloxacin (lev), vancomycin (van), rifampicin (rif), tetracycline (tet) and co-trimoxazole (sxt). susceptibility testing was performed by broth microdilution with interpretation of the results according to nccls guidelines ( ) a map of bacterial resistance in a hungarian region soa . farkas a a , juhasz a b , orosi p a , miszti c c , balogh m d . a kenezy teaching hospital, hygienie, debrecen, hungary , b kenezy teaching hospital, laboratory, debrecen, hungary , c university of debrecen, microbiology lab, debrecen, hungary , d regional hospital berettyóújfalu, laboratory, debrecen, hungary background: regional trends of microbiological resistance pattern constitute basic data and qualifying criteria for effective infection control. purpose: the aim of our study was to establish an internationally compatible regional database in a hungarian county hajdú -bihar. methods: our model is the national nosocomial infections society publications' format from the u.s. published in . it contains data regarding various icu types, ambulatory patients and hospitalised patients. the same format is used for antibiotic utilisation data and device related infections' rates as well. we collected cleaned data of years Á/ from all the microbiological laboratories of our county. results: ciprofloxacin p e. coli . . . Á/above susceptibilities not significantly different from u.s. data were as follows: mr cns, streptococcus pneumoniae /penicillin and rd generation cephalosporin, pseudomonas aeruginosa /piperacillin and enterobacter spp. and escherichia coli /ceftriaxon. conclusions: this database proved to be a very useful tool for choosing primary wards of active surveillance including places for infectious disease physician's visit (icu, rehabilitation unit). additional analysis is needed at an individual institution's level for other heavily used (or useable) antibiotics and bacteria as well (aminoglycosides, beta-lactam Á/beta lactamase inhibitor combinations, nd generation cephalosporins, corynebacteria . to compare epidemiological, clinical, and immunological features of add before and after haart introduction, between the patients (p) diagnosed in Á/ , and the p detected since , in a case-control study. though the mean number of newly diagnosed aids p had a sharp drop in the haart era, from p/year in Á/ to p/year since (p b/ . ), the distribution of add and underlying immunodeficiency showed limited changes. when excluding a greater frequency of tuberculosis (tb) (p b/ . ) and wasting syndrome (p b/ . ), all other add did not show a different frequency before and after . a tendency towards a higher mean cd count at aids disease was noticed: vs cells/ml (p b/ . ), with a significant difference for candida esophagitis , toxoplasmosis, kaposi sarcoma and tb (p b/ . Á/b/ . ). the limited variation of clinical and immunological presentation are attributable to the poor impact of haart before aids recognition: . % of p detected since did not receive haart or had insufficient compliance to antiretrovirals, so that . % of p were aids presenters. during the haart era, an increase of mean age and sexual transmission was found (p b/ . ). notwithstanding the effects of haart on the natural history of hiv disease, the consequences on add distribution and related immunodeficiency were negligible, since most p could not benefit from haart before aids onset. a high clinical suspicion for add should be maintained when facing p with missed or undertreated hiv disease. radata */communication internet platform management of resistance analysis guided haart switch for implementation in clinical practice of hiv-infected individuals soa . paech v, lorenzen t, stoehr a, plettenberg a. ifi, interdisciplinary infectiology and immunology, hamburg, germany purpose: hiv-resistance analyses are indicated to prepare switch of haart in hiv-infected individuals with failure to ongoing haart regimen. specialists at several responsible sites often feel lack of complementary informations if interpretation of resistance analyses is done independent from each other. clinical benefits from resistance analysis assays are sigfnificantly higher for those physicians, who can access external advice from hiv-experts for possible treatment options. the database concept 'radata' (www.radata.de) was developed in germany to generate expert advice for implementation in haart switch. results: fifteen hiv-treatment centres, seven laboratories and high ranked authorities in hiv-medicine contribute to radata database since it is started in january in germany. hiv-infected subjects are eligible to participate at the project after presentation of failure to haart (viral load !/ c/ml). expert advice is generated after all data are evaluated and based on recommendations of Á/ external hiv-experts. observation after therapy switch is scheduled for a period of months. conclusions: radata is a novel database concept with features for evaluation of data and availability of complementary information to participating sites. the project is designed to provide its proficiency to patients and centres from germany and foreign countries. further information will be provided after the number enclosed subjects have enlarged. in vitro effects of hiv infection on abc transporter expression and antiretroviral drug efficacy soa . therefore, we evaluate in primary cultures of human monocytederived macrophages (mdm) and lymphocytes, effects: ( ) of retroviral infection and haart on the expression and activity of p-gp and mrp; and ( ) of specific inhibitors of these host proteins on antiretroviral activities of nrti, non-nrti and ip. results: on the one hand, we evidenced a transitory increase of p-gp mrna expression in lymphocytes and mdm in response to in vitro hiv infection. this was correlated to an increased p-gp cell surface expression and activity, and an increased tnf-alpha production and mrna. in contrast, no significant modulation of mrp was observed. on the other hand, psc and probenecid potentiated in vitro the anti-hiv activity of azt and indinavir. these effects were accentuated when psc and probenecid were combined. conclusion: these results showed that: ( ) hiv infection by increasing abc transporter expression could favorise the efflux of antiretroviral drugs and decrease their pharmacological effects; and ( ) specific inhibitors of these transporters could reverse these deleterious effects. effects of interferon alpha plus ribavirine therapy on frequencies of hcv, hiv and cmv specific cd -t-cell responses in peripheral blood of hiv/hcv coinfected patients after months of treatment soa . methods: two groups of patients with chronic hcv infection were studied: hiv coinfected progressors with antiretroviral therapy and hiv-negative controls. twelve hcv/hiv and hcv patients have already reached months of ifn-alpha'/ribavirine therapy. virusspecific cd -t-cells in peripheral blood were analyzed by ifngamma-elispot-assays using hiv-p , one cmv and three hcv (core, ns , ns ) antigens. results: ( ) at baseline, hcv-specific cd -th -cells frequencies were significantly lower than hiv-and cmv-specific ones; ( ) frequencies of cd -th -cells against hcv as well as against cmv were similar in the two groups; ( ) in hcv'//hiv'/, hcv specific cd -t-cell frequencies did not change between baseline and th month of anti-hcv treatment, decreased in three and increased in only one case. hiv-and cmv-specific frequencies were decreased in seven patients. similar results were observed in hiv-negative group. conclusion: ( ) hcv-specific immune responses might be more prone to tissue compartmentalization than hiv-specific ones; ( ) immune defects induced by hiv infection might not be responsible for the low level of hcv-specific responses observed in hiv-progressors; ( ) ifn-alpha'/ribavirine therapy influence on hcv-and hivspecific cd -t-cell frequencies after months of treatment will be discussed. frequencies of hiv- -p specific th cells (elispot) are correlated with plasma hiv- viral load in a cohort of lt-np and slow progressors soa . martinez v a , alatrakchi n a , costagliola d b , bonduelle o a , agut h c , autran b a , alt study group a . a hopital pitié-salpêtrière, laboratoire d'immunologie cellulaire, paris, france , b faculté de medecine saint-antoine, inserm sc , paris, france , c hopital pitié-salpêtrière, laboratoire de virologie, paris, france background: hiv- -specific t helper- cell responses have been associated with long-term-non-progression (lt-np) in hiv infection but the correlation between frequencies of hiv- -p -specific th cells and viral load has not yet been studied. we prospectively quantified these frequencies by using an ifn-gamma elispot assay in a cohort of lt-np. methods: a cohort of lt-np and slow progressors (infection !/ years and cd counts !/ /mm ) was analysable. hiv- -p specific t cells were analyzed using: proliferation, ifn-gamma eli-spot assays and ifn-gamma production in cell supernatants. results: wide ranges were observed in the frequencies of hiv- -p -specific cd th cells as assessed by elispot ( - sfc/ pbmc) with a median of sfc/ pbmc. these frequencies were negatively correlated with viral load (r /(/ . , p / . ) but not with cd counts and associated with a low level of t cell activation assessed by cd on cd cells (r /(/ . , p / . ). similar results were obtained with t cell proliferation and ifn-gamma production. conclusion: interestingly, the numbers of hiv- -p -specific th cells correlate with plasma viral load, independently of cd counts indicating that: ( ) the defect in hiv- -specific cd th cells does not reflect the global cd depletion; and ( ) these responses are strongly correlated to the control of virus replication. impact of drug Á/drug interactions on therapeutical management of active tuberculosis in hiv infected patients soa . martinez v a , truffot c b , caumes e c , katlama c c , jarlier v b , bricaire f c , jouan m d . a department of infectious and tropical diseases, pitié salpêtrière hospital, institut pasteur, unité de génétique mycobactérienne, paris, france , b department of bacteriology, pitié salpêtrière hospital, paris, france , c department of infectious and tropical diseases, pitié salpêtrière hospital, paris, france , d institut pasteur, unité de génétique mycobactérienne, paris, france since and the use of haart, management of hiv patients with active tuberculosis raised the question of drug Á/drug interactions and therapeutical management of both infections. retrospective cohort study: follow-up of hiv patients with active tuberculosis diagnosed between and . studied data included evolution of tuberculosis and hiv, cd cell counts, plasma hiv viral loads, antituberculosis and antiretroviral regimens. ninety-four percent of patients were treated by quadruple combination antituberculosis drug with rifabutin for five patients. fourteen patients were treated by double antiretroviral therapy of nucleoside reverse transcriptase inhibitors (nrtis) and by triple or more drugs (nrtis and/or nonnucleoside reverse transcriptase inhibitors nnrtis and/or protease inhibitors pis). the median follow-up was months. there was no difference on cd cell counts and viral loads in the two groups and between the patients treated by nnrtis and pis at the diagnosis of tuberculosis, at the time of antituberculosis drug discontinuation and concerning cure rates of tuberculosis. on the other hand, the plasma hiv viral load was significantly better controlled in patients with nnrtis than with pis (p b/ . ). in hiv patients with active tuberculosis receiving haart, antiretroviral combination including nnrtis allows a better control of viral replication than regimen including pis without impact of the use of rifampin or rifabutin. adherence is essential to the effectiveness of antiretroviral therapy. a pharmacy visit would improve the patient advisement. a survey was carried out over a period of months in the u.m.i.t. a self-report was distributed to patients. ninety were evaluated. for %, information's given by the clinician were sufficient and for % the treatment advice cards were useful. however, % of them would like to attend a pharmacy visit. the topics they would prefer to be tackled were drug interactions ( %), side effects ( %) and effect of forgetting ( %). the treatment was well accepted and tolerated for, respectively and % of the patients. the viral load and the cd count were well known by, respectively and %. however, inaccurate pattern of treatment was frequent ( !/ %) and bad adherence was observed: treatment forgotten occasionally ( %), regularly ( %) or inadequate attitude when the treatment was forgotten ( %). number of pills, dose frequency, length of the treatment would be risk factors of nonadherence. for the majority of patients, a pharmacy visit is necessary and beneficial. the first result shows a better understanding of the treatment, an improvement of the adherence and an enhancement of plasma concentration of antiretroviral drugs. in vivo activity of glycopeptides against s. aureus infection in a rabbit endocarditis model: is mic predictive for in vivo efficacy? soa . asseray n, caillon j, lemabecque v, jacqueline c, batard e, potel g, bugnon d. laboratoire antibiologie, faculte de medecine, nantes, france we have studied the in vivo efficacy of vancomycin (v) and teicoplanin (t) against five staphylococcus aureus (sa) strains: two methicillin-susceptible (mssa and ), two methicillin-resistant (mrsa and ) and one glycopeptide-intermediate (gisa ) strain, in a rabbit endocarditis model. mics of v and t (v/t) were . / . , / . , / , . / . , and / , for mssa , mssa , mrsa , mrsa , and gisa , respectively. the animals were randomly infected with one of these strains, then treated for days by v or t. a continuous infusion of v, simulating a mg/kg/ h human dose was used. t was infused as a continuous infusion allowing simulating a mg/kg human dose, following an initial bolus. these regimens achieved clinically relevant serum steady-state concentrations of glycopeptides ( !/ mg/ l). results were as follows: expressed in log cfu/g of vegetations (mean /sd, followed in parenthesis by the number of rabbits used). purpose: to determine the prevalence of the decreased glycopeptides susceptibility among clinical isolates of staphylococcus aureus collected from patients hospitalized in strasbourg university hospital between / / and / / . the susceptibility to glycopeptides of s. aureus isolates collected from hospital environment during approximately the same period was also investigated. methods: the susceptibility to glycopeptides was studied among the mrsa isolates, using: Á/ detection of the decreased susceptibility to glycopeptides using agar plates containing mg/l teicoplanin, Á/ detection of hetero-visa strains using agar plates containing mg/ l vancomycin, Á/ determination of the mics of vancomycin and teicoplanin using the agar dilution and the e -test strips methods. results: thirty-nine percent of s. aureus clinical isolates ( out of strains) are mrsa. no visa or hetero-visa strain was detected. six percent mrsa isolates are teicoplanin intermediate s. aureus strains. in the environment, % s. aureus isolates are methicillinresistant (five out of ). the five strains are all susceptible to glycopeptides. conclusion: the results regarding vancomycin are reassuring. however, the high rate of mrsa and the presence of teicoplanin intermediate s. aureus isolates prove that prevention and control measures need to be improved. comparative investigation of polymerase chain reaction and a conventional methods for detection of methicilin resistant staphylococcus amont clinical isolates soa . kantardjiev tv a , vacheva-dobrevski rs b , panajotov sv a , bachvarova am a , velinov ti a , levterova vs a . a national center of infectious and parasitic diseases, microbiology, sofia, bulgaria , b military medical academy, clinical microbiology, sofia, bulgaria purpose: identification on methicillin resistant staphylococci has a great clinical implication and significant impact of antibiotic therapy. the aim of this study is to compare the disc-diffusion test (ddt), oxacillin agar screen test (oast) and pcr for detection of mec a gene. fifty selective clinical isolates ( staphylococcus aureus and nine s. epidermidis ) determined as methicillin resistant by ddt were enrolled in the study. ddt was performed with oxacillin disk ( mkg/ml) on mueller-hinton agar (mha) without nacl (nccls, ) . oast was performed on mha with % nacl, oxacillin mkg/ ml, t c. these strains was genotypically characterized for the mec a gene presence by pcr method using the mec a - ?-aaa atc cat ggt aaa ggt tgg c- ? and the mec a Á/ ?-agt tct gca gta ccg gat ttg c- ? primers (gibco, brl) . results: in the group of mrs isolates, detected by pcr, positive results were as follow: s. aureus and six s. epidermidis . for six s. aureus isolates ddt and oast were positive; pcr-negative. for two s. aureus and two s. epidermidis isolates pcr was positive; phenotypic methods-negative. conclusions: accurate and rapid detection of mrs is a constant challenge for laboratories. the pcr assay (first time in our country) appears to be more reliable than routine susceptibility testing for the rapid diagnosis of mrsa infections at hospitals, particularly due to the heterogeneous resistance of many strains. . / . ( ) . / . ( ) . / . ( ) . / . ( ) . / . ( ) v . / . * ( ) . / . ( ) . / . * ( ) . / . ( ) . / . ( ) t . / . * ( ) . / . ( ) . / . * ( ) . / . ( ) . / . ( ) distribution and antibiotic susceptibilities of bacteria isolated from suspected urinary tract infections of inpatients in hungary soa . rozgonyi f, csukás z, kamotsay k, szabó d, ostorházi e, berek z, maródi c. institute of medical microbiology, semmelweis university, budapest, hungary between l january and december , a total of , urine samples were cultured % as native urine (nu) and % as uricult-plus (up) (orion diagnostica, finland) . cultivations were negative in % of nu and % of up specimens, while contamination was revealed in % of nu and % of up. in the clinical bacteriologically evaluable positive nu and up cultures, the distribution of the gram-negatives was very similar with the predominance of escherichia coli ( and %) followed by enterobacter spp. the distribution of gram-positives differed significantly according to the types of specimens. nu resulted in % group-d and % group-b streptococcus while up did and . %, respectively. third generation cephalosporins and fluoroquinolons were equally very effective against e. coli strains, while ampicillin inhibited growth of % only. carbapenems, cefepime and the fluoroquinolons were the most active against enterobacter strains. interestingly, trimethoprim'/ sulfarmetoxazole combination could inhibit more than % of enterobacteriaceae strains. piperacillin'/tazobactam ( %), imipenem ( %), and ciprofloxacin ( %) could be the drog of choice against pseudomonas aeruginosa . enterococcus strains were highly sensitive to glycopeptides ( %), nitrofurantoin ( %), imipenem ( %) and amoxicillin'/clavulanic acid ( %). antimicrobial susceptibility levels of escherichia coli isolates cultured from urine at a tertiary care teaching hospital. temporal trend and comparison between community-acquired and nosocomial urinary tract infection soa . nanetti a, manfredi r, valentini r, calza l, chiodo f. uni versity of bologna, infectious diseases, bologna, italy in order to assess the local temporal trend of antibiotic sensitivity of the most common urinary tract bacterial pathogen, all urine-cultured escherichia coli isolates were reviewed as to susceptibility profile, and specimen source (community-versus hospital-acquired infection). when evaluating sensitivity levels of community-acquired pathogens ( Á/ ), a significant resistance rise was limited to cotrimoxazole (p b/ . ) and nalidixic acid (p b/ . ), while a tendency towards increased resistance regarded norfloxacin (p / . ) (fig. ) . when community-acquired e. coli isolates were compared with nosocomial strains (tested in the years Á/ ), a greater susceptibility of community-acquired e. coli isolates was limited to cotrimoxazole versus all other compounds in the year (p b/ . ), while it was extended to amoxicillin, cephalotin, nitrofurantoin and piperacillin in the year (p b/ . ) (fig. ) . on the whole, e. coli showed an elevated sensitivity rate ( !/ % of tested strains) to nitrofurantoin, gentamicin, amikacin, and nd-and rd-generation cephalosporins, while only amoxicillin and piperacillin had a mean resistance rate !/ %, regardless of the community or nosocomial origin. a permanent surveillance of sensitivity levels of the most common pathogens responsible for infectious diseases enables to identify local antimicrobial activity and its temporal variations, and plays a key role in starting empiric therapy, pending bacterial identification and in vitro assays. conclusion: in uti, the antimicrobial agents such as st cg combined with aminoglycosides are recommended as initial treatment as well as the rd cephalosporin generation at monotherapy. in addition, the fluoroquinolones and aminoglycosides are effective in uti. prevalence of resistance mutations to antirretrovirals and relation to virological failure s . garcia f a , suarez s a , alvarez m a , martinez nm a , valera b b , pasquau j b , hernandez quero j a , maroto mc a . a hospital san cecilio, microbiology, granada, spain , b hospital virgen nieves, microbiology, granada, spain purpose: to investigate the prevalence of resistance mutations in the reverse transcriptase (rt) and protease (p) genes of hiv and to relate it with the type of virological failure (vf), we have studied patients ( % naïve or pregnant women, % were first vf, % were second vf, % more than two vf. resistance mutations were investigated using trugene hiv- genotyping kit (visible genetics). results: global prevalence of resistance mutations for rt inhibitors (rti) has been !/ % for m l, d n, k n, m v, l w, t yf, and for l i, m i, l p, a vt, l m for p inhibitors (pi). the prevalence of resistance mutations for the naïve patients studied was very low (a g, v i for rti and l i, m i, m i */all n / */and l p n / for pi). for patients on first vf only k n, m v, t yf (rti) were !/ %, as well as l i, d n, l p (pi); when patients on second vf were studied, then m l, e d, k n, m v, g a, l w, t yf, k qe (rti) and m i, l p, a t (pi) were !/ % prevalence; finally, when patients with more than two vf were studied, the following resistance mutations were !/ %: m l, d n, k r, k n, v i, y c, m v, g a, l w, t yf (rti), and l i, m i, m il, l p, a t, l m (pi). conclusions: the prevalence of primary resistance in the population studied is very low; the prevalence of mutations in the reverse transcriptase and protease genes increase in parallel to the type of virological failure. genotypic resistance in hiv- rna from patient plasma compared with rapid virus isolation and phenotypic resistance in patient pbmcs s . stuermer m, groeschel b, cinatl j, doerr hw. institute for medical virology, university clinic frankfurt, frankfurt, germany objective: to compare hiv- virus isolation in the presence of antiretroviral drugs with plasma hiv- genotyping. materials and methods: hiv- genotyping was performed using the viroseqtm vers. from applied biosystems. interpretation of genotype was done according to international standards. cd -cells were purified from patient plasma and cultivated in microtiter plates coated with anti-cd and anti-cd antibodies in the presence of different concentrations of antiretroviral drugs. virus production was measured using a p antigen assay. phenotypic activity was expressed as % reduction of p concentrations. results: seventeen samples were analyzed. for samples results were obtained from both methods, two samples could not be analysed by phenotyping and four samples not by genotyping. only / samples showed total and / samples partial concordance, / samples showed discordance between the two assays. in discordant samples the genotype gave a definite interpretation. conclusion: hiv- virus isolation and phenotyping from pbmcs may overcome the problem of currently used resistance assays, which analyse only the reverse transcriptase and the protease gene of hiv- . possible mutations in other regions may influence viral fitness and therefore contribute to the growth of the virus population present. the lack of concordance between the two assays is related to the different blood compartments used. the clinical value of resistance tests using pbmcs is under investigation. interleukin- co-operates with a new type i ifn, ifn-tau to inhibit early steps of hiv-i biological cycle s . rogez c a , clayette p a , martin m a , dereuddre-bosquet n a , martal j b , dormont d c . a cea, drm, fontenay-aux-roses, france , b inra, physiologie animale, jouy-en-josas, france , c cea, crssa, ephe, drm, fontenay-aux-roses, france background: type i interferons (ifn) exhibit efficient antiviral activities notably against hiv, but severe side effects restrict their clinical uses. ifn-tau is an ovine or bovine non-cytotoxic type i ifn which displays higher inhibitory effects towards hiv replication than ifn-alpha, particularly in human monocyte-derived macrophages (mdm). the antiretroviral activity of ifn-tau seems to involve antiviral and immunomodulatory mechanisms: il- synthesis is increased in dose-dependent manner in mdm treated with ifn-tau and a specific inhibition of il- biological activity decreases its antiretroviral efficiency. results: after a -h infection, a significant decrease of intracellular hiv rna amount was found in mdm treated with ifn-tau. in parallel, no additive inhibition was observed with ifn-tau during the elongation of proviral dna. these results suggest either an inhibition of hiv nucleocapsid uptake or an immediate hiv rna degradation, and the expression of ?, ?-oas, mxa protein and pkr was then measured. ifn-tau induced the expression of these three host cell factors. the role of il- on these different steps was evaluated and we showed that il- co-operates with ifn-tau during the very early step of hiv biological cycle. conclusion: altogether, these results evidence that ifn-tau uses the same antiretroviral pathway as others type i ifn in mdm, and that il- takes part to its inhibition of early steps of hiv biological cycle. actinomycin-d as a modulator of resistances due to cell-wall active agents like bacitracin (bc) and lysozyme (lz) s . chakrabarty an a , dastidar sg b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india it was observed that development of lzr in the lzr mutants took placed at three different levels and was accompanied with unselected, distinctive and elevated levels of bcr. similarly, bcr in bcr-mutants were also detected at three different levels. although the levels of bcr ( / mg/ml) in the bcr mutants could be raised only by persistent efforts, an increase in the levels of lzr (as cross-resistance) in the same mutants could be easily achieved. a correlation of actinomycin-d resistance with lzr and bcr of the mutant bacteria and the effects of lipase treatment on the same showed a Á/ -fold rise in actinomycin-d resistance of the lzr and bcr mutants of gram-positive bacteria compared with their correspondence wild-types. these findings suggest that lzr and bcr are controlled by several genes accounting for reduced cell-wall and cell-membrane permeability and indirectly, by phenotypic alteration of the lipid content of the cell-wall. thus, the alteration of cell-walls and membranes and a phenotypic extra lipid layer can work in conjunction with the efflux pump mechanisms finally determining the levels of drug-resistance. experimental development of drug resistance to non-antibiotics: a role of alteration of membrane fluidity and efflux systems s . dastidar sg a , mazumdar k a , asok kumar k a , chakrabarty an b . a jadavpur university, pharmaceutical technology, calcutta, india , b department of medical microbiology, calcutta university, calcutta, india drug resistance among clinical strains was studied by selecting mutants resistant to promazine (pr) and methdilazine (md). the results showed that successive step-up mutants of pr and md developed cross-resistance to several unrelated drugs, which in subsequent steps had broader resistance spectra with higher levels of resistance. experiments on the membrane fluidity or permeability of bacterial cells using diphenyl hexatrine (dph), a fluorescent probe on md-mutants showed that three was marked reduction in the membrane fluidity and permeability. when several analogues of the basic phenothiazine structure, e.g. -chlor-methyl-n -methyl-pyrrolidine (cmp), methyl- -methyl- -pyrrolidone- -carboxylate (mmpc), hydroxymethyl-n -methyl-pyrrolidine (hmp) and md with final substitution were tested for antibacterial function on different strains, highest activity was observed with respect to md. with anaerobic bacteria the resistance(s) dependent on efflux pumps showed higher levels of resistance even to md. we have found the non-antibiotic agents triflupromazine, trimeprazine and diclofenac sodium have high degree of activity against vibrios, staphylococci and pseudomonads. the explanation of such a phenomenon in terms of possible efflux pumps will be discussed. csf, plasma and urine pcr in lyme neuroborreliosis s . pícha d a , moravcová l a , lásiková Š a , marešová v a , Ž ïárský e b . a charles university, nd medical school, st clinic for infectious diseases, prague, czech republic , b department of cellular and molecular biology, charles university, rd medical school, st clinic for infectious diseases, prague, czech republic the main reason for high diagnostic value of pcr in neuroborreliosis (nb) is the direct way of spirochete detection. two sets of primers in nested pcr were used: one for plasmide gene encoding ospc protein and second for chromosomal gene s rdna. so far patients with clinically manifested involvement in nb were enrolled into the prospective designed study (being continued). the main including criterion was positive prove of intrathecal specific antibody secretion (in patients) and pcr positivity in csf (in ). all patients were repeatedly examined by neurologist and samples of csf, plasma and urine were taken: ( ) before treatment; ( ) after treatment; ( ) after months. before treatment were patients pcr positive in csf, six in plasma, and in urine. five were parallel positive in csf and plasma and four in all three body fluids. urine after treatment was positive in seven ( %) cases and completely negative after months. the pcr has had relative high sensitivity ( %), but does not rich the sensitivity of antibody index ( %). supported by grant mzcr ; . consumption of imipenem correlates with b-lactam resistance in pseudomonas aeruginosa s . lepper pm a , hö gel j b , trautmann m a , grusa e c . a department of medical microbiology and hygiene, university of ulm, ulm, germany , b department of biostatistics, university of ulm, ulm, germany , c hospital memmingen, central pharmacy, memmingen, germany purpose: in the present study we investigated the monthly consume of three anti-pseudomonas-active antibiotics, namely imipenem, piperacillin/tazobactam (pt) and ceftazidime during a period of years ( Á/ ) . the use of these antibiotics was correlated to the rate of resistance in pseudomonas aeruginosa . results: inspection of the time series for use of imipenem, ceftazidime, and pt, and the corresponding time series for resistance (each available from july to july ) indicates a remarkable coincidence between use of imipenem and resistance against the three antibiotics mentioned. pearsons's coefficient of correlation for the use of imipenem and the resistance against imipenem was . (p b/ . ), between imipenem use and pt resistance was . (p b/ . ), and between imipenem use and ceftazidim resistance . (p b/ . ). we found positive regression coefficients quantifying an association with imipenem use in the same month (p b/ . ) and with the use during the preceding month (p b/ . ). the same was true when checking dependence of ceftadizime resistance (p b/ . ) and pt resistance (p b/ . ) on imipenem use observed during the same month. neither the use of ceftadizime nor of pt could be identified as factors creating resistance to one of the three antibiotics under consideration within a reasonable period of time. conclusion: there might be a strong pressure towards resistance created by carbapenems. this could limit the use of carbapenems for initial empiric therapy. treat hard and fast: short course antibiotic treatment and its relation with patient compliance and effectiveness s . perez-gorricho bpg a , ripoll m b , pechere jc c . a niño jesus hospital, infectious diseases, madrid, spain , b insalud, outpatient consult, madrid, spain , c university of geneve, microbiology, geneve, switzerland 'treat hard and fast ': short course antibiotic treatment and its relation with patient compliance and effectiveness. finding the important implications for the way in which physicians manage patients with mild Á/moderate respiratory tract infections, and the relation of this management with the perception of antibiotic effectiveness, and the compliance with the antibiotic regimen has been the main purpose of the research. in a pan-european market research study of more than patients, designed to determine behaviour to the antibiotic management of mild-moderate respiratory tract infections, patient expectations of antibiotic therapy were identified, particularly those aspects that relate to efficacy and compliance. the study identifies three key drivers of patients perceived antibiotic efficacy: length of antibiotic course, time to onset of symptom relief and time to complete resolution of symptoms. the results demonstrate that once daily treatment for short periods is perceived by patients to be significantly more effective than longer antibiotic courses and thus better meets patient expectations of therapy. in this study, a macrolide, azithromycin, was selected as the drug therapy of shortest course, being the antibiotic with the shortest dosage schedule for common outpatient infections. the perception of efficacy with short course therapy also correlates with overall satisfaction with management by the physician and with patient compliance with antibiotic therapy. purpose of the study: group b streptococci (gbs) remain a major cause of neonatal infections. consensus guidelines have recommended an intrapartum antibioprophylaxis by amoxicillin, which has reduced the incidence of early-onset neonatal gbs infections. however, an increased incidence of beta-lactam-resistant gram-negative neonatal sepsis has been reported. the aim of our study was to analyse the consequences of this antibioprophylaxis on the intestinal microbial colonization of newborns. a study of the fecal flora was carried out on stools samples from days-old newborns divided into groups: group a intrapartum treated mothers (n / ); and group b untreated mothers (n / ). both groups were matched with regards to known factors affecting intestinal microbial colonization: gestational age, type of delivery and feeding. results: colonization by enterobacteria and enterococci was not significantly different and occurrence of amoxicillin-resistant enterobacteria was similar ( / and / in groups a and b, respectively). however, the colonization by clostridia was modified: the number of newborns colonized was significantly less important in group a than in group b (group a: / and group b: / p b/ . ). conclusion: in our study, intrapartum antibioprophylaxis did not affect intestinal colonization by aerobes but reduced significantly colonization by clostridia, potentially anaerobic pathogens. impact of an antibiotic policy restricting the use of b-lactams and macrolides on the incidence of clostridium difficile associated diarrhoea in general medical, renal and elderly patients s . boswell tc a , pacey s b , broomfield s c , westmoreland d c , yates c c . a nottingham city hospital, microbiology, nottingham, uk , b nottingham city hospital, pharmacy, nottingham, uk , c nottingham city hospital, infection control, nottingham, uk the purpose of the study: to investigate the short-term impact of a new antibiotic policy for the treatment of urinary and respiratory infections on the incidence of clostridium difficile associated diarrhoea (cdad) in hospitalised medical, elderly care and renal patients. the results obtained: a policy restricting the use of b-lactams (except parenteral penicillin), and promoting alternative antibiotics including levofloxacin for pneumonia, and doxycycline for non-pneumonic respiratory infections, was launched in july . as a result there was a significant and sustained reduction in use of aminopenicillins, cefuroxime and macrolides, with a corresponding increase in doxycycline and levofloxacin. the incidence of cdad was determined during the st months of the new policy and compared to the last months of the old policy. the incidence of cdad fell from . to . per patients, and from . to . per in-patient days (p b/ . ). in contrast, there was no change in the incidence of cdad in other specialties (surgery, oncology etc.) that had not introduced the new policy. there was no change in the incidence of nosocomial bacteraemia with quinolone-resistant coliforms or mrsa, despite the increased use of levofloxacin. conclusions: hospital-wide reduction of b-lactam and macrolide use in medical patients can result in a significant and immediate reduction in cdad. longer follow-up will determine if this effect is sustained. use of imipenem/cilastatin i.v. (tienam i.v.) for the treatment of low respiratory tract infections in intensive care units s . izzo l a , orsetti r b , boschetto a a , binda b a , della casa u a , caramanico l a , la mazza a a . a department of surgery, universitá degli studi di roma 'la sapienza','p. valdoni', rome, italy , b s. camillo-forlanini, intensive care unit, rome, italy ventilator associated pneumonia (vap) is considered the most frequent infection in the intensive care unit (icu), occurring in Á/ % of patients intubated for longer than h besides nosocomial pneumonia is a common complication in the critically ill surgical or trauma patient. inadequate treatment can lead to the complications of acute respiratory distress syndrome (ards), empyema, and lung abscess. the most important aetiological agents both in vap and in pneumonia which arise as complication in surgical or trauma patients are bacteria, whit a marked predominance of staphylococcus aureus and pseudomonas aeruginosa . the authors present their experience ( cases) on the employment of imipenem/cilastatin i.v. (tienam i. v.) as initial empirical monotherapy at the dose of mg)/ /day or g)/ / day for the treatment of the serious lower respiratory tract infection in an icu. tienam is a well tolerated broad spectrum antibacterial agent that is effective against the majority of gram-positive and gram negative aerobic and anaerobic bacteria including most pseudomonas species. except one patient deceased for causes related to his very poor general conditions and three cases in which has been necessary the addition of an aminoglycoside, in all the other patients the imipenem/ cilastatin (tienam) monotherapy has shown satisfactory clinical and bacteriological responses. clinical auditing of the impact of recommendations on antibiotic treatment s . kinoo j a , david-ouaknine f a , hacquard b a , echard y a , decazes jm b . a centre hospitalier lagny marne la vallée, lagny sur marne, france , b hospital saint louis, paris, france the aim of this study was to assess the impact of curative antibiotic recommendations on suitable prescriptions at lagny-marne la vallée hospital (general hospital, beds). two prospective exhaustive audits were made (all complete hospitalizations, excluding psychiatry, february Á/may and ) of the detailed curative antibiotic prescriptions, before and after distribution of internal recommendations. the same methodology, designed by a multidisciplinary team, was used for both periods. the same antibiotics were available at the pharmacy. the prescriptions were assessed by an infectious diseases specialist and a pharmacist using pre-established criteria: literature recommendations ( audit), internal recommendations ( audit). six hundred and fifty-six prescriptions for patients were collected and analysed in , for patients in . exhaustivity of the recovered prescriptions was over %. patient characteristics, infection sites and microbiological findings were similar for both groups. suitable prescriptions were significantly increased ( Á/ %, p b/ . ). unsuitable prescriptions (economic reasons, too short or too long course, incorrect administration, or underdosage) were significantly reduced. prescriptions for incorrect indications were unchanged and necessary combined treatment not being prescribed, increased. local recommendations improved prescriptions, but efforts have to be done in order to go on the improvement of the practice behaviour. cost-effectiveness analysis of antibiotic therapy in hospitalized patients with copd exacerbations (ae-copd) s . beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy antibiotic costs represent a high burden of total drug costs for hospital administrations. a scientific approach considering also the economic aspects of each therapeutic decision may gain optimal treatment objectives at pondered costs. in our study we retrospectively evaluated the clinical effectiveness and costs of antibiotic therapy in patients with ae-copd. from to , our retrospective study results support previous pharmaco-economic considerations according which in choosing an antibiotic regimen for ae-copd we must take into consideration the expected clinical and microbiological results without forgetting to consider the economic burden of our decisions. significant increase in fungaemia due to non-albicans candida species s . shah pm. klinikum der j.w. goethe universitaet, schwerpunkt infektiologie, frankfurt am main, germany until , predominant candida species in blood cultures was candida albicans . it accounted for . % of all candida species cultured from blood. since then we have observed a gradual increase in number of non-albicans candida species. from , onwards nonalbicans candida species out-number c. albicans . this observation is especially important as non-albicans candida species are generally non-susceptible to azole derivatives and empirical use of azoles in suspected candidaemia should not be recommended. amphotericin b is uniformally active against almost all candida species. echinocandin may be an alternative. see figure below. a search for newer antifungal chemotherapeutics s . chakrabarty an a , dastidar sg b , saha b b , basu l b . a calcutta university, medical microbiology, calcutta, india , b jadavpur university, pharmaceutical technology, calcutta, india fungal infections due to the mucor-rhizopus (m-z) group present formidable problems due to lack of appropriate and effective drugs against them, as seen in increasing number of clinical situations; death due to mycoromycosis is nearly inevitable. we analysed the biological 'weak-spots' of the mucor-rhizopus group and attempted to devise suitable drugs using their weak-spots. we have noted that like many free-living fungi, the m-z fungi are facultatively chemoautotrophic (can grow on simple sources of carbon and nitrogen and a solution of mineral salts), like the human pathogenic chemoautotrophic nocardioform bacteria. we devised a minimal medium based on that of davis and mingioli, supplemented with simple chemical compounds as sole sources of carbon and nitrogen. the key chemical here was diphenylamine with trypan blue (dpa Á/tb) and other similar sources of c and n. we found that while media free of these chemicals (controls) allowed good growth of different strains of m-z fungi, a mixture of dpa Á/tb completely prevented their growth over a wide concentration range. experiments with immunocompromised mice showed that these drugs at the concentrations used are well tolerated; mice experimentally infected with several clinical isolates of m-z fungi and receiving these chemicals showed that these fungi could not grow in vivo. in vitro activity of newer fluoroquinolones against multi-drug resistant salmonella typhimurium s . nolones resistance is being also reported. we have studied the in vitro activity of b-lactams and fluoroquinolones against multi-drug resistant s. typhimurium from human sources. material and methods: fifty multi-drug resistant s. typhimurium were tested against cefazolin, cefuroxime, cefotaxime, cefepime, ofloxacin, levofloxacin, and moxifloxacin, by the agar dilution method according nccls guidelines. results and conclusions: all the strains were resistant to four or more of the following antibiotics: ampicillin, tetracyclines, chloramphenicol, streptomycin, sulphonamides and nalidixic acid. a high proportion of strains were intermediate or resistant to amoxicillin/clavulanate. we found no resistance to cephalosporins. nevertheless, % were intermediate to first and/or second gen. cephalosporins. cefotaxime and cefepime were the most active cephalosporins (mic : . mg/l). though increasing fluoroquinolones resistance has been described among this kind of strains, no resistance to fluoroquinolones was found here. levofloxacin was the most active fluoroquinolone (mic : . mg/l), followed by ofloxacin (mic : . mg/l) and moxifloxacin (mic : . mg/l). high rates of resistance to antibiotics by salmonellae from diarrhoeic children in zliten-libya s . ghenghesh ks a , ben ali m b , abuhelfaia a b , dufani ma a . a faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya , b faculty of arts and sciences, el-ghomes university, biology, el-ghomes, libyan arab jamahiriya salmonellae are major bacterial cause of diarrhoea in libya particularly in children. included in the present study salmonella species isolated from children with diarrhoea in zliten city-libya. the children aged between a few days to years. the organisms were tested for their susceptibility to antibacterial agents using the disc diffusion method. of the isolates examined, ( %) were resistant to ampicillin, ( . %) to amoxicillin Á/clavulanic acid combination, ( %) to cefoxitin, ( . %) to chloramphenicol, ( . %) to doxycycline, ( . %) to gentamicin, ( . %) to nalidixic acid, ( . %) to trimethoprim Á/sulphamethoxazole and none ( . %) were resistant to norfloxacin. a strong relationship was observed between the availability of antibiotics in the pharmacies of the city and resistance of the isolated salmonellae to these drugs. the misuse of the antibiotics by the community may be an important factor (among others) in the emergence of these high rates of resistance by the salmonellae examined. effect of ceftriaxone along with probiotics administration on intestinal ecosystem and betalactamase activity s . bertazzoni minelli e a , benini a a , zoppi g b . a department of medicine and public health-pharmacology section, university of verona, verona, italy , b department of paediatrics, university of verona, verona, italy oral bacteriotherapy during antibiotic treatment is a much debated topic. aim: to study whether different probiotics can prevent imbalance of the intestinal ecosystem (dysbiosis) in children during therapy with ceftriaxone (cx). methods: fifty-one children (mean age . years) with febrile respiratory tract infections were treated with cx mg/kg/day iv, alone (therapy ) and along with the following preparations: saccharomyces boulardii ( ); enterococcus spp. ( ); lactulose ( ); l. casei gg ( ); l. rhamnosus , l. bifidus and l. acidophilus ( ); b. bifidum and l. acidophilus ( ); and a mixture of various lactobacilli and bifidobacteria at high concentrations ( ). faecal samples, collected before and after treatment, were analysed for microflora composition, cx concentration, and beta-lactamase (bl) activity. results: cx causes intestinal dysbiosis. no c. difficile was found. faecal bl increased after therapy in all treated groups. cx alone increased bl activity in % ( / ) of children (no activity before treatment); a higher incidence ( Á/ %) was found in groups and . after therapies , , , , and , bl activity was found in or more children. cx was detected in % of faecal samples. conclusions: the probiotics administration seems to protect against dysbiosis caused by cx and to contain the increase in faecal bl activity. the effects differ according to the probiotic administered and are peculiar to certain bacterial species. these preliminary data need further studies. comparative study of initial and acquired drug resistance in pulmonary tuberculosis in iran s . mansoori d, arami s, mirabolhasani z. nritld, infectious disease, tehran, islamic republic of iran purpose: resistant to anti-tuberculosis agents particularly multiple drug resistant (mdr) is a major obstacle in treatment tuberculosis in the world. between september and march for smear and culture positive pulmonary tuberculosis patients (old / , new / ) pretreatment susceptibility tests of isolated bacilli to inh, rif, emb and stm were performed by standard proportional method and the results were attributed to three groups: (i) newly diagnosed without any history of treatment; (ii) patients with history of treatment for one course; (iii) patients with history of treatment for two or more courses supposed to be mdr cases. the results were collected for each drug individually and different combinations of two, three and four medications. results: resistance to one, two, three and four drugs was significantly increased in group iii comparing to groups ii and i, also in group ii compared to group i. we observed a high rate of primary resistance to inh and stm in groups i and ii and a high rate of mdr (inh and rif resistance) in groups ii and iii. conclusion: the duration of bacilli exposure to antituberculosis agents in the past is a major factor in developing resistance. in contrast to who's guideline, due to high rate of primary resistance especially to stm in our area, we do not recommend addition of stm for treatment of patients whose initial four-drug regimens have been failed (group ii). donors, to understand host interactions with this bacteria, to develop new methods of diagnosis and define new vaccine candidates. nineteen tb patients and seven healthy donors were enrolled in french hospitals. cellular immune responses were evaluated by lymphoproliferation and ex-vivo quantification of specific th cells by elispot-ifn-gamma assays. four recombinant proteins of m. tuberculosis were tested: esat- , b, erp and tb b . and compared with tuberculin. we confirmed that b (but not esat- in our study) gives higher responses in tb patients compared to donors according to the results in proliferation assay (p / . ). in addition, frequencies of th cd specific cells for esat- and tuberculin were statistical different between the two groups (p / . and . , respectively). . % for b and . % for esat- of the patients tested were responders in elispot-assay versus . % for both in proliferation assay. for the new antigens erp and tb b . , no difference was observed between the two groups. in conclusion, b and esat- are recognised by a large number of our patients. they seem to be promising antigens for the development of new methods of diagnosis or for the development of new vaccines. erp and tb b . are not preferentially recognised by tb patients. other exported antigens will be tested. the incidence of tuberculosis (tb) is increasing worldwide. in recent some years, geographical differences in the incidence of tb in former yugoslavia have been observed. an important rise in tb cases was registered in the bordering region of bosnia. it is likely that poorer living conditions, influenced by war and emotional stress, may promote such rising incidence of tb. renal tuberculosis was diagnosed in patients (female , male ) from district brcko in bosnia, during the period of years, Á/ . at the same time none patient had active pulmonary tb lesions, fibrous lesions were noticed in patients, but we did not diagnose any signs of previous pulmonary tb in seven patients. seven patients developed relapse of renal tb after Á/ years of previous treatment. guided by clinical parameters, precisely done renal echosonography enabled early suspicion and searching for renal tb, by radiological and other methods. bacteriological diagnosis was performed by detection mycobacterium tuberculosis on loewenstein Á/jensen medium in patients. pcr as simple, fast and highly sensitive method enabled diagnosis in incipient stadium of disease, so antituberculous therapy could be instituted some months earlier. prompt diagnosis of renal tuberculosis (using pcr, besides standard methods) is necessary, otherwise delayed diagnosis may be dangerous. a study on resistance to first generation anti-tuberculosis drugs in mycobacterium kansasii s . mirsaeidi sm, farnia p, mohammadi f, mansoori sd, jabbari r, taghizadeh r, masjedi mr, velayati aa. national research inistitute of tuberculosis and lung diseases, infectious diseases and immunology (nritld ), tehran, islamic republic of iran purpose: this research has been performed to determine antibiotic resistance of atypical mycobacteria especially mycobacterium kansasi . results: twenty-three pigmented colonies which indicated atypical agents from nritld's mycobacterium culturebank were selected, they then underwent type identification and antibiogram for inh, rif, etb, stm. nine samples were m. kansasi and were other non-mtb, was m. gordonei , m. xenopi , mac, m. bovis , m. tererra , m. asiatioum , m. marinum and . mean age in m. kansasi cases was '/ . year and in non-kansasi cases '/ . year and in whole society ntm was . '/ . . frequency of resistance in kansasi group were to inh ( %), to rif ( %) to etb ( %), to stm ( %) and prevalence of mdr was ( %) and in non-kansasi group frequency of resistance were ( %) to inh, to rif ( %), to etm ( %) and to stm was ( %), to mdr was ( %). conclusion: a significant difference was seen between the age groups of patients who are affected with m. kansasi and non-kansasi (p b/ . ), also in frequency of resistance to first generation anti-tb drugs. m. kansasi is detected as the most common atypical mycobacterium agent in pulmonary infections and attention to antibiogram is recommended before treatment. buruli ulcer caused by mycobacterium ulcerans , is the third most common mycobacterial after tuberculosis and leprosy in west africa. nowadays, the only effective treatment is surgery. it consists in a large excision of the lesions, often followed by a skin transplant. in this study, the effectiveness of rifampin, amikacin and their combination were estimated in the treatment of mice, which were infected experimentally by m. ulcerans . after weeks of treatment with rifampin, amikacin or their combination, no more viable bacilli were found in infected tissues. the animals were kept for other months. among the mice treated with rifampin alone, two mice out of relapsed. the minimal inhibitory concentration of these isolated strains went from . to mg/ml. the dna sequence, obtained from a -pb of the rpob gene from these strains, showed a missense mutations, which affect a ser- replaced by a phenylalanine. this modification on the gene leads to an important inefficacy of treatment when rifampin was used alone. this study showed that rifampin and amikacin have a bactericidal action on m. ulcerans and that a combination of these antibiotics is necessary to avoid the selection of resistant mutants. histopathologic and electron microscopy studies of a severe isolated hiv enteropathy detected in an aids presenter. favorable response to haart introduction or . manfredi r, calza l, chiodo f. university of bologna, infectious diseases, bologna, italy advanced hiv infection was detected in a heterosexual female with a -year history of chronic diarrhoea and severe wasting, as expressed by a body weight of kg, a cd '/ count of cells/ml, and a plasma viraemia of . million copies/ml. a malabsorption syndrome was confirmed by d-xylose test, but repeated pathogen search tested negative at stool examination and light microscopy, scanning electron microscopy (em), and transmission em study of enteric mucosa. em assays detected an ultrastructural modification of duodenal mucosa never reported to date: an extensive thinning of enterocytic microvilli, disappearance of glycocalix, and large vacuolization of the enterocyte cytoplasm. two weeks after starting an indinavir-based haart, diarrhoea disappeared and our patient significantly gained body weight: kg after months, kg after , and kg after year, paralleling a cd '/ increase to cells/ml, and undetectable hiv viraemia. the subsequent -year follow-up confirmed absence of gut disturbances, a stable body weight, a cd '/ count of Á/ cells/ml, and hiv viraemia persistently b/ copies/ml. repeated endoscopy and related histopathologic and em assays documented a notable improvement of mucosal damage, with complete cure reached after years of haart. a direct intestinal localization of hiv may be responsible for severe diarrhoea, malabsorption, and wasting, though the morphological features of hiv enteropathy are still unclear. haart acts favourably also against isolated hiv-related enteropathy. kaposi's sarcoma in a non-hiv immunocompetent adult: relapsing due to the development of a squamus cell carcinoma or . sioula e a , magira ee a , georgopoulou c a , rontogiani d b , gounaris t a . a evagelismos, internal medicine and infectious diseases, athens, greece , b evagelismos, pathology, athens, greece a -year-old heterosexual hiv negative girl was diagnosed with cutaneous kaposi's sarcoma. the disease was started years earlier with the appearance of lesions on the left feet and on the right knee. absolute number of cd and cd were and cells/dl, respectively with a decreased lymphocyte proliferation. human herpesvirus type had been detected in biopsy specimens and she placed on recombinant interferon alpha- b. follow up few months later the lesions decreased in size. two years after the onset of the disease the patient readmitted because of a mass on the left paratracheal region along with mediastinitis. her body temperature was increased. the patient underwent thoracic ct scan, which demonstrated mediastinal well defined soft tissue infiltration associated with mediastinitis and a well-defined mass in the left paratracheal region. the mass biopsy revealed squamous cell carcinoma. several violaceus lesions were observed on the arms, hands and face. severe bilateral lymphedema of the legs with a reddish papules nodules and tumors from . to cm in diameter on the soles, toes and calves were present. this case illustrates the significant relapsing of the cutaneous kaposi's sarcoma soon after the appearance of and the carcinoma and the mediastinitis. a -year-old male patient with insulin-dependent diabetes underwent cardiac surgery for aortocoronary bypass years ago. two weeks after surgery he developed mediastinitis and sternal osteomyelitis caused by methicillin-resistant staphylococcus aureus (mrsa). twice, revisions and plastic surgery for sternal osteomyelitis were performed. the patient received initially treatment with vancomycin. then the patient received intravenous outpatient treatment with teicoplanin for weeks followed by by treatment with fusidic acid and then trimethoprim/sulfametrol. the fistula was closed. four months later he presented again with substernal pain and purulent discharge. the culture revealed the growth of staphylococci which were first mistaken for coagulase-negative staphylococci. after closer investigation these staphylococci were identified as small variant mrsa. computer tomography (ct) revealed multiple mediastinal abscesses. the patient was treated with intravenous linezolid mg bid for days and then switched to oral linezolid mg bid. the oral therapy was pursued for weeks under close surveillance. the patient improved substantially, the purulent discharge disappeared. the mediastinal abscesses were not detected any longer by ct at the end of treatment. the treatment with linezolid was well tolerated. platelets decreased initially but rose to normal values without treatment modification. nosocomial pneumonia due to stenotrophomonas maltophilia in a profound granulocytopenic patient hospitalized for community-acquired staphylococcus aureus severe sepsis or . radulescu a a , sasca n b , lupse m c , tatulescu d c . a university of medicine and pharmac, epidemiology, cluj-napoca, romania , b the teaching hospital of infectious diseases, laboratory, cluj-napoca, romania , c university of medicine and pharmacy, infectious diseases, cluj-napoca, romania objective: to present the diverse opportunistic infections in immunocompromised patients and treatment difficulties. findings: a -year-old women was admitted to the teaching hospital of infectious diseases cluj with a -day history of fever, myalgia, lumbar pain, hemorrhage syndrome. severe sepsis was diagnosed and the conditions that evolved in it were paronychia in a patient with chronic leukemia having prolonged and profound granulocytopenia due to aggressive treatment with ifn. the condition at admission was critical due to trombocytopenia ( b/ platelets/ml) and hemorrhage syndrome. the evolution was favorable under antimicrobial treatment (imipenem), blood and platelet transfusion, intravenous immunoglobulins, granulocyte colony-stimulating factor, antifungal prophylaxis and supportive care. blood and pus cultures revealed mssa. in the th day of hospitalization she developed bronchopneumonia and respiratory failure. the sputum culture was positive for stenotrophomonas maltophilia susceptible to ceftazidime, fluoroquinolones. treatment was unsatisfactory until introducing ticarcilline Á/clavulanate and ciprofloxacine. she had uneventful recovery despite remaining granulocyto-trombocytopenic. conclusions: treatment of infections with emerging agents in immunocompromised patients is difficult, guidance by results of susceptibility testing being misleading with a poor correlation between the tests and treatment outcome. early disseminated listeriosis in a liver transplant recipient (ltr): a rare case due to an in vitro multiresistant strain or . manfredi r, de ruvo n, vivarelli m, bellusci r, montalti r, la barba g, abtueli aden a, cucchetti a, attard l, calza l, cavallari a. university of bologna, infectious diseases, bologna, italy a ltr receiving cyclosporin, azathioprine and steroids, developed an extraordinary episode of sepsis and pleural effusion due to a multiresistant listeria monocytogenes (lm) isolate. a lm strain serov. showing the same, extensive resistance pattern (all penicillins and stand nd-generation cephalosporins), was isolated from multiple blood cultures and pleural fluid weeks after surgery, while stool exam was negative. our p spent her life in countryside and bred some animals, but denied consumption of uncontrolled food. iv cotrimoxazole administration achieved a complete clinical and microbiological cure in days. underlying immunodeficiency may prompt unusual/severe lm infection, but because of its usual community-acquired origin, lm disease remains infrequent in hospitalized p. only seven anecdotal reports of lm infection were described in ltr, Á/ , all but occurring months Á/years after surgery. an early respiratory and systemic infection caused by a community-acquired lm strain which proved resistant to first-choice antibiotics, but had a favorable response to cotrimoxazole (used only once in a ltr with lm sepsis), characterized our episode. an epidemiological survey retrieved the possible source of this usually community-acquired infection. lm should be regarded as an emerging opportunistic pathogen in ltr, and specific risk factors should be seeked. when immunodeficiency is of concern, the unpredictable sensitivity of lm should prompt in vitro assays to adjust antimicrobial therapy problems for discussion hbv Á/hcv and liver carcinogenesis: where does the viral influence end? or . pappas ga. university hospital, internal medicine, ioannina, greece hepatocellular carcinoma (hcc) is a major clinical problem worldwide, usually evolving over a long-standing liver pathology, in the latter stages in the form of cirrhosis. hbv and hcv chronic infection is a common etiology of cirrhosis, and hence, hcc. a number of studies have attempted to clarify the role of these viruses into the progression towards hcc. does their end in the stage of cirrhosis? is progression towards hcc independent of the etiology of cirrhosis (since alcoholic cirrhosis also proceeds to hcc)? do the trials with interferon alfa for patients with hbv or hcv cirrhosis exhibit a favorable result due to the antiviral properties of interferon, or is interferon exhibiting anti-oncogenic potential?, and is hcc cytokine and hormone sensitive (view ongoing trials with somatostatine analogues versus hcc)? (hence, if we treat alcoholic cirrhosis patients with interferon could we have a favorable response?). which patients with hbv or hcv cirrhosis are eligible for interferon treatment?: interferon treatment is a potential hazard for those with thrombocytopenia. how ethical is it to conduct a randomised trial where one leg of cirrhotic patients is left without antiviral therapy? and on the basis of which classification system should the two legs of such a trial be separated? moreover, do viral proteins with oncogenic potential exist (the controversy over the recently discovered hbv protein is still, unresolved)? a major topic awaiting for a major debate. to assess the role of hiv-associated campylobacteriosis (c) according to haart availability, patients with positive culture were identified since . compared with the Â/ hiv-infected p followed in the last decade, no epidemiological differences were shown, save a greater sexual exposure to hiv (p b/ . ). the introduction of haart caused a drop of frequency of c (from . to . episodes per p-year; p b/ . ), and modified clinical features, with disappearance of dissemination and mortality, reported in and patients before (p b/ . ). hiv-related immunodeficiency and disease stage were significantly related to c features before and after haart availability: p b/ . for cd and neutrophil count, p b/ . for aids diagnosis. most cases ( ) were community-acquired, but alimentary or environmental risk factors were never found. ten patients received cotrimoxazole prophylaxis (nine before ; p b/ . ), while no relationship occurred with steroid or antibiotic use, caused cases out of . a % sensitivity was found to quinolones, followed by cephalosporins ( . %), gentamicin ( . %), macrolides ( . %), and cotrimoxazole ( . %). a Á/ -day antimicrobial therapy cured p , but relapses caused by similar strains occurred in patients within Á/ weeks, all in the pre-haart era (p b/ . ). c still occurs in the haart era, probably due to its varied mode of transmission. the frequency of c is greater in hiv-infected patients, but less frequent visceralization, recurrences, and mortality characterized the haart era. objective: to determine the incidence and risk factors for nosocomial viral respiratory infections (nrvi) and involvement of human coronaviruses (hcov) in a neonatal and pediatric intensive care unit. methods: prospective observational study. nasal samples were obtained by cytological brush at admission and weekly thereafter for all hospitalized infants. nasal samples were taken monthly from staff. virological studies were performed, using immunofluorescence for respiratory syncitial virus (rsv), influenza viruses, paramyxoviruses, and adenoviruses; both immunofluorescence and rt-pcr were used for hcov detection. results: during , hcov related nrvi were detected in nn and six in children. three hcov-related outbreaks were observed (february, august and december), associated with a high prevalence of infection in staff. during august outbreak, hcovinfected nrvi were detected over hospitalized infants. seventy-five of hospitalized preterm nn with gestational age under weeks and . % of staff members were infected. risk factors for nrvi in nn were birth weight, gestational age, ventilation, oxygenation and hospitalization length. ninety-two percent of infected preterm nn were symptomatic, mainly with bradycardia and respiratory worsening. conclusions: these data provide additional evidence for a significant role of hcov in nrvi occurring in hospitalized preterm nn. strain typing and screening of dna targets to assess echinococcus sp transmission in new and old geographic endemic foci or . bart jm a , piarroux r a , dia l b , benchikh-elfegoun mc c , vuitton da a , bardonnet k a . a serf, parasitology, besancon, france , b national centre of veterinary study, parasitology, nouakchott, mauritania , c university of mentouri, parasitology, constantine, algeria purpose: cystic echinococcosis is due to echinococcus granulosus. parasite cycles depending on the main intermediate host species involved in different foci have been described promoting mixed infection in the same definitive host. strain typing is a tool to identify the main intermediate host involved via the dogs in the human infection route and to focus the control measures. many dna targets have been used to compare samples and to access the parasite cycle in different countries. but no study has compared the value of each of these targets. eight targets have been tested in mauritania where echinococcosis is an emergent disease, and in algeria where strain typing has never been done. thirty-five cyst samples from human, ovine, camel and bovine have been tested with six nuclear and two mitochondrial targets. results: the two mitochondrial targets and four out the six nuclear targets have allowed to discriminate the different foci. two strains have been found infectious to human : the 'sheep' strain in algeria and the 'camel' strain in mauritania. conclusion: although overlapping geographically sometimes, this raises the question of the respective genetic evolution of the different strains and of their involving in human infection. alveolar echinococcosis in france: an update or . bardonnet k, bresson-hadni s, bartholomot b, gérard a, watelet j, beytout j, saurin jc, piarroux r, vuitton da, who centre collaborating for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the highest prevalence rate for alveolar echinococcosis (ae) in europe has been found in france. in , a french observatory of human ae was done in order to get data that could be used to evaluate presentation, evolution and management of ae. material-methods: french cases were collected for the period Á/ . registration of every case was performed with the subject's agreement. a questionnaire was filled in by referring to the patients' medical files or to practitioners or to patients themselves. completeness of the collection of cases was ensured by multiplying the sources of information. results: two hundred and sixty nine french patients were registered. sex ratio averaged . mean age at diagnostic was . years. . % of diagnosis was performed in 'echinococcosis free' french areas. symptoms, but not always specific liver symptoms, were present at diagnosis in . % of cases. the liver was the main location of lesions in . % of cases. a wide spectrum of management of the patients was observed, accounting for regional differences. conclusion: this french observatory of human ea will facilitate a better management of the disease at the national level. it shows new epidemiological trends, and especially an extension of the endemic area. can coins and paper currency transmit bacillus anthracis ? or . ghenghesh ks. faculty of medicine, al-fateh university, medical microbiology, tripoli, libyan arab jamahiriya anthrax is an often fatal bacterial infection caused by bacillus anthracis . recent events that began in september in us has gained the organism worldwide attention and heightened awareness of and concern about anthrax. many cases of anthrax with a number of deaths have been reported as a result of contact with envelopes, sent through postal mail, containing b. anthracis endospores. a number of studies have shown that currency is colonized with bacterial organisms, that include enteropathogens (e.g. shigella sp.), other enteric flora (e.g. escherichia coli ) and potential pathogens (e.g. staphylcoccus sp. , pseudomonas sp. and bacillus sp.). furthermore, methicillin-resistant s. aureus (mrsa) isolates that produced enterotoxin (seb) and toxic shock syndrome toxin- also been reported. all of these studies do agree on that currency may be considered as a method of spreading potentially pathogenic and pathogenic bacteria in the community. therefore, currency could also be a vehicle for spreading other highly pathogenic organisms that include b. anthracis . in addition, the introduction of the 'euro' could also allow such bacteria greater freedom to travel across the euro zone. the threat of using currency, particularly paper notes, in spreading lethal organisms should be investigated and proper measures to prevent the use of such a method by terrorists should be implemented. salvage of temporary femoral catheters for haemodialysis using antibiotics in ambulatory patients or . gerasimovska v, oncevski a, dejanov p. department of nephrology, clinical centre, skopje, the former yugoslav republic, macedonia the stay of femoral catheters (fc) for haemodialysis is typically short-term for several days. we used fc as a temporary vascular access (va) for a longer period of time in outpatients going on regular ambulatory haemodialysis, who had a problem with their permanent access. we analysed patients who were discharged from hosptal with fc. duration time of fc was between and days (average . days) with cummulative total of days. the incidence of bacteriaemia was . episodes/ catheter days. in six patients we had signs of infection, so according to our protocol we took blood cultures from peripheral vein, and from catheter (at same time) and started with antibiotic therapy (ab) systemically and locally (ab was 'locked' in catheter) with different duration of time. dominant microorganism was staphylococcus coagulasa negative, and much less staphylococcus aureus , and enterococcus.ab that were frequently used were: cefotaxim, vancomycin and ciprofloxacin. at one of six patients we removed catheter at once without trying to save the catheter. catheter tip was sent for microbiological analysis too. criteria for catheter-related bacteriemia (crb) was found in only one patient, and for possible crb in five patients after we removed the catheters. in the absence of clinical signs of infection, ab treatment was not provided for positive tip culture alone or for positive blood culture of the catheter with negative blood culture from peripheral vein. advances in meningitis education or . holt de a , tait mi b , cavanna al b , worgan-brown s a , hart b a . a the meningitis trust, stroud, uk , b the computer-aided learning unit, school of health science, university of wales, swansea, uk background: meningitis remains an important cause of death worldwide despite improvements in diagnosis, treatment and prevention. clinical and lay awareness of the disease relies on education, however educational delivery has changed and the introduction of material suitable for computer and internet application is now necessary. we have developed educational material on cdrom and on the internet applicable both at tertiary university and secondary school level. application: a computer-aided learning program on cdrom, covering all aspects of meningitis has been produced. it is suitable for undergraduate teaching of healthcare professionals from student nurse and doctors to pharmacists. in order to reach school children in a form acceptable to both pupils and teachers, we have developed a curriculum-linked website. these applications are simple to use and can be incorporated into existing courses of study, so that issues raised can be discussed with tutors and group peers. comment: the introduction of new methods of teaching and learning mean that compatible educational material must be produced. we believe that these applications, focusing on meningitis, are the first of their kind and that they offer tutors the opportunity to progress their teaching of the disease both in methodology and content. brivudin compared to famciclovir for improved therapy of herpes zoster: effects on acute disease and postherpetic neuralgia or . potentially treatment-related adverse events occurred in . % of the brivudin recipients and in . % of the famciclovir recipients (p / . ). conclusions: in zoster patients ]/ years, brivudin )/ mg and famciclovir )/ mg showed equivalent effects on prevalence and duration of phn. brivudin is as effective as famciclovir in stopping viral replication in acute herpes zoster. brivudin offers the advantage of a once daily dosage regimen while being as well tolerated as famciclovir. activity of complexes of pt(ii) and pd(ii) with pyridine- -carbaldehyde thiosemicarbazone (hfotsc) (acta virol., , , ) with selectivity index (si) . times higher than that of acyclovir (acv). in order to evaluate virus specific response and structure Á/activity relationships we continue our investigations with three pt(ii) and three pd(ii) complexes. the activity was evaluated against sensitive to acv hsv (strain bja) and resistant strains r- (hsv ) and pu (hsv ) and compared to that obtained against strain victoria (hsv ) infection. si was indicative for activity. the virus specific response was demonstrated by the fact that viruses sensitive to acv were also sensitive to pt(hfotsc) ]cl , while acv resistant viruses were sensitive to [ptcl(fotsc) ]. the structure Á/activity relationship was proved by the fact that the less active against hsv infection was [pd(fotsc)]. influenza diagnosis, treatment, and the impact of new antivirals on current treatment behaviours during influenza outbreaks or . schaetz l a , sessa a b , a hoffman-la roche f. basel, switzerland , b italian college of general practitioners, italy introduction: annual influenza epidemics severely affect individuals, families, health care systems and society. the availability of new and specific antivirals provides an opportunity for better management of influenza. methods: during the / and / influenza seasons, physicians ( Â/ /country) and public ( Â/ /country) in the usa and europe were interviewed to determine perceptions of influenza and behaviours for its treatment. results: patients recognise influenza illness as severe and identify it by symptoms of fever, muscle aches/pains and cough. physicians use these symptoms to diagnose influenza clinically ( % fever, % muscle aches/pains, % cough); their main treatment objective being to reduce complications. antibiotics for influenza treatment are broadly recommended/prescribed by about % of european physicians, whereas currently available antivirals are only recommended by %. the recommendation of antivirals by us physicians increased from % (season / ) to % ( / ) and markedly decreased antibiotic use (from to %). experience from the two influenza seasons shows that influenza antivirals are only used while the virus is circulating and that the volume of use is proportional to the size of the outbreaks. conclusions: experiences in the usa show that with prompt outbreak information antivirals can be used appropriately in times of influenza activity. influenza treatment with oseltamivir: costs and benefits for the individual as well as for society or . objective: to evaluate the effects of treatment of influenza with antivirals (oseltamivir) on health outcomes and costs to patients and society. methods: based on clinical trial data and data from the literature a simulation model has been developed. the underlying clinical pathway covers morbidity and mortality due to influenza and its specified complications. health outcome data and costs were attached to events in the model. the model compares various scenarios, which are defined by treatment schemes within defined populations and other parameters. application of the model is shown using uk unit cost data simulating an otherwise healthy adult population comparing oseltamivir with usual care. results: early treatment results in reduced morbidity, which translates into faster recovery and return to normal activities ( . days). lower morbidity and mortality make this a cost-effective intervention from a societal perspective. the analysis covers more than different scenarios and the incremental cost effectiveness ratios will be discussed. conclusion: antiviral treatment appears to be effective in terms of health outcome and cost for otherwise healthy adults from the perspectives of both the individual patient and society. however, this effect is very sensitive to time when treatment is started and the accuracy of the diagnosis of influenza. oseltamivir is well tolerated by all patient groups or . thakar b a , dutkowski r b , froelich e c , gilbride j a , ward p a . a roche global development, welwyn, uk , b f hoffman-la roche, nutley, usa , c f hoffman-la roche, basel, switzerland introduction: oral oseltamivir, the ethyl ester pro-drug of a potent inhibitor of influenza virus neuraminidase, is licensed for the treatment and prophylaxis of influenza in the usa. patients and methods: safety data [adverse events, laboratory safety evaluations] derived from clinical trials involving !/ subjects (including Â/ children and Â/ high-risk adults) and healthy volunteers in a large study investigating ecg parameters. spontaneous event reports from medwatch or yellow-card reports following use by Â/ individuals worldwide. an observational case-control study of !/ subjects with influenza-like illness treated with oseltamivir. results: oseltamivir was well tolerated in clinical trials; drug-related side-effects were limited to transient gi effects occurring in / : exposed individuals. these resolved spontaneously and caused drop out in b/ % of treated subjects. no effects on ecg parameters were noted at doses ]/sixfold above the licensed regimen. oseltamivir had no adverse effects on pulmonary function. no additional effects were identified among high-risk adults or children, or following prolonged dosing for prophylaxis. occasional reports of liver dysfunction have been documented post-marketing but causal association has not been established. conclusions: oral oseltamivir is an effective and safe antiviral suitable for influenza management in all patient groups. the decision to stop the vaccination against smallpox and the loss of specific immunity of a high proportion of the population made apocalyptic the perspective of a natural or provoked re-emergence of smallpox. therefore, it is important to improve the current capacities to prevent or to treat the orthopoxvirus infections. uracil dna glycosylase (udg) is one viral enzyme indispensable to the replication of poxviruses. udg of the copenhagen strain of vaccinia virus (vv) was characterized with the aim of defining specific inhibitors susceptible to be used as a new class of active antiviral substances on the viruses of the orthopoxvirus genus. the activity of this enzyme was analysed in real time, in an original method, on a pcr quantitative instrument by digestion of amplified dna revealed by fluorescent intercaled molecules. this technique was used to screen and select several active antiviral substances on udg. moreover, the antiviral activity was estimated by the cytopathic effect of the vv on infected vero cells. the cytotoxicity was determined by inhibition of trypan blue exclusion. the specificity of action of each tested compound was estimated by the selective index ( % cytotoxic dose/ % effective dose). two antiviral compounds were selected for their inhibitory effect on udg activity and on vv replication in vero cell culture : ('/)- -iodo- ?-deoxyuridine and -chlorouracil. these compounds are candidates for the chemotherapy of poxvirus infections. objective: to study the efficacy and tolerance of russian antiviral drugs produced from dna in a limited resources context. results: the drug derinat was produced from salmons' milt. mm of dna was Á/ kda, hyperchrome effect !/ %, protein content b/ . %. the conjugation of the dna with fe '/ resulted in a new drug named ferrovir which influences dna and rna synthesis during early stages of hiv- replication by blocking the virus's action on cells' metabolism and reduces cytomegalovirus titre in fibroblast cells for . Á/ . ig tcid . a protective effect of ferrovir against fatal herpes encephalitis mice was found. the drugs are not toxic. ic !/ mg/ml. ec of ferrovir against hiv- was mg/ml. in limited clinical trials patients received mg of drugs twice daily ( Á/ days). administration was well tolerated and no side effects were observed. derinat in . % cases of herpesvirus infection ( patients) improved the healing and shortened duration of illness. hiv-infected patients ( ) treated with ferrovir showed sustained, elevated cd '/ counts and a significant reduction in hiv- viral load (median . ig). the apparent remission was found in patients with concomitant hiv and herpes virus infection. conclusions: antivirals show good antiviral potency against rnaand dna-viruses; are well tolerated by patients and are useful in case of mixed infections; low price makes them accessible to populations with low financial resources. ortho total hcv core antigen assay can aid early prediction of response in patients treated with interferon/ribavirin or . lunel f a , veillon p b , payan c b . a ahu angers, laboratoire de bacterio-virologie, angers, france , b chu angers, laboratoire de bacterio-virologie, angers, france aim: evaluate the predictive value of total hcv core antigen assay and viral kinetics in patients with chronic hcv. methods: one hundred and twenty two patients infected by genotype , , or pretreatment viral load (bdna . , chiron) !/ meq/ml, with no previous treatment, received mu interferon (ifn) during months (m). ribavirin was given with ifn after months therapy, for months in patients with detectable rna. viral load was expressed as log (ui/ml) and hcv ag as log (pg/ml)/ ). results: pretreatment ag values were correlated with viral load (r / . ). we observed a rapid decrease of ag ( . log pg/ml) and viral load ( . log ui/ml) after m in sustained responders (sr). in patients who relapsed (rr) after ifn alone, the fall was less important ( . log pg/ml, . log ui/ml) during m . in sr and rr to combination therapy, the decrease of ag and viral load at m was, respectively, (ag: . and . log pg/ml; rna: . and . log ui/ml). we did not observed significant variation of ag and viral load in nonresponders. the negative predictive value of hcv rna and ag after m of treatment were %, and positive predictive values were and %. after month of ifn alone, the hcv ag decrease was highly predictive of sr, correlated with rna negativation and early reduction of hcv rna ( !/ log). conclusion: early measurements of total hcv core antigen are useful to predict long-term response to treatment. lamivudine in the treatment of acute hepatitis b or . vincenti a, meini m, luchi s, de gennaro m, ricciardi l, moneta s, scasso a. infectious diseases department, infectious diseases, lucca, italy acute hepatitis b is a self-limiting infection, but in some cases its course may be particularly severe. we report a case of a -years-old woman affected by acute hepatitis b treated with lamivudine. on admission in the hospital the alanino-aminotransferase was u/l, the aspartate-aminotransferase u/l, bilirubin , mg/dl, hbsag, hbcigm and hbeag were positive, hbv dna was . copies/ml. during the following days, the levels of ast and alt gradually rose; on the th day prothrombine time was %, bilirubin mg/dl and the patient developed signs of encephalopathy. four plasmapheresis were practiced without benefit, so the patient was treated with lamivudine, mg/day. after days of therapy, lamivudine was discontinued because of the appearance of diffuse maculopapular rash. at this time the results of liver function tests were normal; after four months hbsag and hbv dna were no longer detectable. in our patient lamivudine prevented an acute hepatic failure. our experience suggests a promising role of lamivudine in the treatment of acute hepatitis b, but how long such therapy have to be practiced and in which patients? prospective, controlled, clinical studies using lamivudine in patients with acute-hepatitis b are necessary. the cost-effectiveness of amantadine versus symptomatic care in the treatment of influenza or . morris s a , carman wf b , barber j c . a city university, london, uk , b west of scotland specialist virology centre, glasgow, uk , c alliance pharmaceuticals, chippenham, uk aim: to assess the cost-effectiveness of amantadine versus best symptomatic care in the treatment of influenza in the uk. methods: we constructed an economic model populated with parameters from the published literature. the model structure is the same as that used in the economic evaluation of zanamivir published by the national institute for clinical excellence in the uk. we conducted a cost-utility analysis (incremental cost per qaly gained) of amantadine versus best symptomatic care. the analyses are conducted for all adults (average-risk group) and the at-risk population (high-risk group), based on the prevalence of influenza over an average season and when the virus is circulating. the perspective is that of the nhs. results: in the average-risk group the incremental cost per qaly gained of amantadine relative to best symptomatic care is uk£ , during an average influenza season and uk£ , when the virus is circulating. for high-risk individuals the figures are uk£ , and uk£ , , respectively. the results are sensitive to the hospitalisation rate. conclusions : if the threshold for cost-effectiveness is £ , per qaly gained amantadine represents value for money in the treatment of influenza in a variety of scenarios, including the baseline for both average-risk and high-risk groups when the virus is circulating. background: surveillance studies all over the world have revealed an extraordinary increase in the prevalence of penicillin resistant streptococcus pneumoniae . the newer quinolones are believed to have broad activity against s. pneumoniae . methods: a total of penicillin resistant clinical strains isolated from patients at hacettepe children's hospital, ankara, turkey between and were tested for their in vitro susceptibility to various antibiotics that are commonly used in the treatment of respiratory tract infections. the minimum inhibitory concentrations (mics) of the penicillin, amoxicillin/clavulanic acid, doxycycline, azithromycin, clarithromycin, ceftriaxone, ciprofloxacin, levofloxacin, moxifloxacin and gemifloxacin were determined using the nccls recommended procedure for e -test. results: the range of mics, mic and mic values for all agents tested against the strains are shown in the table. gemifloxacin and moxifloxacin had the highest in-vitro activity among the quinolones tested. all strains tested were susceptible to b/ . mg/ml gemifloxacin, b/ mg/ml moxifloxacin and mg/ml levofloxacin. conclusions: there is some degree of resistance to all the drugs except the newer quinolones which were active against all isolates studied. purpose: stenotrophomonas maltophilia prevalence is growing, mainly in some hospital areas. s. maltophilia is frequently multidrug resistant. fluoroquinolone (fq) resistance varies from one to another study, but in whole resistance is moderate to high. gyra and parc qrdr partial codes have been recently described. we have studied correlations between fq-resistance and mutations in these sequences in s. maltophilia clinical strains. material and methods: gyra and parc qrdr regions from six fqresistant and two fq-susceptible s. maltophilia clinical strains were amplified and sequenced. mics of ciprofloxacin (cfx), gatifloxacin (gfx) and clinafloxacin (cnfx) were determined by the agar dilution method, according guidelines defined by nccls for p. aeruginosa . results and conclusions: mics ranges of cfx, gfx and cnfx for resistant strains were Á/ , Á/ and . Á/ mg/l. susceptible strains had mics of cfx, gfx and cnfx of , . and . Á/ . mg/l, respectively. most susceptible and resistant strains had no significant mutations in the fragments sequenced. only one resistant strain (mic of cfx mg/l) and one susceptible strain (mic of cfx mg/l) had a significant gyra mutation, the same in both strains (ile Á/val). thus, fq resistance in s. maltophilia shall derive from changes in other areas in the topoisomerases or probably from other mechanisms of resistance, such as efflux pumps. purpose: corynebacterium urealyticum is the cause of encrusted cystitis and other inespecific utis and systemic infections. it is frequently multi-drug resistant, with a high rate of resistance to fluoroquinolones (fq). the mechanisms of resistance to fqs have not been described in c. urealyticum . we describe the c. urealyticum parc gene qrdr region and its relationship with quinolone resistance. materials and methods: the activity of ciprofloxacin (cfx), levofloxacin (lfx), gatifloxacin (gfx), clinafloxacin (cnfx) and moxifloxacin (mfx) against c. urealyticum clinical strains was determined following nccls guidelines for enterococci. we amplified and sequenced their parc qrdr by standard methods. results and conclusions: five strains ( . %) were cfx-susceptible (mic . Á/ . mg/l), had mics Á/ mg/l and ( . %) were highlevel cfx-resistant (mic Á/ mg/l). cnfx was -fold more active than cfx. mfx and gfx had mics of and mg/l. all the strains, including the type strain, showed a c to t change at the position referred to wild type s. aureus parc gene, leading to a ser- -phe change, described as the main parc change in fq-resistant s. aureus . this finding suggest that this mutant sequence, as compared with parc sequences from other grampositives, might be the wild-type for this species, and might explain in part its high resistance rate, and its apparent lightness to develop high-level resistance. purpose: during routine surveillance, we identified ciprofloxacinresistant (mic!/ mg/l) pneumococcal isolates and compared clinical details and resistance patterns. results: they were isolated from sputa ( ) and blood cultures ( ) from adults, most with heart or lung disease. hospital admissions were common; half had been inpatients in the previous months. nineteen patients received quinolones in the preceding months, in part reflecting the local policy (introduced in ) of penicillin and ofloxacin for first line treatment of pneumonia. thirteen patients had radiological signs of pneumonia and were pyrexial with raised inflammatory markers. agar dilution mics for quinolones, including norfloxacin with and without reserpine, penicillin and erythromycin were performed. an increase in norfloxacin mics was noted over the period ( Á/ mg/l) to ( mg/l). fluoroquinolone efflux was suggested in three isolates. resistance to moxifloxacin (mic Á/ mg/l) was noted from onwards. all isolates were serotype v and resistant to penicillin (mic!/ . mg/l). thirty-one were resistant to erythromycin (mic!/ mg/l). conclusion: the policy of using quinolones may have contributed to the development of quinolone resistance and this cluster of isolates. the increasing levels of quinolone resistance observed raise concerns about the future use of newer quinolones for the treatment of respiratory infections. s. maltophilia has emerged in the last years as an important nosocomial pathogen, inherently resistant to most of the antimicrobial agents. new quinolones has been proposed as a treatment of choice because their enhanced activity, but several parameters (t a , atmosphere, method) can affect the results of mics. methods: we have performed mics using two different methods (agar dilution and microdilution) and different conditions: and c of temperature; atmosphere of o and co , and incubation times of , and h. a total of strains were assayed with nine quinolones following standard nccls. comparisons were made between results with Á/ and Á/ h using the x -test (a / . ). results: no differences were found between Á/ and Á/ h results with agar dilution, except with atbs in the case of mics at c co . on the contrary, almost all the atbs showed significant differences in the results with and h using microdilution method, at any condition of ta or atmosphere. comparison of mics (p values, significance level / %) with incubation times of and h at different procedure conditions. the incubation time is a parameter that seems to affect significantly the results of mics of quinolones when microdilution method is used, whereas only few differences can be encountered with the agar dilution method. results: breakpoints were used as proposed by nccls . during the study period the pneumococci resistance was noted as follows: % to pc, % to em and % to sxt. the rank order of activity of the five fqs against multi-drug resistant pneumococci was: cip (mic : mg/l), ofx (mic : mg/l), lvx (mic : mg/l), grx (mic : . mg/l), tvx (mic : . mg/l). conclusions: in romania, fluoroquinolones represent alternative treatment to beta-lactams and macrolides for first-line empirical treatment for respiratory tract infections caused by pneumococci but, continued vigilance for emerging resistance to fqs is further indicated. introduction and material/methods: susceptibility testing (semiautomated broth microdilution method, sensititre, trek diagnostics, usa, following nccls recommendations) was performed with six different quinolones to streptococcus pneumoniae isolates with a ciprofloxacin-cip */mic ]/ mg/l collected in two consecutive sauce$ surveillances in spain ( Á/ / Á/ ). nccls resistance (r) breakpoints were used ( ]/ for ofloxacin-ofl and levofloxacin-lev; ]/ for sparfloxacin-spa; ]/ for gatifloxacin-gat */and moxifloxacin-mox), but for gemifloxacin-gem */where ]/ was used. results were as follows. conclusions: for cip-r isolates gem and mox were the most active agents. gem was the only agent not influenced by cip mic increase regarding prevalence of r , with % resistance for strains with cip mic ]/ mg/l. $sauce is an acronym standing for 'sensibilidad a los antimicrobianos utilizados en la comunidad en españ a' (susceptibility to the antimicrobials commonly used in the community in spain) and is the spanish word for the willow tree. in vitro activity of gatifloxacin and seven other antibiotics against respiratory and urinary tract pathogens from the community. first results of the basic */study ps grimm h, on behalf of a european multicenter study group, institute for med. microbiology, weingarten, germany a total of centers in austria, belgium, france, germany, italy, portugal, spain and switzerland are involved in the basic study (bacterial annual susceptibility information collection). the mics of gatifloxacin (gati), ciprofloxacin (cipro), clarithromycin (clari), benzylpenicillin g (pen), amoxicillin (amox), amoxicillin/clavulanic acid (augm), cefuroxime (cur) and cefixime (cix) were determined using the microdilution method. each center is requested to investigate strains each of the following species: s. pneumoniae (spn), s. pyogenes (spy), s. aureus (sau), e. faecalis (efa), m. catarrhalis (mca), h. influenzae (hin), e. coli (eco), k. pneumoniae (kpn), p. mirabilis (pmi) and p. aeruginosa (pae). so far approximately strains are enrolled. some important mic %/percentage resistance were as follows: from the oral antibiotics tested gatifloxacin has the highest activity and broadest spectrum against all relevant respiratory and urinary tract pathogens. gatifloxacin is a promising alternative for therapy of respiratory tract bacterial infections. in vitro activity of gatifloxacin against bordetella pertussis in comparison with erythromycin, ciprofloxacin and levofloxacin ps bourgeois n, pangon b, ghnassia jc, doucet-populaire f, de versailles ch. microbiologie, le chesnay, france purpose of the study: bordetella pertussis infections are far more common in adults and adolescents than is generally estimated. however, they are often not recognised. infected or colonised adults can act as a reservoir of infection, passing it to children. fluoroquinolones are currently recommended for the treatment of respiratory tract infection in adult patients, which is usually empirical. gatifloxacin is a novel -methoxyquinolone, with a potent activity against both gram-negative and -positive bacteria. the in vitro activity of gatifloxacin was compared with those of erythromycin, the drug of choice for both treatment and prophylaxis of pertussis, ciprofloxacin and levofloxacin, against clinical isolates strains of b. pertussis including erythromycin resistant strains. results: we used the agar dilution method on mueller Á/hinton medium supplemented with % horse blood to determine the mic of each antibiotic. gatifloxacin (mic , . mg/l) was as active as ciprofloxacin and levofloxacin (mic , . mg/l) against both sensitive erythromycin (mic , . mg/l) and resistant erythromycin (mic , !/ mg/l) strains. conclusion: gatifloxacin may be an effective drug in the treatment or prophylaxis of adults with suspected or confirmed pertussis. ex vivo serum activity (killing rates) after gemifloxacin mg versus trovafloxacin mg single doses against ciprofloxacin-susceptible andresistant streptococcus pneumoniae ps calvo a a , giménez mj b , alou l a , gó mez-lus ml a , aguilar l b , prieto j a . a microbiology department, universidad complutense, madrid, spain , b glaxosmithkline, medical department, tres cantos, madrid, spain serum bactericidal activity was measured ex vivo after single dose administration of gemifloxacin (gem) mg and trovafloxacin (tro) mg to healthy volunteers in a randomized, cross-over phase i trial. blood samples were collected h (cmax) after dosing and serum killing rates were determined against a serotype penicillin (pen) Á/ciprofloxacin (cip) susceptible strain (s ) (mics of . , , . and . mg/l for pen, cip, gem and tro) and a serotype pen Á/cip resistant strain (s ) (mics of , , . and . mg/l for pen, cip, gem and tro). tubes with . ml of serum sample and . ml broth ( % todd Á/hewitt'/ % hbss) were incubated over h at c. final inocula was cfu/ml. mean colony counts for samples and controls (k ) are shown in the figure: gem exhibited higher colony counting decrease of the initial inocula, versus tro, for both strains. after h incubation, the initial inocula decrease obtained with tro and the cip susceptible strain was similar to that obtained with gem and the resistant strain, showing a lower influence of cip mic increase in the ex vivo bactericidal activity of gem versus tro. urine bactericidal activity after administration of gemifloxacin and trovafloxacin single doses in a phase i study ps garcía-calvo g a , parra a a , giménez mj b , ponte c a , aguilar l b , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain urine bactericidal activity after o.d. administration of gemifloxacin (gem) mg and trovafloxacin (tro) mg, was assessed in six adult males in a cross-over phase i trial. urine killing rates (ukr) against escherichia coli atcc (mic mg/l of . and . for gem and tro) and s. saprophyticus atcc (mic mg/l . and . for gem and tro) were performed with samples collected at , Á/ , Á/ and Á/ h. a . ml of iso-sensitest broth and . ml of bacterial logarithmic growth were added to ml sample, giving a final inoculum of cfu/ml. colony counting was performed after , , and h incubation. percentages of initial inocula reduction (iir) were calculated. mean urine concentrations measured by bioassay were (mg/l): . , . and . for gem, and . , . and . for tro. against e. coli , an iir of . % was obtained after h incubation with all samples except with tro at Á/ h. against s. saprophyticus an iir of % was obtained after h incubation with all samples except with tro at Á/ h, where bacterial regrowth was found. the maintenance over h of gem urine antibacterial activity suggests its efficacy in the treatment of uncomplicated cystitis. influence of the decreased susceptibility to ciprofloxacin on gemifloxacin versus levofloxacin efficacy in experimental pneumococcal pneumonia in guinea pigs ps garcia-olmos m a , parra a a , gimenez mj b , garcia-calvo g a , ponte c a , aguilar l b , soriano f a . a fundacion jimenez diaz, medical microbiology, madrid, spain , b glaxosmithkline, medical, madrid, spain the efficacy of ciprofloxacin (cip), levofloxacin (lev) and gemifloxacin (gem) in the treatment of pneumococcal pneumonia was assessed in a guinea pig model using three strains (s) with mics (mg/l) of , and . (s ), , and . (s ) and , and (s ) for cip, lev and gem, respectively. intraperitoneal treatments started h after s. pneumoniae intratracheal inoculation, and continued t.i.d up to four doses. ten animals were included in each group. doses (mg/kg) used were , and for cip, lev and gem, respectively, in order to mimic auc - h and cmax obtained in humans after standard doses. animals that survived h after inoculation were sacrificed and colony counts were performed in lungs ( the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of nosocomial pneumonia. our aim was to study the pharmacokinetic Á/pharmacodynamic appropriateness of lfx mg iv bid in the treatment of six inpatients with ventilator-associated pneumonia (vap) ( / years; m Á/ f; / kg). blood and urine samples were collected in steadystate conditions at appropriate intervals. lfx concentrations were analysed by hplc. the aetiological agent was identified in all the cases and its in vitro sensitivity to lfx was always assessed. the results obtained mean values ( /sd) of the major pharmacokinetic parameters were: cmax, . / . mg/ml; vdss, . / . l/kg; t / b, . / . h; cl, . / . ml/min/kg; auc -t, . / . mg/ml h. cumulative urinary excretion was . / . %, confirming that lfx clearance is mainly renal. clinical cure and microbiological eradication were obtained in all the patients after a Á/ day therapy. a suprainfection due to acinetobacter anitratus insensitive to lfx occured in case. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/ ; auc/mic !/ ) in all the cases. the conclusion reached the findings suggest that lfx mg bid iv may be considered effective in the treatment of vap caused by sensitive bacteria. comparative pharmacokinetics of levofloxacin in patients with lower respiratory tract infections (lrti) being treated with sequential therapy ps pea f a , brollo l a , lugatti e b , di qual e a , dolcet f b , talmassons g b , furlanut m a . a institute of clinical pharmacology and toxicology, dpmsc, university of udine, udine, italy , b division of pneumology, sm misericordia hospital, udine, italy the purpose of the study levofloxacin (lfx) is a fluoroquinolone whose activity against both gram-negative bacilli and gram-positive cocci enables its use in monotherapy for the treatment of lrti. our aim was to study the pharmacokinetic Á/pharmacodynamic appropriateness of a standard switch lfx iv/os regimen ( mg iv od for Á/ days followed by mg os od for Á/ days) in the treatment of seven inpatients with lrti ( / years; m Á/ f; / kg). blood samples were collected in steady-state conditions at appropriate intervals. lfx plasma concentrations were analysed by hplc. the aetiological agent was identified in / cases and its in vitro sensitivity to lfx was assessed. the results obtained absolute oral bioavailability was . / . , with a cmax of . / . vs . / . mg/ml after iv and oral administration, respectively. no significant difference in the main pharmacokinetic parameters was observed between the two routes. the major pharmacodynamic parameters of fluoroquinolone efficacy were significantly higher than the proposed threshold (cmax/mic !/ ; auc/mic !/ ) in the two assessable cases. all the patients were clinically cured after a Á/ day therapy. the conclusion reached the ad interim findings show that lfx mg od may guarantee per os an exposure similar to that achievable after iv administration, suggesting that sequential therapy may be considered effective in the treatment of lrti. levofloxacine in the exacerbations of copd due to pseudomonas ae ps micheletto c, tognella s, pomari c, dal negro r, ospedale orlandi, div.pneumologia, bussolengo, italy development of antibiotic resistance in bacteria is a problem of great concern. gram-negative bacteria, including multidrug-resistant (mdr) pseudomonas aeruginosa (ps), are responsible for a significant proportion of episodes of copd exacerbations, particularly in elderly ( ). aim was to check the susceptibility to common antimicrobial treatments against ps strains isolated from bronchial secretions in patients with severe exacerbations of copd. methods: microbial investigations were conducted on specimens: spontaneous purulent sputum ( . %), and tracheobronchial aspirates ( . %, collected with a protected specimen brush). results: fifty-seven ps pathogen strains ( cfu) were identified ( . %) and tested over a -month period: ps. aeruginosa . %; ps. putida . %; ps. fluorescens . %, and burkholderia cepacia . %. the assessed susceptibility to most common antibiotics was: levofloxacine ( %), ciprofloxacine ( %), ipenem cil. ( %), ceftazidime ( %); amikacin ( %), and piperacillin'/tazobactam ( %). a much lower susceptibility was found for ticarcillin Á/clavulanic acid ( %), gentamicin ( %), and netilmicin ( %). ( ) at present, levofloxacine proves the most effective antimicrobic option for treating copd exacerbations due to ps infection; ( ) a much more efficient policy of antibiotic prescribing should be promoted in order to prevent the selection of resistant strains in these cases. these results confirm the excellent 'in vitro' activity of levofloxacin against nosocomial gram-negative pathogens, including the esbl producing strains ( % of escherichia coli , e. cloacae and k. pneumoniae were inhibited at . mg/l). levofloxacin was more rapid than ciprofloxacin to determine a bactericidal effect particularly against s. maltophilia . moreover, considering the favourable pk/pd profile, levofloxacin can represent a valid therapeutic option for the treatment of severe gram-negative nosocomial infections. moretti f, quiros-roldan e, casari s, chiodera a, viale p, carosi g. university of brescia, institute of infectious and tropical diseases, brescia, italy a -year-old man, ivdu, hiv positive was attended in aur hospital for fever and toracic pain. a x-chest radiography revealed a round lesion of cm near the lingula with central hyper-diaphan area. lymphocytes cd '/ count was cells/mm and viral load . cp/ ml. hospital stay rhodococcus equi was found in cultures of peripheral blood, faecal and sputum specimens. antibiotic treatment with oral rifampin ( mg/qd) and with intravenous imipenem ( mg tid) was started. due to the persisting fever, immodificated radiography and negativity for p. carinii , mycobacteria and bacteria in bal coltures, imipenem was substituted by parenteral vancomycin ( mg bid). after days, because of persisting fever and increase of the diameter of the lung lesion ( cm) vancomycin was sustituted by oral levofloxacyn ( mg bid), continuing rifampin. after a days course of levofloxacyn therapy the fever remitted. the patient was discharged with levofloxacyn ( mg bid) and rifampin and, after months of follow-up, a radiological control pointed-out a remarkable resolution in the lung lesion. we may suppose that levofloxacyn can be effective for the treatment of r. equi infection, even if more studies (particularly controlled studies) are necessary. liberti a a , izzo b a , loiacono l b , calabria g a , patarino t a , izzo e a . a ii department, naples, d. cotugno hospital, italy , b iii department, naples, d. cotugno hospital, italy the increasing prevalence of salmonella typhi strains with reduced susceptibility of chloramphenicol had prompted the search for other antibiotics with the same efficacy.quinolones are a class of antibiotic with an activity in vitro and in vivo against enteropathogenes. we inwestigated the use of levoxacin in two regimens of treatment of typhoid and paratyphoid infection. patients and methods: thirty-two adult patients were incluted in this study from september to april ; patients had positive culture for s. typhi and six had positive cultures for s. paratyphi . all isolated were fully susceptible to levoxacin. we compared treatment with levoxacin for days, mgr b.i.d. (group , patients), with treatment for days, mgr b.i.d. (group , patients). clinical cure was defined as defervescenze of fever by day of treatment, with an absence of complications and no clinical relapse during the followup. results and conclusion: the clinical cure rate was % ( patients) for group and % ( patients) for group ; the difference in these rates was not statistically significant. the blood cultures of all patients were sterile by day of treatment and remained so until the th month of follow-up, no subjects had clinical or microbiological relapse and all stool cultures remained negative, also. the two regimens of treatment was good tollerated and no adverse event was registered; it was concluded that levoxacin treatment for days in enteric fever is not necessary the mulidrug-resistence of s. typhi led to the use of quinolones as the first-line drug in the treatment of enteric fever. pefloxacin in the treatment of patient with acute infectious diarrhoea ps troselj-vukiae tvb a , strahinja v b , poljak i a , stojanoviae d c , nikoliae n d . a department of infectious disease, university hospital center, rijeka, croatia , b glaxosmithkline, marketing, rijeka, croatia , c dept. rijeka, institute of public health, rijeka, croatia , d dept. rijeka, maritime academy, rijeka, croatia the purpose of the study was to investigate clinical and bacteriological efficiency in and days pefloxacin treatment and to compare it with symptomatic therapy. the results obtained in patients treated with pefloxacin the therapy was clinically effective already in the third day while in the control group this happend in the th day. bacteriological eradication was noted in patients ( %) of the first and patients ( %) of second group in the th days of the treatment. they all had negative cultures and weeks after pefloxacin protocols were completed. only patients ( %) in control group had negative stool cultures in the th day of the treatment and all of them weeks after it ended. there was no statistically significant difference in clinical (p / . ) and bacteriological (p / . ) efficiency between and days pefloxacin treatment protocols. both protocols significantly differed in clinical (p b/ . ) and bacteriological (p / . ) eradication from the control group. the conclusion reached is that the efficiency of pefloxacin (quinolones) in the treatment of acute infectiuos diarrhoea and justifies their use in the more severe forms of the disease. dalhoff a a , ullmann u b , schubert s b . a bayer ag, wuppertal, germany , b university of kiel, institute of med. microbiology, kiel, germany background: the antibacterial activity of moxifloxacin (mxf) was compared to levofloxacin (lev), amoxicillin (amx), clarithromycin (cla) and erythromycin (ery) in an in vitro model. method: pharmacokinetics in bronchial mucosa (bm) and serum (s) following single oral doses of mg mxf or cla and mg lev, amx or ery were simulated using a one compartment model. bacteria tested staphylococcus aureus (nos. , ), streptococcus pneumoniae (nos. , ). aliquots were taken ( Á/ h) and plated on to brain heart infusion agar for enumeration. results: s. pneumoniae was eliminated by all agents studied. significant differences were apparent with s. pneumoniae and the s. aureus strains: objective: to compare the safety and efficacy of once-daily moxifloxacin with once-daily ceftriaxone in the treatment of cap in hiv-infected patients (pts). methods and results: in a retrospective survey, oral moxifloxacin ( mg daily)/ Á/ days) was compared to standard regimen of i.v. ceftriaxone ( g daily)/ Á/ days) for treatment of cap in hiv'/ pts. adults pts with clinical signs and symptoms of cap and consistent chest x-ray findings were included. pts had a median age of years (range Á/ and % were male). demographic characteristics were similar in both treatment groups; pts received mxifloxacin and pts ceftriaxone. clinical success rates were % for moxifloxacin and % for ceftriaxone. at a post-study evaluation approximately weeks later, moxifloxacin-treated pts and ceftriaxone-treated pts had relapsed. the adverse events reported were comparable for both treatment groups. there were four-related adverse events ( gi, headache) for moxifloxacin-treated and ( gi, skin) for ceftriaxonetreated pts. conclusion: the results of this study show that moxifloxacin as oral therapy is as effective and well tolerated as i.v. ceftriaxone in the treatment of hiv'/ pts with cap. therapy with moxifloxacin was not associated with any significant clinical or laboratory abnormalities. these data suggest that once-daily oral administration of moxifloxacin is potentially convenient and cost-effective alternative therapy for cap in pts with hiv infection. moxifloxacin in the treatment of acute maxillary sinusitis after first-line therapy failure or acute sinusitis with high risk of complications ps gehanno p a , berche p b , perrin a b , arvis p c . a ent department, bichat claude bernard hospital, paris, france , b microbiology department, necker enfants malades hospital, paris, france , c bayer pharma, medical affairs department, paris, france the efficacy and safety of moxifloxacin (mxf) mg once daily for days was evaluated in the treatment of acute maxillary sinusitis after first-line therapy failure, or acute sinusitis with high risk of complications. in this prospective, multicenter study, a total of patients with acute bacterial sinusitis confirmed by sinus x-ray were valid for efficacy analysis: one hundred and seventy five patients ( . %) had an acute maxillary sinusitis which failed to respond to a previous antibiotic therapy given for a mean duration of . days, and ( . %) had an acute sinusitis with high risk of complications (frontal: , pan-sinusitis: and sphenoid: ). ninety two patients ( . %) were microbiologically valid. clinical cure and continued clinical cure rates at Á/ and Á/ days post-therapy were . and . %, respectively. clinical cure rates at Á/ days post-therapy were . and . % in sinusitis after first-line therapy failure and in sinusitis with high risk of complications, respectively. bacteriological eradication rate during therapy (day Á/ ) was . %. at Á/ days post-therapy. bacteriological success rates were . % in patients who failed to respond to a previous antibiotic and . % of patients who had sinusitis with high risk of complications. . % of patients experienced drug-related adverse events, abdominal pain ( . %), nausea ( . %) being the most frequently reported events. mxf was rapidly effective and a well-tolerated treatment for this kind of infection. neisseria gonorrhoeae with decreased susceptibility to penicillin and ciprofloxacin: novel mutation patterns in the gyr a and par c genes of ciprofloxacin resistant isolates and plasmid profile of penicillin resistant isolates of n. gonorrhoeae in india (delhi) ps chaudhry u, saluja d. dr. b. r. ambedkar center for biomedical research, university of delhi, delhi, india commercial sex workers (csws) serve as the most important reservoir of gonorrhoea. periodic monitoring of the antimicrobial susceptibility profile of neisseria gonorrhoeae in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. in india, such a surveillance of the in vitro antimicrobial susceptibility of n. gonorrhoeae was established in . significant increasing trend of penicillin and ciprofloxacin resistance with high mic of Á/ and Á/ mg/ml, respectively were found over the years ( Á/ ) . the molecular basis of ciprofloxacin resistance, i.e. mutations in the gyr a and the par c genes of isolates, were analyzed. four isolates (with an mic of mg/ml for ciprofloxacin) harbored triple mutations (ser- to phe, asp- to asn and val- to leu) in the gyr a gene. the third mutation of val- to leu, lies downstream of the quinolone resistance determining region of the gyr a and has not been described before in gonococcus. in addition, these isolates had a phe- to tyr substitution in the par c, a hitherto unknown mutation. the alterations in the par c gene were seen in these isolates only in the presence of changes in the gyr a gene and comprised amino acid changes at codons , , , , and . the presence of b-lactamase plasmid among the penicillinresistant isolates was determined by their plasmid profiles and further confirmation was carried out by a pcr based protocol. our findings suggest that emergence of penicillin and ciprofloxacin resistance in n. gonorrhoeae isolates from a major std center in india, indicates the need for the increased awareness and prudent use of antimicrobials. in vitro activity of newer antibiotics against methicillin-resistant staphylococcus aureus ps gutierrez zufiaurre mn, sanchez hernandez j, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: mrsa are frequently co-resistant to a number of structurally unrelated antibiotics. more than % mrsa are resistant to gentamycin, ciprofloxacin, macrolides and clindamycin. newer antibiotics active against multi-drug resistant grampositives have been developed. we have tested the in vitro activity of newer antibiotics against genetically-characterized, high level ciprofloxacin resistant mrsa. material and methods: thirty-six ciprofloxacin-resistant, gyra/grla mutant mrsa clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), gatifloxacin (gfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method, according nccls guidelines. results and conclusions: all the strains were resistant to cfx, , % were lfx-susceptible and . % were gfx-susceptible. nevertheless, mics of lfx and gfx for all susceptible strains was in the highest extreme of the susceptibility range. mfx was the most active quinolone. almost all the strains were high-level er-resistant with constitutive mlsb phenotype. tl did not improve significantly its behaviour, although it was -fold as active as er against the only er-susceptible strain. va, lin and syn had excellent activity against all the strains. they showed a very homogeneous behaviour against all the strains that were included in a range of Á/ mg/l of lin and va and . Á/ mg/l. sanchez hernandez j, gutierrez zufiaurre mn, munoz-bellido jl, garcia-rodriguez ja. hospital universitario de salamanca, microbiología, salamanca, spain purpose: corynebacterium urealyticum is the etiologic agent of encrusted cystitis and inespecific utis, and can be also involved in systemic infections. c. urealyticum is frequently multi-drug resistant, so only glycopeptide antibiotics and tetracyclines have high susceptibility rates, while fluoroquinolones resistance rates vary significantly. we have tested the in vitro activity of linezolid, telithromycin, synercid and newer fluoroquinolones against multi-drug resistant c. urealyticum clinical strains. material and methods: sixty-four c. urealyticum clinical strains were tested against levofloxacin (lfx), ciprofloxacin (cfx), moxifloxacin (mfx), erythromycin (er), telithromycin (tl), linezolid (lin), synercid (syn) and vancomycin (va). mics were determined by the agar dilution method according guidelines defined by the nccls for enterococci. results and conclusions: results confirm the high resistance rate to older fluoroquinolones and macrolides, with !/ % cfx resistance and % er-resistance. lfx was more active (mic mg/ml). mfx was the most active fluoroquinolones (mic mg/ml). tl improve its behaviour in respect to er (range . Á/ mg/ml). va, lin and syn had excellent antimicrobial activity. no resistant strains were found. mic were , . and mg/ml, respectively. mics were similar for all the strains independently of their resistance to other antibiotics plasma concentrations, urinary excretion and bactericidal activity of linezolid ( mg) versus ciprofloxacin ( mg) in healthy volunteers after a single oral dose ps wagenlehner fme a , wydra s a , onda h a , kinzig-schippers m b , sö rgel f b , naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, (ibmp), nürnberg-heroldsberg, germany purpose of the study: in a randomized cross-over study volunteers received a single oral dose of mg linezolid versus mg ciprofloxacin to assess plasma concentrations (up to h), urinary excretion (by hplc), and urinary bactericidal titers (ubt) up to h. the mean maximum plasma concentration of linezolid was . mg/ ml and of ciprofloxacin . mg/ml. the cumulative renal excretion (mean) of parent drug was %/ % for linezolid/ciprofloxacin. ubts were determined for a reference strain and five gram-positive clinical uropathogens with the following mics (mg/ml) for linezolid/ciprofloxacin: s. aureus atcc ( / . ), s. aureus (mssa) ( / ), s. aureus (mrsa) ( / ), s. saprophyticus (msse) ( / . ), e. faecalis ( / ), e. faecium ( / ). results: median ubts measured within the first h for linezolid were : for enterococcal strains and : to : for the four staphylococal strains. median ubts for ciprofloxacin were : for the two enterococcal strains, : to : for the two ciprofloxacin susceptible, : . for the two resistant staphylococcal strains. areas under the ubt Á/time-curve showed statistically significant differences only for the two ciprofloxacin resistant staphylococcal strains in favour of linezolid. conclusion: linezolid exhibited the same bactericidal activity against ciprofloxacin resistant as susceptible strains. linezolid should be tested for treatment of complicated uti due to gram-positive uropathogens in a clinical trial. whitehouse t a , cepeda ja a , tobin purpose: we performed pharmacokinetics within a double-blind, randomised trial comparing linezolid and teicoplanin in intensive care patients with known or suspected gram-positive infection. they received either mg linezolid intravenously -hourly, or mg teicoplanin -hourly for the first three doses and once daily thereafter (or every rd day if renally impaired). blood samples were collected to create serum pharmacokinetic profiles. linezolid was quantitated by hplc and teicoplanin by fluorescence polarization immunoassay. results: twenty two patients were studied in the linezolid group (m Á/f : , mean age years [range Á/ years]). median treatment duration was . days (range Á/ ). eighteen patients were treated with teicoplanin (m Á/f : , mean age years [range Á/ ]) for median days (range Á/ ). steady state peak concentrations (mean / sd) for linezolid and teicoplanin were . / . and . / . mg/l, respectively. trough concentrations at day were . / . mg/l for linezolid and . / . mg/l for teicoplanin. recommended breakpoints of staphylococcus aureus are mg/l for linezolid and mg/l for teicoplanin. accumulation occurred in one -year-old linezolidtreated patient with impaired renal function. conclusion: current recommended dosing regimens for linezolid and teicoplanin are generally appropriate in the critically ill, though a more detailed analysis is required. stamos g, lebessi e, ioannidou s, paleologou n, kallergi k, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of the study was to investigate the susceptibility of methicillin resistant staphylococcus aureus (mrsa) isolates from a -bed paediatric hospital to quinupristin/dalfopristin (q/d, streptogramin combination) and linezolid (lzd, oxazolidinone). material: we performed a retrospective analysis of mrsa strains, isolated from patients hospitalized in miscellaneous medical departments [neonatal unit ( ) , surgical wards ( ), orthopaedic wards ( ), oncology unit ( ), other wards ( ) and outpatient clinic ( )], during a -year period ( Á/ ) . the sources of isolation were pus ( ), throat ( ), nasal ( ), bronchial ( ), skin ( ), stool ( ) ear ( ) and other ( ) specimens. all isolates were sensitive to glycopeptides, while . % were resistant to gentamicin and . % to erythromycin. methods: the sensitivity testing was performed by a disk diffusion method (bbl sensitivity disks, becton dickinson), according to nccls guidelines. the breakpoint zone diameters for lzd and q/ d were ]/ and ]/ mm for susceptibility and / and / mm for resistance, respectively. results: all isolates were proved to be susceptible to both antibiotics. the mean inhibition zones were . mm for lzd and mm for q/d. conclusions: lzd and q/d are very promising antimicrobial agents, showing excellent activity against mrsa clinical isolates. the prudent therapeutic use is strongly recommended to avoid the emergence of resistance. in vitro activity of streptogramins and oxazolidinones against streptococcus pneumoniae clinical isolates ps stamos g, lebessi e, paleologou n, psatha m, sanida p, zaphiropoulou a, foustoukou m, 'p. and a. kyriakou ' children's hospital, microbiology, athens, greece the purpose of this study was to evaluate the in vitro activity of linezolid (lzd), a member of oxazolidinones and the streptogramin combination quinupristin/dalfopristin (q/d) against clinical isolates of streptococcus pneumoniae from a tertiary care paediatric hospital. material: a total of pneumococcal isolates exhibiting reduced susceptibility to common antibiotics were included in the study. the strains were isolated from middle ear fluid ( ), eye ( ), nasal ( ) blood ( ) and other ( ) cultures during the last years. the percentages of the isolates that were resistant to penicillin, erythromycin, cotrimoxazole and clindamycin were . , . , . and . %, respectively. methods: the susceptibility testing was performed by a standard disk diffusion method (bbl sensitivity disks, becton dickinson). in case of marginal results or intermediate sensitivity to quinupristin/ dalfopristin, the mic was determined using the e -test method (ab biodisk). results: all isolates were found to be sensitive to lzd. q/d was very active as well, except for two isolates that exhibited intermediate susceptibility, showing cross-resistance to macrolides and clindamycin, as well. conclusions: the new antimicrobial agents show excellent activity against resistant to common antimicrobials pneumococcal isolates, but the clinical use is not suggested, unless no other therapeutic solutions are available. linezolid is a new oxazolidinone with excellent activity against gram-positive organisms including glycopeptide-resistant strains of staphylococci and enterococci. in icu linezolid has been used for the treatment of severe gram-positive infections in a control trial. the susceptibility pattern of all gram-positive isolates from icu patients has been studied. methods: a total of specimens from icu patients were processed, were from patients enrolled in the antibiotic trial. all methicillin-resistant staphylococcus aureus (mrsa), coagulase-negative staphylococci (cons), enterococcus sp and methicillin-sensitive staphylococcus aureus (mssa) were tested. the break point for linezolid was mg/l and for teicoplanin mg/l. isolates were tested for susceptibility by e -test. results: linezolid ( isolates) mic / (mg/l) were as follows: mrsa (n / ) . / , cons (n / ) . / . , enterococcus sp (n / ) . / . , mssa (n / ) . / . teicoplanin ( isolates) mic / (mg/l) mrsa (n / ) . / . , cons (n / ) / . , enterococcus sp (n / ) . / . , mssa (n / ) . / . . all grampositive isolates were inhibited by concentrations of linezolid below the breakpoint including eight strains of staphylococci resistant to teicoplanin. conclusions: linezolid was highly active against grampositive isolates. resistance to teicoplanin was similar to other reported series. there was no emergence of resistance to linezolid. mrsa colonization is often a limiting factor for discharge from icu. clearance of mrsa is seldom achieved with conventional glycopeptide treatment. the oxazolidinone, linezolid, has excellent soft tissue and respiratory tract penetration and might be expected to eradicate carriage in some patients. we recently performed a doubleblind randomized trial in icu patients with known/suspected gram positive infection. received intravenous linezolid, mg b.d., and patients received teicoplanin mg o.d. mrsa clearance was assessed at end of treatment (eot), at -and days follow-up. results: in the linezolid and teicoplanin groups, and were known to be colonized with mrsa at study entry, respectively, while and were not. detection of clearance of mrsa colonization at eot was % for linezolid vs % for teicoplanin group (x b p b/ . ), at days it was % vs % (x b p b/ . ), and at days % vs % (ns). conclusion: short-term mrsa clearance can be achieved in significantly more patients treated with linezolid however this was not maintained at days, either because of incomplete initial eradication or recolonization. further analysis will follow when molecular typing of the isolates is completed. penetration of linezolid into bone, fat and muscle during hip arthroplasty ps lovering am, bannister gc, zhang j, macgowan ap, southmead hospital, bcare, bristol, uk there are limited data describing the concentrations and penetration of linezolid (lzd) into tissues, such as bone, that can be used to guide therapy for non-vascular infections. here we report the concentrations and penetration of lzd for bone, fat and muscle in comparison with cefamandole (cmd). twelve patients received mg lzd as a min infusion and mg cmd as a bolus injection immediately before hip arthroplasty. bone, fat, muscle and blood samples were collected at timed intervals after the infusion and assayed by a validated hplc method. for bone, peak levels of both agents occurred min after administration with mean levels of lzd . mg/kg versus cmd . mg/kg, decreasing to lzd . mg/kg versus cmd . mg/kg at min. correction for blood concentrations gave penetration of lzd % versus cmd % at min and lzd % versus cmd % at min. for fat and muscle, peak levels occurred min after infusion. mean levels were lzd . mg/kg versus cmd . mg/kg (fat) and lzd . mg/kg versus cmd . mg/kg (muscle). correction for blood concentrations gave penetration of lzd % versus cmd % (fat) and lzd % versus cmd % (muscle). we conclude that linezolid exhibits rapid penetration into bone and associated soft tissues achieving levels in excess of the mic for sensitive organisms; with a similar distribution and penetration profile to agents currently used for treatment of infections in these tissues. bacqué e a , barrière jc a , berthaud n b , desmazeau p b , dutruc-rosset g b , dutka-malen s b , ronan b b . a aventis pharma, chemistry, paris, france , b aventis pharma, disease group, paris, france xrp is a new oral streptogramin composed of semi-synthetic synergistic components in a / (w/w) association: rpr ( d-( -morpholino)methyl- d, g-dehydro pristinamycin i e ) from pi a and rpr [( r )- -deoxy- -fluoro pristinamycin ii b ] from pii b by original synthetic routes. the association has antibacterial activity against staphylococci including methicillin */mls b -resistant strains (mic range: . */ mg/ml), streptococci (mic : . mg/ml), pneumococci including multidrug resistant strains (mic : . mg/ ml), enterococci including vancomycin-resistant strains (mic : mg/ ml), m. catarrhalis and neisseria spp. (mic : . mg/ml), h. influenzae (mic : mg/ml), legionella spp. (mic : . mg/ml) and anaerobes (mic range: . Á/ mg/ml). xrp is generally bactericidal at the concentration of Á/ )/the mic against s. aureus , s. pneumoniae , h. influenzae and demonstrates consequent pae ( . Á/ !/ . h at Á/ )/mic, following an exposure of . Á/ h). mutants of s. aureus to xrp were isolated at low frequencies ( . )/ ( Á/ . )/ ( ) at )/ and )/mic while no mutant could be isolated at )/mic. these results suggest that xrp ( / w/w) is a promising compound for the treatment of community-acquired infections. ex vivo evaluation of rpr /rpr (xrp ), a new oral streptogramin ps berthaud n, diallo n. aventis pharma sa, infectious disease group, paris, france the intra cellular activity of xrp , was assessed in j murine macrophages containing ingested staphylococcus aureus (three strains) or l. pneumophila (one strain). at the concentration of )/ the mic, growth of s. aureus was strongly inhibited after a -h period of incubation (d log cfu/ml vs t controls range: (/ . Á/(/ . , according to the strain tested). at the concentration of )/ the mic, growth of intracellular l. pneumophila was inhibited after a Á/ -h period of incubation (d log cfu/ml vs t controls about (/ . and (/ . at and h, respectively). rpr and rpr alone had also inhibiting effect on bacterial growth (d log cfu/ml vs t controls after h of incubation about (/ . and (/ . , respectively). the bactericidal activity of xrp was also assessed against slowly growing s. aureus , under experimental conditions mimicking those observed in patients with an infected indwelling device (first step of infection: adherence to inert support; declared infection: biofilm model). under the experimental conditions, xrp demonstrated a rapid and potent bactericidal effect against s. aureus adherent to an inert support or included in biofilm. this effect was observed at each of the three concentrations tested ( , , and , and )/ mic, respectively). in vivo evaluation of xrp (rpr /rpr ), a new oral streptogramin ps berthaud n, huet y, aventis pharma sa, infectious disease group, paris, france the oral efficacy of xrp , was assessed in staphylococcus and pneumococcal murine infections. mice were challenged ip ( )/ , and )/ times ld ). abscesses were established by intramuscular injection of about bacteria into the right thigh of mice. pneumonia was established by intranasal injection of about bacteria. mice were treated twice a day for (staphylococcus aureus septicaemia and abscesses), (s. pneunomoniae septicaemia) or days (s. pneumoniae pneumonia). six to days post infection, for septicaemia and abscesses the results were expressed as ed , whereas for pneumonia they were expressed as the dose yielding an average survival time (ast) significantly longer than that of the untreated infected controls. xrp was efficacious in the treatment of experimental infections in mice caused by mls b -sensitive and -constitutively resistant s. aureus (ed range: Á/ and Á/ mg/kg/administration in septicaemia and abscess models, respectively). it was also efficacious in the treatment of infections caused by s. pneumoniae whatever the serotype and the resistance profile of the strains tested (ed range: Á/ mg/ kg/administration in septicaemia, ast: mg/kg/administration). these results suggest that xrp might be effective for the treatment of staphylococcal and pneumococcal community-acquired infections. xrp (rpr /rpr ), a new oral streptogramin: bactericidal activity and pharmacokinetics in a model of streptococcus pneumoniae mouse pneumonia ps berthaud n, huet y, diallo n. aventis pharma sa, infectious disease group, paris, france the bactericidal activity against streptococcus pneumoniae of xrp , a new oral streptogramin composed of two semi-synthetic synergistic components in a / (w/w) association (rpr , pristinamycin i derivative and rpr , pristinamycin ii derivative), was assessed in lungs of mice with pneumonia. mice were inoculated intranasally with about cfu of strain - (mls bresistant). eighteen hours later (t ), animals received xrp ( mg/kg p.o). the administration was repeated , , , and h afterwards. to study the influence of varying the ratio of pi to pii component administered on activity and pk parameters, ratios of rpr to rpr ranging from / to / were also administered under the same conditions. after an oral unitary administration at mg/kg, xrp , as well as ratios of rpr to rpr ranging from / to / , demonstrated strong and quick bactericidal activity in lungs. lung levels of rpr and rpr were generally equal or two times higher than blood levels. resulting rpr /rpr ratios in blood and lung, although not in accordance with the initial ratio administered, were synergistic for Á/ h in blood and for Á/ h in lungs explaining the activity observed. conclusion: based on in vitro data, telithromycin is a good candidate for the treatment of rti. in vitro evaluation of abt- , telithromycin and azithromycin against streptococcus pneumoniae , moraxella catarrhalis , haemophilis influenzae and methicillin-resistant staphylococcus aureus ps steele-moore l, berg d, barnes i, couch k, klein j, holloway w. christiana care, infectious disease, wilmington, us macrolide resistant streptococcus pneumoniae (sp) is a worldwide concern predominantly because these isolates tend to be multiply drug resistant. new agents with increased activity against these pathogens are clinically important. the ketolide class of antimicrobial agents demonstrate excellent in vitro activity against sp, even those that are macrolide resistant. the in vitro activities of the ketolides abt- (a) and telithromycin (t) were compared to azithromycin (az) against clinical isolates of sp, moraxella catarrhalis (m.cat), haemophilis influenzae (h.flu) and mrsa. organisms tested: strains of sp (including az resistant), h.flu, mrsa and m.cat. microdilution mic tests were performed following nccls recommendations using freshly prepared plates containing haemophilis test medium for h.flu, cation adjusted mueller-hinton broth (camhb) with laked horse blood for sp and camhb for m.cat and mrsa. the new ketolides, a and t had superior activity against sp including the az resistant strains (mic s: a / . mcg/ml, t / . , az / ). all compounds had excellent activity against m.cat while none demonstrated activity against mrsa. h.flu activity was comparable among a, t and az. these new ketolides are not currently approved by the fda; however t has been approved in europe. ló pez h, vidal gi, salomó n jm, scaglione m, zitto tr. centro de infectología, infectious diseases, buenos aires, argentina objective: we evaluated the impact of the initial treatment failure rate, hospitalisation and costs in outpatient treatment of adult cap in argentina comparing amoxicillin, clarithromycin and telithromycin. method: a probabilistic model was implemented in outpatient treatment of cap. we estimated an initial treatment with amoxicillin, clarithromycin or telithromycin. we assumed expected clinical cure at . , . and . %, respectively. for those patients with failure treatment we evaluated a second-line of antibiotics (amoxicillin followed by clarithromycin and clarithromycin followed by new fluorquinolone) or hospitalisation. patients with telithromycin and failure treatment must be hospitalised without a second line of outpatient treatment. costs of cap included drug's costs by days, medical visits, chest radiographies, analysis and hospitalisation. results: we estimated treatments in patients and first-line drug failure in , and patients with amoxicillin, clarithromycin and telithromycin, respectively. costs in outpatient treatment were: hospitalisation $ , and $ , . ; second-line drug $ and $ ; second-line hospitalised $ . and $ with amoxicillin and clarithromycin, respectively and hospitalisation with telithromycin $ . conclusions: telithromycin showed lower clinical failure, hospitalisation and costs in cap. some studies suggest shortening cap telithromycin treatment to days helping adherence to treatment and decreasing costs even more. the new macrolides */a good alternative of tetracycline in the treatment of mediterranean spotted fever ps popivanova nip a , petrov aip a , boykinova obb a , kazakova zkk a , baltadjiev agb b . a medical university, infectious disease, plovdiv, bulgaria , b department of anatomy, medical university, plovdiv, bulgaria mediterranean spotted fever (msf) caused by rickettsia conorii appears an endemic disease for some regions in bulgaria. frequently the disease has a severe course with multiple organ lesions. the early and adequate treatment is of extreme importance for the outcome of the disease. searching an alternative antibiotic treatment of this disease we considered macrolides for their good cell and tissue penetration and dose-dependent bacteriostatic and bactericide effect. we treated msf patients with doxycycline )/ mg/day, patients with clarithromycin )/ mg/day, as well as patients with midecamycin )/ mg/day and midecamycin acetate mg/kg/day. as a surrogate marker for treatment evaluation the effect on the febrile syndrome was used. our findings showed that by the th day of treatment the fever normalized in . , . and . % of the patients treated with doxycycline, clarithromycin and midecamycin, respectively. for the same period the patient fever decreased below c in , and . %, respectively. the intoxication symptoms were influenced within the same period equally in all treated patients. conclusions: we suggest that the new macrolides appear a good alternative of tetracycline on patients with msf. erythromycin resistance of gram /'// cocci in bulgaria. benefits of the new macrolides in the treatment of respiratory infections ps popivanova ni a , yovtchev ip b , dobreva md c , argirova ta c . a medical university, infectious disease, plovdiv, bulgaria , b medical university, ear-nose-throat disease, plovdiv, bulgaria , c medical university, microbiology, plovdiv, bulgaria in the recent years we tested erythromycin sensitivity of species of gram /'// cocci isolated from throat and nose secretions, ear and eye effusions, sputa, cerebrospinal fluid and blood cultures, vaginal and urethral secretions, urine and fecal samples from patients with inflammatory diseases of the listed organs and systems. staphylococcus aureus , staphylococcus coagulase /(//, streptococcus â-haemolyticus , enterococcus were isolated. of these microorganisms s. aureus was the most abundant. our resistograms revealed sensitivity of the gram /'// cocci in . and . % for and , respectively, and resistance of . and . %, respectively. in the tests with midecamycin and midecamycin acetat the same microorganisms showed sensitivity of . % and resistance of . %. the clinical findings showed excellent effect of the new macrolides including clarithromycin and azalides */azithromycin. we conclude the resistance of gram /'// cocci and especially of s. aureus to erythromycin increases very quickly and has reached dramatical extent. by now, the new , , and -membered ring macrolides and azalides show high antibacterial activity and good clinical effect. pappas ga, liberopoulos e, tsiara s, elisaf m, tsianos e. university hospital, internal medicine, ioannina, greece quinupristin/dalfopristin (q/d) is a novel injectable streptogramin antibiotic which initiation in therapeutics was hailed as an important step towards treatment of vancomycin-resistant enterococcus faecium (vref) species. initial reports concluded in an excellent response of vref to q/d. reports of q/d-resistant strains of e. faecium have emerged, both in usa and europe. we report two cases of e. faecium bacteremia in which the responsible isolate was not sensitive to q/d. the first patient was a woman with acute leukemia and septicemia. e. faecium was cultured from blood samples: the species was resistant to almost all antibiotics, exhibiting sensitivity only to tetracycline (!), while its sensitivity to q/d was indermediate. the second patient was a man with endocarditis, in whom blood cultures isolated e. faecium sensitive to a number of antibiotics, including ciprofloxacin and vancomycin; still the sensitivity to q/d was indermediate. q/d has not been officially introduced to the antibiotic arsenal of greek medicine. moreover, the drug has never been used in our hospital, not even experimentally. other e. faecium species isolated in our hospital have been sensitive to q/d. how much hope can be put on a drug that seems to be partly useless before its initiation? increasing reports of v/ q-resistant strains of e. faecium from all over europe raises fears that the v/d story might well end before it begins. varadinova t a , diakov t a , karagiozova d a , genova p a , pardon p b , baudry c b , quideau s b . a sofia university, virology, sofia, bulgaria , b et institut du pin, centre de recherche en chimie moleculaire, universite bordeaux , bordeaux, france vegetable tannins posses a wide range of biological activities. the aim of the present study was to evaluate the cytotoxicity of five purified vegetable tannins against mdbk cells. the maximal nontoxic concentrations (mnc) and the concentrations required to inhibit cell growth by % (cc ) were evaluated after , and h periods of action. mnc values after h indicated that compounds stimulated cell surveillance when applied in concentrations lower than . mm. cc values indicated: (i) a decrease in cytotoxicity after h as cc were up to times lower than those observed after the h period, and (ii) a re-increase in cytotoxicity when the period of action was prolonged up to h as cc were times lower than those observed after the h period. these data thus appear to reveal the capability of the investigated natural polyphenolic products to stimulate cell surveillance in a time-dependent manner. antibacterial effect survey of enoxolone on periodontopathogenic and capnophilic bacteria isolated from specimens of patients with periodontitis ps salari mh a , kadkhoda z b , sohrabi n a . a tehran university of medical sciences, pathobiology, tehran, islamic republic of iran , b tehran university of medical sciences, dentistry, tehran, islamic republic of iran objectives: most of the microorganisms associated with periodontitis are capnophilic and anaerobic bacteria. purpose of this study was to detection of antibacterial effect of enoxolone on periodontopathogenic and capnophilic bacteria. methods: in this study periodontopathogenic and capnophilic bacteria were isolated from specimens of patients with periodontitis by culture method. an anti-bacterial activity of enoxolone against these microorganisms was investigated by minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) methods. results: based on our findings the mic, mbc and lethal does (ld ) of enoxolone for actinobacillus actinomycetemcomitans , eikenella corrodens and capnocytophaga were ' , , ', ' , , ', and ' , , ' mg/ml, respectively. conclusion: our results show enoxolone has antibacterial effect on a. actinomycetemcomitans , e. corrodens and capnocytophaga spp. sakalova stn. medical university, microbiology, grodno, belarus we have synthesized diamides of dicarboxylic acids, with components such as -nitrothiazole benzolsulphamides and triazol. all the above compounds exhibited bacteriostatic activity towards some microorganisms. for further studies of bacteriostatic activity of amides and diamides of dicarboxylic acids, as well as for determination of 'structure Á/activity' relationship, we have synthesized a range of monoamides. antibacterial activity of the synthesized compounds was studied in vitro by agar dilution methods. for this purpose, approximately various gram-positive and -negative microorganisms including clinical strains of staphylococcus aureus , bacillus subtilis , serratia marcescens , escherichia coli , proteus morganii , micrococcus lisodeicticus , staphylococcus epidermidis , shigella sonnei , salmonella typhimurium , yersinia enterocolitica . minimum inhibitory concentration was expressed in mg/ml. nitazole was used as a comparison substance. analysis that new derivatives of -nitrotiasole have high antibacterial activity relative towards certain microorganisms included strains obtained from infection department's patients. results will be shown. microbial susceptibility to the essential oil of ziziphora clinopodiodes lam. ps purpose of the study: antimicrobial activities of essential oils vary from oil to oil and from one micro organism to another. the antimicrobial and chemical properties of essential oil from ziziphora clinopodiodes lam. has not been studied and hence the present study was planned to evaluate those properties against a series of micro organisms viz, escherichia coli , staphylococcus aureus , pseudomonas aeroginosa , klebsiella pneumonia , bacillus subtilis , bacillus licheniformis , streptococcus faecalis , candida albicans and saccharomyces cerevisiae . results: z. clinopodiodes lam. essential oil was found to have remarkable antimicrobial property against all the microorganisms but p. aeroginosa . the oil exhibited its best antimicrobial activity within a maximum of min. seventeen components were identified by gas chromatography and mass spectrometry (gc and gc/ms) analysis of the oil, out of which pulegone ( . %), neomenthol ( . %), cyclohexene, -methyl- -( -methenyl)trans ( . %), , -cycloheptadien- -one, , , -trimethyl ( . %), piperitone ( . %), and limonene'/ , -cineole ( . %) constitute major parts of the oil. conclusion: monoterpenes seem to have antimicrobial role. it seems necessary to explore antimicrobial properties of new harmless antimicrobial agents from natural sources as substitutes for common chemical drugs. methanol extract of carpobrotus edulis enhances killing of methicillin resistant staphylococcus aureus phagocytosed by thp- human monocyte derived macrophages and promotes the release of modulators of cellular immunity ps although alkaloids from the family mesembryanthemaceae have anti-cancer activity, species of this family have received little attention. because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, we have studied a crude methanol extract of carpabrotus edulis , a common plant found along the portuguese coast, for properties normally associated with plasma membrane active compounds. the results of this preliminary study show that the extract is non-toxic at concentrations that prime thp- human monocytederived macrophages to kill ingested methicillin resistant staphylococcus aureus and promote the release of lymphokines associated with cellular immune functions. the extract also induces the proliferation of thp- cells within day of exposure and days earlier than that induced by phytohemagglutinin. similar results were obtained with monocyte-derived macrophages isolated from human peripheral blood. the active component or components of the plant extract may be exploited as intracellular active anti-bacterials as well as modulators of cellular immunity. enhancing of erythromycin production by saccharopolyspora erythraea nur with common and uncommon oils ps hamedi j a , malekzadeh f a,b saghafi-nia ae b . a department of biology, faculty of science, university of tehran, tehran, islamic republic of iran , b shafe-e-sari co., antibiotic production co., teheran, iran enhancing effect of various oils on the erythromycin production by saccharopolyspora erythraea nur were evaluated in a complex medium consisting soybean flour and dextrin as the main substrates. the biomass, erythromycin, dextrin and oil concentrations, and ph value were measured on a daily basis. also, the kinds and frequencies of fatty acids of the oils used were determined. saturated fatty acids in the shark oil were higher than that of vegetable oils used. erythromycin concentration in the melon (cucumis melo var. inderus cultivar mashhad ) seed oil containing medium was . times higher than that of the control medium (without oil) and . times higher than that of rapeseed oil containing medium. erythromycin concentration in the other oil containing media, including rapeseed, soybean, shark (carcharhinus dussumieri ), and safflower oils was . , . , . , . time higher than that of control medium, respectively. the melon seed oil had the least enhancing effect on the biomass production, and thus decreasing the cost of the biomass separation. can varicella be eliminated by universal childhood vaccination ? */ epidemiological and economic data from germany ps wutzler p a , banz k b , neiss a c , goertz a d , bisanz h d . a institute for antiviral chemotherapy, university of jena, jena, germany , b outcomes international, basle, switzerland , c institute of medical statistics and epidemiology, technical university, munich, germany , d glaxosmithkline, pharma, munich, germany purpose: universal varicella vaccination in childhood is expected to reduce substantially the number of uncomplicated cases of chickenpox and to decrease the number of complicated cases requiring hospitalization. to generate fundamental data for decisions of the health authorities epidemiological and health-economic data were collected in two large studies. using an age-structured decision analytic model the benefits, costs and cost-effectiveness of a varicella immunization program over a period of years were assessed. results: it could be shown, that the vast majority of varicella cases occur in children aged less than years. in . % of the cases a severe course was assessed. overall incidence of complications was estimated to be . %. a routine varicella vaccination program targeting healthy children could prevent . % of varicella cases and over major complications per year provided the coverage rate would be %. under these conditions the elimination of varicella is predicted to be achievable within Á/ years. a combined measles, mumps, rubella and varicella vaccine is expected to provide the required coverage. conclusions: routine childhood varicella vaccination appears to be a highly efficient strategy to significantly reduce the sizeable burden of varicella and leads to significant savings from both societal and payer's perspective. bulgakova va, balabolkin ii, sentsova tb. scientific center of child health, russian academy of medical sciences, scientific research institute of pediatrics, moscow, russian federation objective: to estimate efficacy of vaccine influvac at children with allergic diseases. methods: twenty children aged Á/ years with allergic diseases received vaccine influvac (solvay pharma). for the control group of children, with allergic pathology did not receive this vaccine because of an intolerance of chicken protein ( children). results: all vaccinated children for the observable season of months did not get influenza. general and aboriginal reactions to a vaccine did not occur. in control group for the observable season two children were ill with influenza and four children with acute respiratory virus infection ( %). among vaccinated children there was an increase in titre to a protective level ( : and above) to all to three strains of influenza days after injection. vaccine influvac can be recommended for an immunisation against influenza of children with an allergic pathology because of efficacy and absence of side effects. ló pez h, zitto tr, vidal gi, cánepa mc, salomó n jm, scaglione m. centro de infectología, infectious diseases, buenos aires, argentina objectives: our study examines the possible economic impact of the influenza on health working adults in argentina, and the intervention cost saving with immunization. methods: this is a theoretical study based on a mathematical model. population data was published in s national statistics. the global incidence of influenza infection was estimated at %. we have estimated the direct cost on influenza infection (outpatient visits, drugs and hospitalization) and indirect cost (work absenteeism and productivity loss) and projected net saving for the Á/ year-old vaccinated group. the vaccine effectiveness was estimated at and %. the price of vaccine was $ each. results conclusion: influenza vaccination is effective in diminishing cases of flu and reducing working-day loss. its a safe and cost-effective vaccine. ló pez h, zitto tr, cánepa mc. centro de infectología, infectious diseases, buenos aires, argentina flu infection is a major cause of illness and one of the most common cause of work absenteeism, increasing institution costs, healthcare provider visits, use of drugs, and decreasing work productivity. vaccine against flu has an effectiveness between and %, in health adults. objective: evaluate the impact of flu-like respiratory tract infections in a health institution staff during year, comparing vaccinated with not-vaccinated groups. methods: we evaluated all causes of absenteeism along year ( ), based on the written note made by the professional who has evaluated ill people, selecting flu-like respiratory infection causes. we evaluated age, working days loss related to illness, and cost on vaccinated and not-vaccinated groups. results: one hundred and sixty eight of the total staff ( people) were vaccinated, of them had flu-like infection, resulting in working days lost. for not-vaccinated group, people had flu and lost days. lost cost for vaccinated group was of $ , and for notvaccinated group, $ . conclusion: we observed a decrease in working days loss and money waste related to flu-like infections on the vaccinated group. because of safety and effectiveness of vaccine against flu, the implementation of vaccination will be cost-effective for all institution staff. we studied functioning of the interferon system in children with atopic bronchial asthma (ba) at the age of Á/ years. the control group included healthy children. we investigated the interferon status (method of grigoryan s.) and serum concentrations of ifngamma (ifng) (elisa). there was a decrease in the ifn-producing ability of leukocytes to the synthesis of ifna at % and ifng at % of children with ba. serum level of ifn of children with ba during all period of illness is compared to the children without predisposition to atopy ( . / . and . / . pg/ml accordingly) was significantly decreased. production of ifna increased after using viferon (recombinant a b ifn and antioxidants). decreased ability of gammainterferonogenesis in the most children was not affected by the action of immunomodulators. there was shown interferon system's dysfunction in the development of atopy and increasing predisposition to respiratory infections and to persistent of atypical infections in children with ba. harxhi a, pilaca a, pano k. university hospital center of tirana, infectious diseases, tirana, albania congo-crimean haemorrhagic fever is viral disease with a high rate of mortality that is caused by a nairovirus, bunyaviride species. this is a zoonotic disease, which affects sporadically humans and is geographically distributed even in eastern europe and balkan. during the months of may and june , in northeast of albania were reported eight cases of haemorrhagic fever. serologic tests performed in the laboratory of reference in thesaloniki, greece confirmed the diagnosis of congo-crimean haemorrhagic fever. in the mean time, who reported the outbreak in southwest kosovo of cases suspected for haemorrhagic fever from which were confirmed laboratory as congo-crimean haemorrhagic fever. we are describing here the clinical history of one of eight cases with cchf in albania. from the epidemiological point of view this case was considered peculiar, as it was the only one hospitaly acquired, and due to the gravity of the haemorrhagic syndrome was admitted at the intensive care unit at infectious diseases service, university hospital center of tirana. results: in the study were included patients ]/ years of age with documented hbsag-carrier ]/ months (average age */ . years, male */ %, female */ %). hbv dna in serum was tested by qualitative and quantitative pcr (commercial test-system ampli-sens hbv). hbeag, hbeab, hbsag were detected by elisa (hoffmann la roche). hbv dna by qualitative pcr was detected in % patients, by qualitative pcr was detected in % patients in the concentration ]/ copies/ml, in . % ]/ copies/ml, in . % ]/ copies/ml, in . % ]/ copies/ml ( fig. ). hbv dna level distribution among the hbsag carriers. elevated ( . the upper limit of normal) alt level was determined in . % of the hbv dna negative and . % of the hbv dna positive patients. hbeag was detected in . % of the hbv dna positive patients and had not been determined in the hbv dna negative patient. eleven percent of patient had the combination of the biochemical, serological and virology criteria, which are typical for active chronic hepatitis b (hbsag-carrier !/ months, hbv dna ]/ )/ copies/ml, elevated alt). conclusion: in smolensk % of the hbsag-carriers have viral replication confirmed by qualitative pcr. eleven percent of them have active chronic hepatitis b. kandemir o a , polat a b , kaya a a . a medicine of faculty, mersin university, clinical microbiology and infectious disease, mersin, turkey , b medicine of faculty, mersin university, pathology, mersin, turkey the exact potential of nitric oxide in the pathogenesis of chronic viral hepatitis is not known. the elevated nitric oxide production is assumed to be responsible for the pathological changes in many inflammatory conditions, mainly via peroxynitrite, a potential oxidant that is produced by the reduction of superoxyde anion with nitric oxide. the intensity and the distribution of the immunohistochemical staining of intrahepatic inducible nitric oxide synthase were studied in the biopsy specimens obtained from patients with viral hepatitis and patients with elevated transaminase levels from other etiology. hepatic inducible nitric oxide synthase staining was significantly more intense in the viral hepatitis group (p / . ). inducible nitric oxide synthase staining levels correlated well with the severity of the viral hepatitis using the knodell's liver histological activity index (r / . , p / . ). among the viral hepatitis group, the pathological distribution of the inducible nitric oxide synthase staining favored the periportal regions whereas less staining was observed in the bile duct and parenchyma regions. as nitric oxide mediated nitration of hepatocellular proteins is found to be elevated in the inflamed hepatic tissues and it well correlated with the severity of the disease, we suggest that inducible nitric oxide synthase can possibly have a critical role in the pathogenesis of chronic viral hepatitis. there were patients under observation who were divided into two groups, the first of patients (eight chronic virus hepatitis; three chronic virus hepatitis'/steatosis; four steatohepatitis; three chronic cryptogenic hepatitis; there were men and four women aged from to . the second group consisted of patients, eight with chronic virus hepatitis; four with steatohepatitis; six with chronic cryptogenic hepatitis. there were men and three women aged from to . diagnosis was confirmed with the help of clinical data, biochemical tests, serological markers, psr-diagnostics and ultrasound examination and computer tomography of the abdomen. in the first group of patients the treatment with ursofalk was administered at the dosage of mg/kg of body mass from month to years with improvement in general condition of the patients: heaviness, pain under the right rib, nausea and skin itch have disappeared. in all the cases, improvement in the biochemical blood analysis took place during treatment. the average index of alt activity was before */ . u/l, after */ . u/l and ast */ . and . u/l; alp */ . and . u/l; ggt */ . and . u/l; chol */ . and . mmol/l; tg */ . and . mmol/l. in the second group of patients the treatment was carried out with various hepatoprotectors during the courses from to months. before the average index of alt activity was . u/l, after */ . u/l; ast */ . and . u/l; alp */ . and . u/l; ggt */ . and . u/l; chol */ . and . mmol/l; tg */ . and . mmol/l. treatment of patients suffering from hepatitis of viral and other aetiologies with ursofalk produces a positive effect on both clinical symptomatic and biochemical indices. remission was more stable during a long period of taking the preparation. the hepatoprotective effect of ursofalk during the years was sustained for the whole of the period of the treatment. after stopping, an acute attack of cytolytic syndrome was observed. with other hepatoprotectors we did not get any improvement in clinical scene of the disease or in biochemical indices. shavlov nm, kletsky sk. minsk, belarus hsv- and - have possibility to damage different organs and systems. sometimes they cause damage of the liver, which resemble viral hepatitis. the etiology of such hepatitis may be confirmed only by results of liver biopsy. we have diagnosed cases of herpetic hepatitis: eight children and four adults. clinical course was different. in five cases the acute beginning took place: high temperature, the jaundice at the Á/ day (the level of bilirubin was Á/ mkmol, especially direct), cholestasis, the pain in upper right part of abdomen. the ascites was found in three patients with acute hepatitis during st week from the beginning of disease. in seven cases the beginning was gradual. the temperature was subfebrile, prolonged; malaise and moderate pain in upper part of abdomen were constant complaint. the jaundice was moderate; bilirubin increased until mkmol. the level of alt was moderately increased ( Á/ times). the blood analysis showed moderate leukocytosis with neutrophilia, and increased sre. the serological markers of hepatitis a, b, c, d were negative in all cases. hsv- and - were found in the blood. the diagnosis was defined by the results of hystochemical investigations, when the viruses were found in liver bioptat, and confirmed with the results of specific treatment. specific damage of liver cells was found: protein dystrophy and specific inclusions in cell nucleus. in all cases the treatment with acyclovir were given. the results we have observed during st week: the temperature became normal, the jaundice decreased and bilirubin was normal during Á/ days. in one case the recidive took place weeks later after treatment. the second course of acyclovir with intron a gave good results. nesic z a , delic d b , prostran m a , vuckovic s a , stojanovic r a . a department of pharmacology, school of medicine, university of belgrade, belgrade, yugoslavia , b clinical center of serbia, institute for infection and tropical disease, belgrade, yugoslavia the large number of unsolved cases of acute and chronic hepatitis has most probably the viral etiology. in mid s, two independent groups of authors reported a new human hepatotropic virus, with flavivirus like genomes, hepatitis g virus (hgv). the aim of this pilot study was to determine the prevalence of hepatitis g viral infection among patients at high risk of exposure to blood and blood products, as well as to evaluate if the risk of hgv infection was higher among them than in the general population. immunoenzyme test on microtitration plate for detection antibodies against hgv e antigen in plasma or sera (r&d systems, minneapolis, usa) was used for evidencing anti-hgv igg antibodies in sera. anti-hgv antibodies were detected in the control group (blood donors) in . % ( / ) patients. prevalence of anti-hgv antibodies among i.v. drug users was evidenced in . % ( // ), in hemophiliacs . % ( / ), in patients acquiring multiple blood transfusion . % ( / ), in hemodialyzed patients . % ( / ) and in patients with transplanted organs . % ( / ). our results suggest that patients exposed to blood or blood products have a higher risk of hgv infection than general population. evaluation of ortho total hcv core antigen assay in assessment and follow-up of patients treated for chronic hcv ps lunel f, veillon p, payan c. chu angers, laboratoire de bactério-virologie, angers, france an assay to quantitate 'total' hvc core antigen (hcv ag) in serum or plasma, may reflect viral load, has been developed by ortho-clinical diagnostics. methods: we evaluated hcv ag with two quantitative assays for hcv rna: bdna . (bayer) and monitor . (roche). we studied samples from untreated patients and from patients with chronic hcv treated with ifn or ifn/ribavirin. results: correlation of ag and quantitative assays was high (r / . for bdna . and . for monitor . ). no difference between the levels of rna and ag among hcv genotypes (r / . Á/ . ) was found. ag values, before treatment, were significantly lower in sustained responders (sr) than in other groups ( . log versus log, p b/ . ). in patients treated with ifn or combination therapy, we found very good correlation between decrease and negativation of ag and viral load: log iu/ml decline after m of interferon was significantly correlated with the negativation of hcv ag and sr. thirty-eight/ of sr had a rna load decrease !/ log iu/ml and / had a negativation of hcv ag after m . conclusion: total hcv core ag appears to be a new tool for monitoring patients with hcv infection. hepatitis c virus rna and hcv core antigen kinetics predict the efficiency of interferon-alfa and ribavirin therapy in naive patients infected by hcv genotype or ps fifty-five patients infected by genotype or were treated with a primary dose of (if hepatitis c virus (hcv) rna b/ meq/ml) or million units of interferon alfa- b (ifn) thrice weekly for months. ribavirin was added at month (m), until m if hcv rna was found positive after m of ifn. the viral kinetic was assessed during the follow up by serial measurements of hcv rna (bdna . and monitor . ) and using a new assay from ortho-clinical diagnostics which is able to quantify total hcv core antigen. sustained virologic response was observed in % of the patients ( / ). after month of ifn treatment, sustained responders had a fall of hcv rna and hcv core antigen higher than non-responders ( . / . log ui/ml versus . / . log ui/ml, p b/ . , for hcv rna) and ( . / . log (pg/ml)/ ) versus . / . log (pg/ml)/ ) p b/ . , for hcv core antigen). after month of ifn, the positive and negative predictive values of sustained response were, respectively, and . % for hcv rna negativation and . and . % for hcv antigenemia negativation. these results suggest that both kinetics of viral load and antigenemia are highly predictive of sustained response. theodorou m, petinelli i, pontikaki d, mela c, blana a, papanastasiou a, toliopoulos a, stavrakaki m, sagkana e. microbiology department, western attika general hospital, greece, egaleo, greece greece has accepted a big number of economic immigrants lately. we investigate the prevalence of hepatitis b/c as well as the epidemiological features that might influence the public health. . economic immigrants from: albania , eastern europe and from asiatic-african countries , visited our hospital to be checked in order to get a health certificate to obtain the green card. they where tested for hepatitis b/c. the serological markers were determined by immunoenzymatic method. all hbsag('/) and anti-hcv were further tested for hbv dna and hcv rna by competitive rt pcr. hcv rna('/) were genotyped by strip hybridization immunoassay. in albanians . % were hbsag('/), . % hbv dna('/), . % anti-hcv('/) and . % hcv rna('/). in east europeans . % were hbsag('/), . % hbv dna('/), . % anti-hcv('/) and . % hcv rna. in asians-africans, . % were hbsag('/) and % hbv dna('/). in pakistanis, . % were anti-hcv('/) and . % hcv rna('/). of the rest of asians-africans, . % were anti-hcv('/) and . % hcv rna('/). albanians: higher prevalence of hbv infection ( . %). greek blood donors: % pakistanis: hcv infection is % (predominance of a type), general greek population: %. public health services in greece and europe must take appropriate measures. el zawawy la a , mohamed on b , ali sm a , eissa me a , allam sr a . a faculty of medicine, parasitology, alexandria, egypt , b high institute of public health, microbiology, alexandria, egypt the purpose of the study was to investigate the influence of schistosomal suppression on the antibody response to hepatitis-b vaccine (hbv) and to study if the vaccine has any protective effect on experimental () infection. the results obtained revealed that infection reduced the serum antibody level against hbv. parasitological and histopathological findings showed significant protection against infection. the conclusion reached was; in order to reduce the incidence of virus-b infection especially in schistosomiasis endemic areas, public health officials should evaluate a policy for regulation of hbv booster vaccination to enhance the population immunity against hepatitis-b infection. cooper e, fisher t, shingadia d. newham general hospital, family clinic, london, uk sustained anti-retroviral combination chemotherapy requires excellent adherence to the regimen so as to suppress viral replication sufficiently to delay the emergence of resistance. if chemotherapy were taken to scale, e.g. in africa, erratic adherence might soon lead to multi-resistant circulating virus. we reviewed our experience in a well established london family clinic with a team including community nurses. we reviewed the records of the african immigrant children, aged Á/ , treated with anti-retrovirals exclusively at our centre throughout . whereas had undetectable hiv rna within the year, only four had undetectable rna throughout the year. four failed therapy through proven resistance mutations, but nine were considered through circumstantial evidence to have rising viral loads primarily because of poor adherence. three were known to have stopped taking drugs for extended periods. the three boys over years were unreliable in adherence, but the one girl in this age-group was fully adherent. our preliminary assessment is that for the children in our families, despite a team approach and home visits, nonadherence to haart may be twice as common as selection of a dominant viral mutant as a primary cause of failure to sustain viral suppression. quiros-roldan e, moretti f, castelli f, el-hamad i, carosi g. the prevalence of hiv related lipodystrophy-syndrome depending on the definition and severity of lipodystrophy ranges from to %. we have retrospectively reviewed the medical records of african patients followed. the characteristics are shown in the . % of africans had triglycerides !/ mg/ml and . % had cholesterol !/ mg/ml, none had both metabolic alterations. glycemia !/ mg/ml was observed in . % patients. it is interesting highlight that in any the africans morphological changes were noted and all of them showed weight stable. although the low prevalence of metabolic alterations may be attributed to the different ethnic alimentary behavior if self-body perception by african is not as accurate as by caucasian on the estimation of the body changes have to be investigated. alabaz d a , alhan e a , yaman a b , evliyaoglu n a , kocabas e a , aksaray n a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey hepatitis a virus (hav) infection is usually asymptomatic in children. however, it may occasionally cause a severe disease with high morbidity and mortality, and loss of school or business days. in a previous study, we have shown that every one of two to three school children from upper social classes living in adana carries high risk of hav infection. it is well known that maternally transmitted anti-hav antibodies interfere with hav vaccination. in an effort to determine the optimal age for hav vaccination, babies ( % girls and . % boys) born in our hospital were prospectively followed up at least months for the presence of maternal antibodies to hepatitis a (anti-hav igg). anti-hav igg titers were measured from the blood specimens obtained at birth from the mothers and from the offsprings at months, , , , , , , , and . the prevalence of positive anti-hav at birth ( %) was similar to those of hav seroprevalance studies carried out in adults in our area. the disappearance of antibodies occurred between the st and st month of life. the prevalence of anti-hav igg among children aged , , , , and months were , , , , and %, respectively. in light of these findings, we suggest that hepatitis a vaccination be given after months of age. earlier vaccination may be ineffective due to interference with maternally transmitted anti-hav antibodies. ghaderi b, alaghebandan r, rastegar lari a. department of microbiology, iran university, tehran, islamic republic of iran diarrhoea is a major public health problem in developing countries. amoebiasis is one of the most common causes of diarrhoea in iran as an endemic area for amoebiasis. little, however, is known about the extent of the condition in our society. the aim of this study is to determine socio-demographic and clinical characteristics of patients with intestinal amoebiasis. during july and august , we collected patients with diarrhoea among patients who visited at a referral hospital in shahriar area (in countryside of tehran), iran. thirty out of patients ( %) had intestinal amoebiasis and were followed up prospectively until the resolution of the illness. nineteen of ( . %) patients were male and the remaining of . % was female. the patients were aged Á/ with mean of . years. most of the patients ( %) were below years of age and the peak of occurrence was between the age of and years. watery diarrhoea with abdominal cramps was the main clinical feature. seventy percent of patients were resident in urban area and the remaining ( %) in rural area. average family income was low and all patients were in low socioeconomic level. water supplying system for all patients was pipeline water. low socioeconomic level associated with poor personal hygiene was the most important factor for highly prevalence of this problem in our society. also it seems that food plays important role in transmission of protozoa then water. the new strategy for allele identification of the genes coding for pertactin and pertussis toxin subunit s in bordetella pertussis ps bordetella pertussis strains demonstrate a significant polymorphism in toxin s subunit and pertactin, which are major protective antigens of the organism. monitoring the changes in prevalence of particular alleles of genes coding for these proteins in local b. pertussis populations is an essential issue in cases of the observed decrease of vaccination effectiveness. we have developed a new method for allele identification of these genes, which eliminates the necessity of dna sequencing. the approach is based on the identification of the number of repeats or the presence of specific nucleotides in the polymorphic regions or residues, respectively, of the genes and utilises products of their full or partial pcr amplification. the nucleotide heterogeneity in each polymorphic site is analysed either by the differential digestion of the amplicons or by the arms (amplification-refractory mutation system) methodology. numbers of repeats in particular regions of the genes are revealed by the size analysis of the adequate pcr products or their restriction fragments. in all cases the presence, size or pattern of dna molecules obtained is visualised by the agarose gel electrophoresis. the preliminary analysis of the recent and archival b. pertussis strains identified in poland was performed using the described approach. the presented strategy provides a much easier, faster and more cost-effective than dna sequencing mean to study the polymorphism of the major b. pertussis antigens. vaccination coverage and history of vaccine preventable infectious diseases among students in second year of medicine and pharmacy of tours university ps borderon jc a , hamed a b , ragot s b . a centre hospitalier universitaire, tours, france , b médecine préventive universitaire, tours, france the purpose of this study was to determine the level of infectious risk in students who will be exposed to patients. information was obtained by a questionnaire for each student, and by checking medical records for immunization coverage and vaccine preventable infectious diseases. answers could be specified for students, of whom females (f) and males (m). the number of non-immunized students was against diphtheria: two, tetanus: three, pertussis: four, poliomyelitis: two, hepatitis b: six, and hepatitis a: , respectively. among the students non-vaccinated against measles, (nine f and five m) had no history of that disease. among ( f and m) non-vaccinated against rubella, ( f and seven m) had no history of that disease, uncertain in seven others (six f and one m). the date of vaccination was often late regarding recommendations. fifteen students had no history of varicella. one student had not received bcg vaccination. fifty-eight students had received two, and three bcg vaccines. post-bcg tuberculin skin testing was missing after first bcg, second and third bcg. the date of the first tuberculin test was often one or several years after bcg vaccination. adverse effects of vaccination were rarely reported: two cases of fever (dt polio, measles); three cases of local reaction (dt polio, dtp polio). one case of contraindication for influenza vaccine: egg allergy. the survey shows failure in immunization coverage actually recommended in health care students. objectives: to present the morbidity of rabies and evaluate the efficiency of our prophylaxis scheme in lasi county. material and method: we made a retrospective study of the rabies cases in the patients admitted in our unit in a th-year period. we have analysed all the clinical, epidemiological and biological aspects. results: in a -year period, cases of rabies were admitted in the clinical infectious diseases hospital lasi. the highest incidence was for Á/ */eight cases ( %); the highest yearly cases were three cases in and . most of the patients were male ( %), came from suburban areas ( patients). eight cases occurred in may Á/june, wild animals were involved in half the cases (fox, wolf). for patients, no prophylaxis was performed and an incomplete course in four cases. the period of time to the appearance of the first symptom was Á/ days. the prophylaxis scheme led to a good protection. conclusions: in lasi county, rabies is a problem with a prevalence of . %/year. trends in the use of antimicrobials in riyadh in were analyzed. data was obtained from a survey of randomly selected families of school children aged Á/ years in a -month period in . one hundred and ninety-nine ( . %) students were on antibiotics in the month preceding the study; ( %) received antibiotics for the diagnosis of pharyngitis; ( %) students antibiotics were prescribed by a physician; and in ( %) the duration of antibiotics was less than week. this study shows a major problem in antibiotics prescription in our community and also the need to establish effective antibiotics policy in general practice to limit the potential emergence of drug resistance bacteria in the community. mir s a , cura a a , erdogan h a , guler s a , sengul gn a , koyu a a , ozinel ma b . a department of pediatrics, ege university medical faculty, izmir, turkey , b department of microbiology, ege university medical faculty, izmir, turkey antibiotic susceptibility spectrum of childhood urinary tract infection agents are geographical variation. the current antibiotic regimens and the selection of antibiotics for prophylaxis should be re-evaluated periodically. the objective of our study was to determine the local resistance rates to antibiotics and to give a direction for the selection of antibiotics in uti treatment. we evaluated urinary culture assays retrospectively, sent from pediatrics and pediatric surgery inpatient and outpatient clinics of our hospital during the last months, and investigated the isolated pathogens and the resistance rates to antibiotics. in addition, the data obtained were compared with of years ago. with respect to the resistance rates to antibiotics of uti pathogens, the resistance rates of e. coli for carbapenems, aminoglycosides and third generation cephalosporins were , and %, respectively as before, but the rates for ampicillin increased from to % and for tmp-smx it increased from to %. we concluded that the resistance profiles to antibiotics should be reviewed every years at least and thus the selection of proper antibiotics would lessen the morbidity as well as the medical expenses. chanturidze tk, tsiklauri r. ministry of labor, health and social affairs, public health, tbilisi, georgia purpose: since infectious diseases (id) are increasing in georgia. this study is aimed to reveal economic barriers of effective id control by assessing financial contribution to id from public and private sources, household's total spending on health and their capacity to pay. sources: ) national household expenditure and revenue survey. ) who fair financial contribution methodology. ) meta-analysis. results: . % of population leaves under the poverty level; % out of total household expenditure (average gel; us$ ) % is spent on food */non-subsistence income covers expenditures on goods and services including health; % of population refuses health services because of inability to pay; public spending on health comprises % of total health expenditures; public spending on id control is below gel per capita; almost all private spending goes to id treatment and equals to . gel per patient. conclusions: insufficient public spending on id control transfers the burden to the population with extremely low capacity to cover health expenses. refusal to utilize health services, and incomplete treatment and increases the threat of id spread and drug resistance. government should increase the allocations to id from public sources for effective id control in georgia. antimicrobial consumption trends in children's university hospital ps ratchina s a , averchenkova l b , jarkova l a . local surveillance of antimicrobial (am) consumption is essential to promote the rational use of this group of drugs. the purpose of this study was to analyze the trends of am use in the children university hospital in and . data on am usage were obtained from the hospital drugstore requests in the -beds multi-ward children university hospital. consumption was expressed as the number of ddd per bed-days (b Á/d). the total am consumption figures were similar in and ( . and . ddds/ b Á/d, respectively) with notable differences in am prescribing patterns. penicillin consumption increased from . to . ddds/ b Á/d mostly due to amoxicillin. the overall aminoglycoside usage remained comparable ( . vs. . ddds/ b Á/d) though amikacin has considerably replaced gentamicin. there was a sevenfold increase of ciprofloxacin ( . vs. . ddds/ b Á/d) along with the evident decrease of tetracycline and co-trimoxazole consumption found ( . vs. . ddds/ b Á/d and . vs. . ddds/ b Á/d, respectively). the tendency to prescribe more effective in respect of the local resistance data and/or more safe am was detected in comparing with that can be explained by the introduction of the local guidelines for infectious diseases management in . clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis */a pilot study ps the aim of this pilot study was to determine the efficacy and tolerability of clarithromycin in the treatment of chronic prostatitis caused by chlamydia trachomatis (ct). fifty-two patients older than years of age with diagnosed chronic chlamydial prostatitis were enrolled. the presence of ct in expressed prostatic secretion or urine specimen voided immediately after prostatic massage was confirmed by isolation on mccoy cells and lugol staining. the majority of patients suffered from suprapubic pain and pain in the groin. twelve patients had no clinical symptoms. according to rectal palpation, prostate gland was normal in patients, tender and soft in and firm in five patients. clarithromycin was administered orally mg twice daily for days. simultaneously the patients' partners received mg orally twice daily for days. clinical efficacy and tolerability of administered clarithromycin were evaluated Á/ days and Á/ weeks after the end of treatment. bactericidal efficacy of administered drug was evaluated Á/ weeks after the end of treatment. the eradication of ct was achieved in out of patients, while patients were clinically cured. two patients had nausea and elevated serum transaminases. in asymptomatic patients, the eradication of ct was achieved in of patients who reported no side effects. this pilot study has shown an excellent efficacy and tolerability of clarithromycin in the treatment of patients with chronic chlamydial prostatitis. women with diagnosis of urinary tract infections (uti) often demonstrate vaginal colonisation with alpha-haemolytic escherichia coli strains. in the present study we decided to evaluate a distribution of virulence genes encoding for cytotoxic necrotizing factor type (cnf ), p-fimbriae, s/f c-fimbriae aerobactin (aer), and afa genes in alpha-haemolytic e. coli strains isolated from gynaecological material in our region and to compare the detected sequences in clinical isolates of other diagnostic groups. of alpha-haemolytic e. coli strains, were isolated from urine, from gynaecological specimen, and were faecal strains. e. coli strains were tested for the production of haemolytic phenotype on blood agar plates. the amplification of virulence factors was performed by pcr according to previously described protocols (le bouguenec et al., ; blanco et al., ; and yamamoto et al., ) . we found that all gynaecological alphahaemolytic strains were positive for cnf , (p b/ . compared to % of urine strains, and p b/ . compared to % of faecal strains). similarly, sfa/foc specific dna sequences were found in % of gynaecological isolates (p / . compared to % of urine strains and p / . compared to % of faecal strains). from this point of view, the female genital tract seems to be a potential reservoir of these uropathogenic e. coli strains. azithromycin in the treatment of pelvic inflammatory disease caused by chlamydia trachomatis ps administered in hospitalized patients with chlamydial pid. the diagnosis was made prior to hospitalization. microbiological analysis of urine, blood and swab specimens collected from endocervix, vagina and urethra confirmed c. trachomatis to be the single suspected causative pathogen of pid. the presence of c. trachomatis in swab specimens from endocervix was examined by dnk/rnk hybridization. azithromycin was administered Á/ days after samples for microbiological analysis were collected in dose of )/ mg iv for days. clinical efficacy and tolerability of therapy were assessed Á/ days after the end of therapy and clinical and microbiological analysis Á/ weeks after completion of therapy. the eradication of c. trachomatis and normalization of gynecological findings were achieved in and disappearance of subjective symptoms in patients. no side effects and deviations from normal values in hematologic and biochemical blood parameters were recorded. this study showed high bactericidal efficacy, rapid clinical effect and good tolerability of once-daily administration of mg azithromycin for days in the treatment of patients with pid caused by c. trachomatis . altindis m a , cevrioglu s b , aktepe oc a , cetinkaya a . a kocatepe university school of medicine, microbiology, afyon, turkey , b kocatepe university school of medicine, obstetrics and gynecology, afyon, turkey diagnosis of the causative organism of the vaginitis is usually based on clinical criteria. a standardized, laboratory based and rapid diagnostic test for the identification of these organisms is desirable. to determine the laboratory method that best predicted the causative organism, we calculated the sensitivity, specificity and predictive value of positive and negative test for clinical criteria, an oligonucleotide probe test (affirm vpiii-bd usa) and compared them with the combination of positive vaginal culture and gram-stained vaginal smear. we evaluated consecutive women aged Á/ years, attending for vaginal discharge. vaginal swab specimens were used for culture of gardnerella vaginalis , trichomonas vaginalis and candida sp, preparation of a vaginal smear for gram-stain interpretation and wet mount evaluation and affirm test. affirm detected g. vaginalis in ( %), candida sp in three ( . %) women and no trichomoniasis case found by any methods. the sensitivity and negative predictive values of affirm test and clinical signs were same ( %) in identifying of bacterial vaginosis. however, affirm test was more specific ( vs %) and also has higher positive predictive value ( vs %) than clinical signs. we did not evaluate the results for patients with candidiasis because of less number. according to these results affirm-microbial identification tests are objective and specific for the rapid diagnosis of the bacterial vaginosis. comparison of efficacy of single dose of tinidazole with standard dose of metronidazole in giardia lamblia infection (preliminary report) ps fallah m a , moshtaghi aa b . a university of medical sciences, parasitology, hamadan, islamic republic of iran , b university of medical sciences, pediatrics, hamadan, islamic republic of iran objectives: giardia lamblia is the most common intestinal protozoa in developing countries. treatment of the infection with metronidazole, the drug of choice, requires a long course of therapy and produced some side effects. the object of this study is to determine efficacy and side effects of tinidazole in g. lamblia infection. this is a preliminary report of an ongoing trial. methods: a randomized controlled clinical trial was carried out and subjects with g. lamblia infection were treated with tinidazole or metronidazole. tinidazole mg/kg single dose and metronidazole mg/kg three times a day for days were given orally to and children, respectively. parasitological cure was documented when there were consecutive negative stool examinations at Á/ weeks after therapy. results: twenty-one of individuals treated with tinidazole and of children treated with metronidazole had parasitological cure. cure rates between two groups were not significant statistically. no major side effects were observed except one case in metronidazole group who had mild headache and abdominal pain for days. conclusions: we concluded, tinidazole at the dose has efficacy equal of metronidazole in the treatment of g. lamblia infection. because of single dose prescription, short course of therapy and good compliance of patients, this preparation is preferred to metronidazole in giardial infection. ebeid fa, seif el-din sh. theodor bilharz research institute, pharmacology, cairo, egypt b-carotein was given in different doses starting from . to mg/kg body weight (b.w.) for different groups of albino mice days before infection with s. mansoni . infection of animals was done by body immersion using egyptian strain of s. mansoni cercariae/mouse. forty-nine days after infection the animals were sacrificed and hepatic and mesenteric worms were extracted and determined. ova count in liver and intestinal tissue and the total number of worms/animals were also determined in experimental groups comparing with infected control animals. the results indicated marked decrease in number of worms and ova count in both liver and intestine comparing with control ones. this reduction increased significantly with increasing dose. it was concluded that b-carotein could be used as a prophylactic agent against s. mansoni infection. barisic z, babic-erceg a, borzic e, zoranic v, carev m, kaliterna v. department of microbiology, public health institute, split, croatia the aim of this study is to determine frequency of pseudomonas aeruginosa urinary tract infection (uti) in outpatient's population in south croatia and to suggest optimal antimicrobial treatment for these patients. during months long observation period, from total number of examined urine specimens, significant bacteriuria was found in specimens. p. aeruginosa was the sixth most common isolate, it was isolated from specimens ( . %). these specimens were taken from different patients. susceptibility testing was performed by disk diffusion method, and the following results were obtained: resistance to cefibuten occurred in . % patients, to norfoxacin in . %, to ciprofloxacin in . %, to gentamicin in . %, to netilmicin in . %, to amikacin in . %, to ceftazidime in . % and to imipenem in . % patients. p. aeruginosa strains showed better susceptibility to tested parenteral antibiotics than to antibiotics for oral use which complicated treatment in outpatients. the best susceptibility was shown to imipenem, but this drug is inappropriate for use in outpatients setting, so the best choice for treatment p. aeruginosa uti in our outpatients is treatment with ceftazidime, and the second choices are aminoglycoside drugs amikacin and netilmicin. silan l a , breza j b , krcmery v jr. c . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school, bratislava, slovakia , c department of pharmacology, st. elisabeth cancer institute, bratislava, slovakia we studied the clinical efficacy of oral treatment with ciprofloxacin/ cpf/ alone and combined with clarithromycin in patients with complicated urinary tract infection/cuti/ with or without an indwelling catherer. patients were randomly allocated to mg cpf/cpf group/ or to mg cpf plus mg cam/combination group/ for days. evaluation was done on day according to the criteria advocated by the japanese urinary tract infection committee. in patients with a urinary catherer, the combination achieved a higher complete bacterial elimination rate / %/ and clinical efficacy rate / %/ than cpf alone / and . %, respectively/. while no significant difference was found in the bacterial elimination rate between the two groups, the clinical effect of the combination / %/ was superior to that of cpf alone / %/ in patients with an indwelling catherer. the better clinical efficacy of the combination may partly be attributed to the antibiofilm effect of cam in the clinical setting. the results also indicated that difficulties still remain in the treatment of cuti in patients with an indwelling catherer. in conclusion, clinical study suggested that cam might have an inhibitory action on biofilm formation in the clinical setting. combination of cam with other appropriate antimicrobial agents may have a favorable effect on the treatment of cuti. vesicoureteral reflux and urinary tract infections */the management of primary vesico-eureteral reflux in children ps the children studied presented with primary vesicoureteral reflux at derer s universitz hospital in bratislava between and . seven hundred and sixty patients, boys and girls, suffering from primary vesicoureteral reflux in age from months to years were tested and systematically analyzed outcomes data for seven treatment alternatives. key outcomes identified were probability of reflux resolution, likelihood of developing pyelonephritis and scarring, and possibility of complications of medical and surgical treatment. available outcomes data on the various treatment alternatives were summarized and the relative probabilities of possible outcomes were compared for each alternative. conclusions: increased of urinary tract infection, vesicoureteral reflux nephropathy includes early diagnosis, appropriate evaluation, effective atb therapy, and surgery indicated. the main determinants of renal damage are bstruction, age, sex, predisposition on renal scarring, reflux grade and laterality, therapeutic delay, individual susceptibility, bacterial virulence and immunogenetic status. ) the one and only absolute indication for surgical management is failure of medical therapy to prevent chronic recurrent pyelonephritis, renal injury or other reflux complications and eliminations of the reflux condition will minimize their likelihood. genetically conditioned immunopathogenic mechanisms are involved in the pathogenesis of the chronic recurrent pyelonephritis in patient suffering from vur. for most children we recommended continuous antibiotic prophylaxis as initial treatment-medical therapy is based on the principle that reflux often resolves with time, and antibiotics maintain urine sterility and prevent infections while the patients awaits spontaneous resolution. ) vur predispose an individual to renal infection, the immunological and inflammatory reaction caused by a pyelonephritic infection may result in renal injury or scarring. silan l a , breza j b . a department of internal medicine, derer s university hospital, bratislava, slovakia , b department of urology, comenius university school of medicine, bratislava, slovakia elderly patients with uti are believed less likely to be cured by antimicrobial therapy than younger patients. the reasons for this poorer outcome have not yet been clarified. we have investigated the efficacy of antimicrobial therapy in elderly patients with complicated uti. five hundred patients, men and women, who had complicated uti/ symptomatic and symptomatic and were Á/ years of age, were treated with one of three different drugs, one was a never quinolone and two were oral cephems. multivariate logistic regression analysis of treatment outcome revealed that the clinical response was significantly related to general underlying diseases and diseases of the urinary tract, but not to age, symptomatic or asymptomatic uti, or infection site such as the kidney or bladder. we concluded that the clinical effectiveness of an antimicrobial agent was not directly related to age, and that urological examination for underlying disease and control of them is quite important for effective treatment and control of complicated utis, especially in elderly patients. the study on the frequency and antimicrobial resistance of escherichia (e ) coli in urine isolates of patients admitted to maribor teaching hospital in and ps rebersek gorisek j a , baklan z a , unuk s a , novak d b . a department for infectious diseases, teaching hospital maribor, maribor, slovenia , b department for microbiology, regional institute of public health maribor, maribor, slovenia purpose: the aim of the study was to determine the frequency and antimicrobial resistance of escherichia coli isolated from urine samples of patients admitted to maribor teaching hospital in and . the frequency and the antimicrobial resistance were compared between years and . methods: in the prospective study going on between and , all urine isolates from patients at maribor teaching hospital were collected and analysed. urine cultures were done using the modified sanford method. the susceptibility testing was performed by disk diffusion method according to nccls. results: in the year , urine isolates and in the year , urine isolates were analysed. e. coli represented . % of urine isolates in and . % of urine isolates in . e. coli resistance rates (%) to amoxycillin was . in the year and . in the year ; to amoxycillin/clavulate was . and . ; to cefalotin was . and . ; to cefaclor . and . ; to trimethoprim sulfamethoxazole was . and . ; to ciprofloxacin was . and . ; to gentamicin was . and . . conclusion: compared to , the frequency of e. coli isolated from urine samples is similar to that in the year . the resistance to amoxycillin, cefaclor and gentamicin is stable. the resistance to trimethoprim sulfamethoxazole and ciprofloxacin is increased and the resistance to amoxycillin/clavulate and cefalotin is decreased. prevalence of the resistance to metronidazole, furazolidone and nitrofurantoin in helicobacter pylori clinical strains ps de la obra sanz p a , roman jl a , lomas e a , villar h b , lopez-brea m a . a hospital de la princesa, microbiology, madrid, spain , b hospital de san agustin, microbiology, aviles, spain the objective of this study was to determine the prevalence of metronidazole, furazolidone and nitrofurantoin resistance in helicobacter pylori clinical isolates. methods: a total of strains of h. pylori were included in this study. all these were tested against metronidazole, and against furazolidone and nitrofurantoin by an agar dilution method. resistance was defined as: metronidazole, mic !/ mg/l; and mic !/ mg/l for furazolidone and nitrofurantoin. results: sixty-eight strains were resistant to metronidazole ( . %). the mic and mic values were and mg/l, respectively. three of strains ( . %) were furazolidone resistant (mic / mg/l), two of these strains were metronidazole resistant (mic / mg/l) and they had mic of mg/l for nitrofurantoin. the mic and the mic were . and . mg/l, respectively for furazolidone. only one of the strains ( . %) was nitrofurantoin resistant (mic mg/l), this strain was metronidazole resistant (mic mg/l) and had a mic / mg/l for furazolidone. the mic and the mic were . and mg/l, respectively for nitrofurantoin. conclusion: the low frequency of furazolidone and nitrofurantoin resistance, compared to metronidazole suggests that the furazolidone and the nitrofurantoin may be good alternatives to metronidazole for treatment of h. pylori infections. antimicrobial resistance of campylobacter isolated from human origins ps zhukhovitsky vg, drabkina iv. department of bacteriology, botkin hospital, moscow, russian federation the purpose of the study was to determine the antimicrobial resistance of thermophilic enteropathogenic campylobacter spp. (tec) isolated from human under acute diarrhoea in in moscow. among tec strains c. jejuni and five c. coli were identified. the antibiotic tested by disk diffusion test on mueller-hinton agar with sheep blood were ampicillin (a), amoxycillin/clavulanate (ac), imipenem (i), meropenem (m), erythromycin (e), clarithromycin (cl), tetracycline (t), doxycycline (d), gentamicin (g), azithromycin (az), chloramphenicol (ch), lincomycin (l), ciprofloxacin (c), nalidixic acid (na). the resistant rate of the tec isolates was highest for na ( %) followed by a ( %), t ( %), d ( %), n ( %) and cl ( %). a moderate resistance rate was obtained for a ( %), ch ( %), az ( %), ac ( %). none of the isolates demonstrated resistance to i, m and g and four of isolates ( %) were sensitive for all the antibiotics tested. mic to na was estimated as mg/l. among na resistant tec strains ( %) were identified as c. jejuni and one ( %) as c. coli . among c. jejuni and c. coli na resistant rate was and %, respectively. one na resistant c. coli and nine na resistant c. jejuni were resistant to ciprofloxacin. ring c, atanassova v. department of food hygiene and microbiology, school of veterinary medicine, hannover, germany aim of the study: poultry meat is known to be often contaminated with salmonella and other foodborne pathogens and thus has to be considered as a possible source for human infections. the aim of the study was to monitor the resistance of salmonella isolates from poultry meat of different european countries to various antibiotics. material and methods: from september to december a total of samples of frozen poultry meat from france, germany, italy, spain, the netherlands and portugal were examined for the prevalence of salmonella using classical cultural detection as well as rflp-pcr. all isolates were tested for their sensitivity towards ampicilline, kanamycine, ciprofloxacine, tetracycline, trimethoprim, sulfamethoxazole, nalidixic acid and erythromycine using standard procedures. results: from . % of all examined samples salmonella spp. were isolated. of these isolates . % were characterized as salmonella , . % as s. hadar and . % as s. typhimurium . nearly % of all isolates were resistant to erythromycin. resistance towards four or more isolates was observed in several cases. discussion: the consumption of poultry meat, if insufficiently prepared, has still to be considered as a major source for human infection with salmonella spp. the question arises whether the resistance of the isolates to various antibiotics is of clinical importance in the treatment of the patients. objective: to provide insight into the epidemiologic situation of salmonellosis for the nis area (the largest area in serbia, with inhabitants */ , ). methods: the material was processed at the institute for public health (epidemiology and microbiology divisions). isolation of microorganisms was performed on apparatus for rapid identification (vitek-biomerieux) and by applying elisa tests and classical microbiological methods. results: in the period Á/ , salmonella laboratory confirmed cases were reported. the greatest number of diseased in the Á/ years group. the most frequent isolated salmonellae were: s. enteritidis ( . %) and s. typhimurium ( . %), s. hadar , s. agona , s. virchow , s. infantis , s. derby , s. enteritidis showed the greatest sensitivity to antibiotics with the infrequent resistance to ampicillin and trimethoprim-sulfamethoxazole. s. typhimurium showed the greater resistance to the wide spectrum of antibiotics and some isolates were resistant to all antibiotics tested. the less common types of salmonella were sensitive to all antibiotics except trimethoprimsulfamethoxazole and ampicillin. conclusion: specific resistance to some antibiotics was related to serotypes. typhoid fever */retrospective study of cases in lebanon ps tohme a, abboud j, ghayad e. hôtel-dieu hospital, internal medicine, beirut, lebanon objectives: to present epidemiological and clinical features of typhoid fever in lebanon. methods: fifty-two patients were seen at hotel-dieu hospital of beirut between and . diagnostic criteria were positive blood culture for s. typhi or paratyphi and/or a somatic o agglutinin titer ]/ / as determined by the widal test with symptoms suggestive of typhoid fever. we also present an epidemiological study of cases registered by the ministry of health during the same period. results: among the cases, % of the patients' ages were between and years and % were less than years. the overall male to female ratio was . and % of cases were seen on january, february and % on august. among the patients, young adults were the most affected. average duration of symptoms before the diagnosis was / days. the main presenting symptoms were: fever ( %), diarrhoea ( %), abdominal pain ( %) and headache ( %). complications were noted in % of cases and digestive complications were the most prevalent. leucopenia was not a helpful diagnostic marker. s. typhi was the most frequent ( %) serotype identified. resistance to ampicilline was %, to cotrimoxazole and chloramphenicol % for each. the mortality rate was %. conclusion: typhoid fever is still an endemic disease in our country and the occurrence of resistant strains of s. typhi will favor ceftriaxone or fluoroquinolones in the treatment. maaloul i a , hammami b a , zambaa f a , elleuch r a , hammami a b , -ben jemaa m a . a chu hedi chaker, service des maladies infectieuses, sfax, tunisia , b chu habib bourguiba, laboratoire de microbiologie, sfax, tunisia although its not very frequent, the psoas abscess is not an exceptional entity. in order to specify its clinical, biological, radiological and evolutionary features, a retrospective study has been led in our service, on a period of years (january Á/december ). on the whole, cases have been listed. they were men and women. the age average was years (extreme Á/ years). the study did not find any underlying diseases, except diabetes mellitus for three patients. the clinical symptoms were dominated by fever with abdomino-lumbar aches ( cases), and psoitis (eight cases). biology showed an inflammatory syndrome in all cases and a hyperleucocytosis in cases. the diagnosis of psoas abscess, evoked on clinical data, has been confirmed by the imagery data: ultra-songraphy ( cases), ct scanning (six cases), magnetic resonance imaging (three cases). the tubercular etiology has been confirmed in six cases, among which two were associated to escherichia coli (one case) and to brucella melitensis (one case). the other etiologic agents were dominated by staphylococcus aureus (eight cases), b. melitensis (two cases), e. coli (one case), bacteroides fragilis (one case), streptococcus anginosus (one case), fusobacterium nucleaticum (one case) and candida glabrata (one case). all patients received an anti-infectious treatment adapted to the micro organism in question. a drainage of the abscess has been realized for patients (percutaneous: nine cases, surgical: six cases). the evolution was favourable for patients. however, two patients had a relapse after stopping the treatment. conclusion: the diagnosis of the psoas abscess, difficult on the clinical data, is based on the imagery techniques (us, ct, rmi). the percutaneous drainage guided by the imagery is recommended (in an etiological and therapeutic aim). associated to an adapted antibiotherapy, it allows to defer a surgical drainage. zezoski mbz, nikolova o, gavriloski pmg. medical center, infectious diseases, prilep, the former yugoslav republic, macedonia purpose: to make a list of the most frequent abdominal changes in patients with human brucellosis. materials and methods: there were new patients with human brucellosis, between and . diagnosis was made using standard clinical, biochemical and serological investigations (bab, wright, coomb's, rvk, -mercaptoethanol, elisa igm and igg), and specially ultrasound examination of the abdomen and retro peritoneum. results: weight loss is the most frequent change, presented in ( . %) patients. follow atypical abdominal pain in ( . %), vomiting in ( . %), diarrhea in ( . %), enlarged liver in ( . %), enlarged spleen in ( . %) and hepatic lesion with increased ast and alt in ( . %). conclusion: although frequent, abdominal changes seldom could be missed in patients with human brucellosis. we recommend routine ultrasound examination with standard biochemical test for liver function, due to avoid unnecessary complications. osteoarticular complications are common in brucellosis. the most common site of involvement is the sacroiliac joint. the osteoarticular complications such as, sacroiliitis and spondylitis are diagnosed with radiologically. in the present study, we aimed to determine the severity (grade) of sacroiliitis by using some laboratory parameters such as esr, crp and tube agg test. seventy-two ( male, female) patients with brucellosis were included in the study. osteoarticular involvement was present in patients. the most common osteoarticular finding was sacroiliitis in the patients ( %). twenty ( ) healthy subjects were formed the control group. there was statistically significant difference between patients and controls regarding esr, crp, and tube agg test (p / . , . , . , respectively). in addition, sacroiliitis has an effect on esr and crp. there was a positive correlation between the grade of sacroiliitis and the value of crp (p / . , r / . ). in conclusion, it has been suggested that, crp may be used as an auxiliary or a secondary parameter in grading sacroiliac joint involvement in brucellosis. magira ee a , papandreoy s a , gounaris t a , spirelis ma a , tasopoulos g a , anagnostopoulou m b , paniara o b , gounari p a , sioulal e a . a evagelismos, internal medicine, athens, greece , b evagelismos, micro- a -year-old greek farmer was admitted to the hospital because of painful scrotal swelling, hepatosplenomegaly, lumbar pain and lowgrade fever accompanied by profuse sweating. his life style included occupational animal exposure ingestion of raw milk and dairy products. the laboratory data were within the normal ranges. focal hypoechoic right testicular lesions, swelling of the concurrent epididimis along with an increase in the vascularity of the right testis were seen on an echo examination. these findings were consisting in unilateral epididimo-orchitis. a ct scan of the lumbar spine area showed a decrease of the signal intensity localized in the anterior aspect of l vertebral body at the diskovertebral junction involving the subchondrial parts of the l and s vertebrae standard tube agglutination test was positive for antibodies to brucella melitensis (titer !/ / ). cultures of blood specimens were positive for b. melitensis . the patient had been given to a combination of antibiotics with doxycycline, streptomycin and rifampin. a remarkable improvement of his clinical condition was showed weeks later. this case illustrates the following point: in areas in which brucellosis is endemic when scrotal abnormalities are seen the possibility of genitourinary tract complications of brucella should be considered. pappas ga a , akritidis nk b , mastora m b , tsianos e a . a university hospital, internal medicine, ioannina, greece , b 'g. hatzikosta ' hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and forms of complications associated with brucella infection. patients and methods: we studied the most recent patients, in all larger series approaching , diagnosed as suffering from brucellosis, and assessed the presence of signs and symptoms of arthritis and spondylitis, or other forms of bone involvement. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures or cultures from other media. results: osteoarticular complications were noted in patients, presenting with arthritis, and presenting with spondylitis. eight patients presented with genitourinary complications, either orcheoepididymitis (four patients), or hematuria resolving with treatment (four patients). meningitis was present in two patients. gastrointestinal complications (vomit and diarrhea) were present in three patients, while one patient presented with ascites. respiratory tract complications, in the form of pneumonia (four patients) or bronchitis (three patients) were noted in seven patients, while one patient with pneumonia exhibited pleural fluid. skin rashes, of macular type, were present in three patients. no patient presented with complications from the heart. hematologic complications were frequent, in the form of severe (one patient) or moderate (two patients) pancytopenia, isolated thrombocytopenia (three patients), or lymphocytosis (eight patients). akritidis nk a , pappas ga b , mastora m a . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scope: to determine the incidence and modes of bone and joint involvement in the course of brucellosis. patients and methods: we studied the most recent patients, in all larger series approaching , diagnosed with brucellosis, and assessed the presence of arthritis and spondylitis. the diagnosis of brucellosis was based on serology or isolation of brucella species from blood cultures. results: twenty-three patients exhibited a form of osteoarticular involvement. arthritis was present in patients, most often involving the knees, but also the hips, elbows, even smaller joints as intephalangeal joints of the hand. synovial fluid, when aspirated, was often characterised by an intense mononuclear infiltrate. spondylitis was present in patients, most often involving the lumbar spine, but also the thoracic spine. the characteristic erosion on the upper anterior crest of the vertebral body was visible in plain x-rays in three patients, while mri and bone scan were helpful in other cases. discussion: osteoarticular involvement in the course of brucellosis is the most common focal presentation of the disease. acute brucellosis is often accompanied by bone and joint ache, especially of the lumbar spine, still frank involvement in the form of arthritis and spondylitis is not rare. arthritis usually presents in the acute form of the disease, while spondylitis tends to be characteristic of a chronic form of the disease, often necessitating prolonged use of antibiotics. bosilkovski m, krteva l, caparoska s, grozdanovski k, sajn b. clinic for infectious diseases and febrile conditions, medical faculty, skopje, the former yugoslav republic, macedonia one hundred and twenty-six patients with brucellosis were studied prospectively. seventy-eight ( %) of them had osteoarticular involvement. peripheral arthritis in ( %) patients was the most frequent, followed by spondilitis in ( %), sacroiliitis in ( %), rarely bursitis, tendinitis and osteomyelitis. the overall male to female ratio was : . their average age was (sd ) years. direct contact with animals was the reason for acquisition of the illness in % of patients, in % alimentary or aerogenous route was incriminated, and in % the route of aqisition was unknown. the average duration of the symptoms from the onset to establishing the diagnosis was (sd ) days. the main presenting symptoms were joint pain ( %), sweating ( %), fatigue ( %) and fever ( %). hepatomegaly was present in %. in % of patients, involvement of some other system was evident. comparison with patients, who did not have osteoarticular illness, showed that patients with osteoarticular involvement had significantly more often joint pain, fatigue, weight loss and more prolonged duration of symptoms before the diagnosis was established. doxycycline and chloroquine as combination therapy for chronic q fever endocarditis ps calza l, attard l, manfredi r, chiodo f. division of infectious diseases, university of bologna, s. orsola hospital, bologna, italy introduction: endocarditis is the main clinical manifestation of chronic q fever, occurring in about Á/ % of all reported cases, and it is diagnosed almost exclusively in patients with either cardiovascular abnormalities or an immunocompromised condition. case report: a -year-old caucasian male patient with biological prosthetic aortic valve was first hospitalized because of an interstitial pneumonia. six months later, our patient was re-admitted owing to intermittent fever, chills and weight loss. echocardiographic study showed a small vegetation of mm in diameter on left cusp of aortic valve. serology for coxiella burnetii revealed a complement-fixing igg antibody titer to phase i antigen of more than : , consistent with chronic q fever endocarditis. antimicrobial therapy with i.v. doxycycline and oral chloroquine was started, leading to a clinical and echocardiographical recovery. therapy was continued by oral doxycycline and chloroquine, and the patient remained asymptomatic during a -year follow up. conclusion: the optimal treatment of q fever endocarditis has not been well established: the most effective antimicrobials are fluoroquinolones and rifampin, but chloramphenicol, doxycycline and trimethoprim are also useful. the role of chloroquine in combination with doxycycline seems to be promising, because chloroquine may increase the lysosomal ph, enhancing the doxycycline bactericidal activity. akritidis nk a , pappas ga b , mastora m a , liappis e a , tsianos e b . a 'g. hatzikosta ' hospital, internal medicine, ioannina, greece , b university hospital, internal medicine, ioannina, greece aims and scopes: to review the incidence and the forms of lower respiratory tract infection in patients suffering from q fever, and their clinical and radiological characteristics. patients and methods: twenty-seven patients diagnosed as suffering from q fever, were assessed for the presence of lower respiratory tract infection. the diagnosis was confirmed serologically. results: thirteen patients expressed lower respiratory tract pathology, as confirmed by clinical examination and chest x-ray. in of these patients the main cause of admission was respiratory tract symptoms, ranging from dry cough to hemoptysis. chest x-ray was pathological in patients: patients had lobar pneumonia, two of them multiple nodular opacities, and one of them bronchopneumonia. hepatitis was a common finding. all patients were treated with tetracycline. discussion: although coxiella burnetii infection is acquired via the respiratory tract, it is paradoxical that symptoms attributed to the lung are not invariably positive. q fever pneumonia is an atypical pneumonia that usually follows a benign course. diangostic suspicion is usually raised by the epidemiologic pattern and the accompanying mild hepatitis. pleural effusion is not a common finding. the usual radiologic appearance of q fever pneumonia is that of a lobar or segmental pneumonia. one important aspect of q fever pneumonia is its common presentation in the form of multiple nodular opacities often necessitating the exclusion of malignancy. ( ), culture ( ), and serology'/culture ( ). there were Á/ cases per year, mainly in october ( %). a history of exposition to hare was present in / ( %) and to marmot in / ( %). skinning ( / %), animal contact ( / %), bite ( / ) and wound during bait preparation with frozen meat for hunting ( / ) were noted. the initial clinical presentation was ulceroglandular ( %), glandular ( %) and pneumonic ( %). the involved nodes distribution was axillary ( / ), cubital/axillary ( / ) and cubital ( / ). median incubation period was days (range Á/ ); time to consultation days (range Á/ ), and time for effective treatment days (range Á/ ). an initial diagnosis of tularemia was made presumptively in %. effective antibiotic regimen used was aminoglycosides in % ( / ), and tetracyclines in % ( / ). note that intravenous netilmicin was used in cases. complication rate was % ( / ) with one death ( %), and was associated with delay in effective treatment ( !/ days of illness) (p b/ . ). conclusion: in our area tularemia occured mainly in male population, during autumn, with a short incubation period and history of hare contact. delay before appropriate treatment increased the complication rate. bompolaki i, doukakis s, triantafillidou d, polimili g, kastanakis m, nikiforakis k, vittorakis e, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece a severe frontal and/or retroorbital headache represents the most common neurologic manifestation of murine typhus. other neurologic manifestations as confusion, stupor, nuchal rigidity and in severe cases delirium, extreme agitation or coma appear less commonly. eightyfour patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi, were studied from our team. seventy-four patients ( %) presented headache and nine patients ( %) presented confusion. one patient ( . %) presented nuchal rigidity in combination with severe headache and confusion giving us the suspicion of meningitis. in this case a lumbar puncture was performed emergently and the cerebrospinal fluid (csf) was examined. the findings of csf were proteins: mg/dl, wbc: /ml and glucose: mg/dl and its culture was negative. all patients were treated with a specific anti-rickettsial treatment. the outcome of murine typhus was favorable for all patients ( %). no one patient presented neurologic sequelae. conjunctivitis usually accompany rickettsial diseases such as rocky mountain spotted fever, epidemic typhus and murine typhus. eightythree patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january and the first semester of . the clinical examination of these patients revealed the presence of conjunctivitis in / patients ( . %). in the same time these patients referred retroocular pain and mild photophobia. this study showed that in murine typhus the injection of conjunctivae is rather common. almost a quarter of the patients presented conjunctivitis despite the fact, that this ocular manifestation is less severe than in other typhus and spotted fevers. eighty-three patients with compatible clinical status of murine typhus and high serological titers of antibodies against rickettsia typhi , were studied from our team, during a period of time between january and the first semester of . three blood samples were obtained from each patient for the study of their hematological abnormalities. the first sample was obtained on admission, the second sample weeks after the first, the third sample, month after the second. on admission / patients ( %) presented anemia, / patients ( %) presented leukopenia and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. two weeks later anemia was presented in / patients ( %), / patients ( %) presented leukopenia, / patients ( %) presented leucocytosis and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. one month later / patients ( %) had anemia and / patients ( %) presented thrombocytopenia. the mean value of hematocrit, white blood cells and platelets was . g/dl, . )/ and )/ /ml, respectively. our study showed that early thrombocytopenia and anemia are frequent in murine typhus and that white blood cells count is usually normal. renal function in murine typhus: a study of cases ps doukakis s, polimili g, triantafillidou d, kastanakis m, vittorakis e, palla k, kastanakis s. first medical department, 'saint george ' general hospital, chania, greece the clinical course of murine typhus is usually uncomplicated and the mortality rate is low ( b/ %). advanced age and prolonged interval before administration of a specific anti-rickettsial treatment are correlated with severity of the disease. renal function in murine typhus is usually unaltered except in elderly patients with prolonged hypotension. eighty-three patients with compatible clinical status of murine typhus and high serological titres of antibodies against rickettsia typhi, were studied from our team, during a period of time between january and the first semester of . three blood samples were obtained from each patient for the study of their renal function. the first sample was obtained on admission, the second sample approximately weeks after the first, the third sample, taken from the half of the patients, was obtained one month after the second. on admission / patients ( . %) presented acute renal failure. the outcome of murine typhus was favourable for all patients ( %). the four patients who presented acute renal failure reversed after the administration of anti-rickettsial treatment and careful administration of fluids. in murine typhus the induction of hypovolaemia insufficiently corrected by normal homeostatic mechanisms may lead to prerenal azotaemia. in these cases the immediate onset of an antirickettsial treatment and the correction of hypovolaemia are essential for the rapid clinical improvement of the patient. experimental ocular toxoplasmosis: clinical, histopathological, immunological and therapeutic studies ps el zawawy lae a , hammoda na a , allam sr a , ali sm a , galal as b . a faculty of medicine, parasitology, alexandria, egypt , b faculty of medicine, ophthalmology, alexandria, egypt the purpose of the study was to investigate clinical, histopathological, immunological and therapeutic features of an experimental model of ocular toxoplasmosis in sensitized and non sensitized rabbits and to assess the influence of treatment by interleukin (il- ) on ocular lesions. the results obtained was that 'toxoplasma' retnochoroiditis developed in both groups of rabbits with more pronounced effect in non sensitized animals. administration of il- improved ocular lesions in both groups with more evident effect in sensitized rabbits. immunological findings were consistent with clinical and histopathological observations. the conclusion reached was that; ocular lesions were manifested in non sensitized rabbits more than in sensitized ones. il- revealed a significant impact on improving the host defense against toxoplasm infection in eye. immunotherapy with il- would open the way for a new range of treatment based on immunomodulation. express-diagnostic of streptococcus antigen for the adequate antibiotic therapy in patients with pharyngitis ps pertseva to, konopkina li, kireeva tv. dsma, internal medicine , dniepropetrovsk, ukraine the purpose of the study: evaluation of effectivness of streptococcus express-diagnostic for the adequate antibiotic therapy in patients with pharyngitis. results: we deal with clinical and microbiological comparison in patients with pharyngitis. using of streptococcus antigen express diagnostic in swabs from the backside of pharynx allowed to get positive results in the seven cases ( . %). following cultural study has confirmed these results. positive test was more probable in patients with pronounced fever (more than c), headache, weakness and in cases associated with chronic tonsillitis. isolated streptococcus pyogenes was susceptible to ampicillin, claritromicin, erytromicin, azytromicin, , clindamicin, ceftriaxon, levafloxacin, oxacillin, cefuroxim, roxytromicin. conclusion: using of the express diagnostic of streptococcus antigen allows to restrict groundless prescription of antibiotic therapy in patients with other types pharyngitis (i.e. viral, candidal etc.). alabaz d a , turgut m a , kocabas e a , tumgor g a , yaman a b , alhan e a . a division of pediatric infectious diseases, cukurova university, adana, turkey , b department of microbiology, cukurova university, adana, turkey chickenpox is a common viral infection that usually follows a benign, self limited course in healthy children. the most common complication in children with varicella is superimposed cutaneous infections with pyogenic bacteria (streptococcus pyogenes and staphylococcus aureus ). varicella gangrenosum, a necrotising soft tissue infection complicating the vesicular eruption of chickenpox, is rare. here we present three cases with necrotising soft tissue infections following chicken pox. these children were admitted because of common crusted lesions and necrotising soft tissue infection over the neck, the back, and the inguinal area. they all had the contact history and ensuing vesiculopapular rush. these infections were caused by group a streptococci in two cases, and s. aureus in one case. after instituting of appropriate antibiotic therapy, each patient underwent a surgical exploration with fasciotomies and debridement. widespread use of varicella vaccine may decrease invasive infections in children, adolescents, and adults, thus decreasing the burden the disease with its complications impose up on the family and the society . cefprozil is a second generation cephalosporin. the aim of this open, multicentre, non-comparative study was to investigate the efficacy and safety of cefprozil in the treatment of streptococcal tonsillopharyngitis. fifty-eight patients were clinically assessed for signs and symptoms of streptococcal infection. laboratory confirma-tion was sought using three tests; culture, rapid strepto test and estimation of antistreptolysin (aso). one or more tests were done in of the patients. treatment was for days, mg/kg per day in children, mg per day in adolescents. patients were again clinically assessed on the th Á/ th day. the results showed clinical success in patients ( . %) and in of the who had laboratory tests ( . %). two patients had treatment withdrawn because of nausea and abdominal pain ( . %). of the patients with laboratory tests at least one test was ositive. the most helpful was the strepto test giving the quickest positive result in % of those tested ( / ). culture was positive in % of those tested ( / ). the aso test was of limited value. in conclusion, cefprozil showed high clinical efficacy and safety in the treatment of streptococcal tonsillopharyngitis. tsiara s, militadou g, milionis c, elisaf m. internal medicine department, university of ioannina, ioannina, greece streptococcus group b agalactiae (gbs) is a rare pathogen for healthy male adults. we present an old man in whom (gbs) was isolated in blood cultures. case report: a -year-old man was admitted to the hospital in order to investigate osteolytic lesions in the lumbar spine. two weeks before, he experienced severe low back pain radiating to the right leg and fever arising to . c with chills and rigors. a gbs was isolated from blood cultures and treatment with penicillin was initiated. a spine ct scan revealed osteolysis in the t and s vertebrae and the patient transferred to us. he was a previously healthy man. he received only antihypertensive therapy. on admission he was afebrile with arthralgias and myalgias. on clinical examination there was tenderness on the right pleurospondylic angle radiating to the right leg. laboratory data on admission: hb: . g/dl, wbc: . )/ /l, esr: mm/h. biochemical values, serum protein electrophoresis, rectal examination and a prostatic specific antigen (psa) were normal. a bone marrow aspiration and biopsy were negative. a transoesophageal ultrasound revealed vegetation on the right cusp of the aortic valve, with low grade regurgitation. an mri of the lumbar spine revealed infectious myositis with concomitant osteomyelitis involving the l , l vertebrae without any evidence of osteolytic lesions. a thorough investigation did not revealed any underlying immunosuppressive disease. treatment with vancomycin and gentamycin iv was initiated and the patient discharged from the hospital in excellent health after weeks. discussion: gbs infections usually occur in neonates and pregnant, or in adults with underlying disease as diabetes mellitus or immunosuppression. the most common site of infection is soft tissue. we present this case because gbs infections are rare in the elderly in the absence of underlying disease. common sites of involvement are soft tissues, while bone, joints, and heart valves account to Á/ % of the involved organs. although our patient had more than one site of involvement he responded well to medical treatment without surgical debriment or heart valve replacement. objective: to evaluate the safety and efficacy of rhugm-csf in combination with broad-spectrum antibiotics for the treatment of ccnf. methods and results: a retrospective review of all patients (pts) with ccnf treated with antibiotics'/rhugm-csf at our hospital from january to december was performed. five patients were identified with the diagnosis of ccnf. ages ranged from to years; there were three women and two men. dental infection was the most common source of ccnf in %. one patient had acute tonsillitis leading to ccnf. all cases studied experienced infection of the neck with spread into the submandibular, submental, sublingual, retropharyngeal, and parapharyngeal spaces. all infections were polymicrobial. diabetes mellitus was a co-morbidity in one case. all pts were treated with dual antibiotic coverage (vancomycin'/meropenem), rhugm-csf ( mcg/kg/daily given s.c.) and aggressive wound care. rhugm-csf was given for Á/ days. in spite of the severity of the infection all pts recovered and do not experienced local or systemic complications. discussion: ccnf is a severe bacterial infection of the cervical fascia resulting in extensive fascial necrosis with widespread undermining of the surrounding tissues. prompt antibiotic therapy combined with rhugm-csf resulted in a % overall survival in our experience. to our knowledge, this is the first report describing the successful treatment of ccnf with use of broad-spectrum antibiotics combined with rhugm-csf. the following case adds to the clinical manifestations and course of meningococcal disease. a previously healthy -year-old girl presented acutely with high fever purpuric rash including conjuctival haemorrhages and hypotension. the child had also neck stiffness. a presumptive diagnosis of meningococcal septicaemia was made and treatment with penicilline, chloramphenicole, fluids and inotropes was initiated. laboratory investigations showed wbc: /ml, hb: . g/dl, hct: . %, esr: mm in the first hour, pt: s, aptt: s. neisseria meningitidis group b was isolated from the blood cultures. csf obtained after antibiotics were started did not grow n. meningitidis . the patient had an adverse outcome. she died after h of hospitalization. this patient developed a fulminating meningococcal septicaemia. shock is a clinical diagnosis arising from the failure of compensatory mechanisms that maintain perfusion of vital organs at the expense of non vital. septic shock results from loss of circulating plasma volume due to increased vascular permeability and maldistribution of intravascular volume. in young children there is a prevalence of serogroup b meningococcal disease which can be explained by the immaturity of the immune system and by the fact the group b capsule synthesis is known to inhibit alternative complement pathway activation. this case emphasizes the need for further protection against n. meningitidis group b. . results: forty-eight patients were included in this study. the etiological agents were: viral (n / , . %, mean ('x ) age / years), streptococcus pneumoniae (n / , . %,'x age / ), neisseria meningitidis (n / , . %, 'x age / ), s. viridans (n / , %, 'x age / ), p. multocida (n / , age ). the peak incidence of bacterial meningitis was in winter (pneumococcal %, meningococcal %, s. viridans %) .the cerebrospinal fluid (csf) findings in viral meningitis were 'x white cells / /mm , 'x pmn / %, 'x glucose csf/ serum . , 'x protein mg/dl and in bacterial meningitis were 'x white cells / /mm , 'x pmn / %, 'x glucose csf/ serum / . , 'x protein / mg/dl, gram stain was positive in %, culture was positive in %. all pneumococcal and meningococcal strains were susceptible to penicillin. the case fatality rates for pneumococcal and meningococcal meningitis were and . %, respectively. conclusions: the cases of bacterial meningitis were according to typical epidemiological features of age and season. the case fatality rate of pneumococcal meningitis appear to be high regardless of susceptibility to penicillin. none had received pneumococcal vaccine prior to becoming ill. diagnosis and therapy of meningococcal meningitis */trend and particularities of a 'romanian model' ps lintmaer i, moroti r, popescu a, popescu c. institute of infectious diseases matei bals , unit , bucharest, romania background: newer diagnosis methods and antimicrobials are expected to change the management of menigococcal meningitis (mm) and to improve its prognosis. objectives: to determine the changes in the diagnosis methods and therapy of mm patients in a infectious diseases hospital. to compare mm management in bucharest with literature data. methods: retrospective rewiew of mm in adult patients hospitalized over a -year period. our results were compared with other studies made in the s, taken from medline. results: there were episodes of mm during the study period ( episodes in Á/ and episodes in Á/ ) . we noticed a defined diagnosis increase and increased blood culture specificity. the antimicrobial monotherapy was maintained but penicilin was replaced by ceftriaxone. hhc was replaced by dexamethasone in pathogenic therapy. we noticed a shorter length of treatment and a reduced lethality. the most important differences between our results and other studies are: monotherapy regimens are less frequent and therapy lengths are longer; however, prognosis is similar. conclusions: the mm management has been modified in the last Á/ years: prognosis is improved, but the changes do not bring clear cost/ effective benefits. tiouiri th, kilani b, amari l, zouiten f, kanoun kf, ghbontini a, ben chaabene t. rabta hospital, infectious diseases, tunis, tunisia objectives: in order to study epidemiological, clinical and therapeutical characteristics of the infective endocarditis (ie). methods: we reviewed all the cases of ie fulfilling the duke criteria. data were collected during a -year period ( Á/ ) in the unit of infectious diseases. results: one hundred and eight cases were identified. the mean age was . years. sex ratio was . . eighty-five ie ( . %) occured in patients with native valve, and ie ( . %) with prosthetic valve. fever was the most common sign, % had a congestive heart failure, . % had cutaneous signs. the most common primary focus of ie was orthodontic. blood cultures were positive in % of cases. in one case, serological test identified rickettsia conori . streptococci and staphylococci were isolated in . and . %, respectively. echocardiography detected abnormalities in . % of cases. rheumatic heart disease was the most predisposing condition. empirical therapy was based on combination of b lactam with aminoglycoside. recovery was obtained for patients. cardiac surgery was performed in cases. overall mortality rate was . %. conclusion: ie affects young persons. prevention needs eradication of acute rheumatic arthritis. a major outbreak of legionnaires' disease in spain: diagnostics aspects ps guerrero c a , toldos cm a , yagü e g b , ramírez c a , rodríguez t a , -segovia m a . a hospital morales meseguer, servicio de microbiología, murcia, spain , b departamento de microbiología, facultad de medicina, universidad de murcia, murcia, spain objective: to evaluate the value of different methods (serological tests, culture of respiratory secretions, blood cultures and urinary antigen testing) for the diagnosis of legionella pneumophila pneumonia during an outbreak in spain. results: we have studied patients from a recent outbreak of legionellosis in murcia (spain). the diagnosis was achieved in patients. urinary antigens were positive in patients. in the patients with urinary antigen negative the serological response was demonstrated by indirect immunofluorescence (ifa) in patients. all blood cultures processed were negative. sputum samples were obtained from patients, of these l. pneumophila was isolated only in six patients. in all of them direct immunofluorescence test (dfa) was positive. conclusions: although the serological diagnosis was the most sensitive method the urinary antigen testing was of great value in the rapid diagnosis of the legionella's outbreak in murcia. the isolation of l. pneumophila by culture showed a poor sensitivity probably because of the low severity of the illness. purpose: to evaluate chloramphenicol for an initial empiric antibiotic treatment of purulent meningitis in adults. study group: one hundred and twenty patients hospitalized for the diagnosis purulent meningitis in the department in years Á/ , males and females, age range Á/ years, mean age . years. children up to years were not included. method: a retrospective analysis of the study group focused on antibiotic treatment both initial and changes during treatment. results: chloramphenicol was used as an initial antibiotic in ( %), rd generation cephalosporin in ( %), penicillin in ( %), ampicillin in five ( %) and other antibiotic in five ( %), respectively. during treatment chloramphenicol was switched for rd gen cephalosporin in seven of patients with streptococcus pneumoniae meningitis and in five of patients with meningitis of unknown etiology. the reason for the change was non-improving csf formula in three, persisting csf culture positivity in two and persisting coma in seven patients. conclusion: because of repeated necessity to switch chloramphenicol for rd gen cephalosporin during treatment of purulent meningitis of pneumococcal and unknown etiology the initial treatment strategy was changed in . third gen cephalosporin is now used as a first choice antibiotic, what is in consent with international recommendation of treatment. to evaluate and compare groups treated initially with chloramphenicol and with rd gen cephalosporin will need several more years. low prevalence of multi-drug resistant mycobacterium tuberculosis in jerez de la frontera-cadiz (spain) ps alados jc, aller ai, de miguel c, de francisco jl, calbo l. hospital del sas-jerez , microbiologia, jerez de la frontera, cadiz, spain introduction and aim: previous reports indicate that multi-drug resistance mycobacterium tuberculosis (mtb) is an worldwide problem. the aim of this study was to review the resistance of mtb to the first-line antimycobacterial agents in our area. material and methods: over a period of years ( Á/ ), strains of mtb isolated from non-treated patients with tuberculosis ( strain in , in , in and in ) were studied. these isolates were tested for in vitro drugs susceptibility to isoniacid-i, rifampicin-r, streptomycin-s and ethambutol-e using the bact/ alert method (organon teknica) as described by the manufacturer. results: our results showed that . % ( / ) strains were resistant to one or more drugs. single drug resistances were detected on nine strains to i ( . %), one to r ( . %), two to s ( . %), one to e ( . %). three mtb strains were resistant to more than one drug but only one was multi-drug resistant (i'/r).the incidence of i-resistant strains over the period fell from % in to . % in . conclusions: ( ) multi-drug resistance is not an important problem in our area. ( ) isoniacid resistance was declined to an admissible level. to investigate the anti-tuberculosis drug resistance pattern of pulmonary tuberculosis isolates in southern taiwan, an area with higher tuberculosis incidence and mortality than other regions of the island, we performed a hospital-based surveillance at a southern taiwan medical center from to . the combined drug resistance rates to at least one of five first-line agents was . %, and to both isoniazid and rifampin (multi-drug resistance, mdr) was . %, indicating high resistance rates compared with those reported in the who/iuatld global project and in northern taiwan. the resistance rates to two second-line drugs, cycloserine, and kanamycin, were . and . %, respectively. a significant decreasing trend in resistance rates to all drugs except streptomycin was observed during the -year period. though combined drug resistance rate may not be the most accurate tool as it includes previously treated cases which inflates the resistance rate, the observation of trends in the susceptibility of pulmonary tuberculosis in accompany with the increasing percentages of tuberculosis patients receiving complete treatment course and the decreasing percentages of cases lost of follow-up in kaohsiung after the institution of new governmental regulations for case management in suggest the usefulness of intervention programs. lipid profile in patients with multidrug resistant pulmonary tuberculosis ps extrapulmonary tuberculosis may sometimes present with confusing clinical manifestations. a -year-old female patient was admitted with a history of recurrent supra-sternal abscess for year. mri confirmed the presence of sternal osteomyelitis and an anterior mediastinal mass. the diagnosis of tuberculosis was proved by histologic examination and acid-fast stain. the patient was treated with first-line agents, which isoniazid, rifampin, pyrazinamide, and ethambutol. tobacco smoking as a factor of the decrease of chemotherapy effectiveness and of the development of the drug resistance in patients with pulmonary tuberculosis ps shprykov as a , zhadnov vz a , shprykova on b . a medical academy, department of tuberculosis and lung diseases, nyzhny novgorod, russian federation , b medical clinic for infectious # , laboratory of bacteriology, nyzhny novgorod, russian federation studies of the effect of smoking on the results of chemotherapy of patients with tuberculosis of lungs. intensive tobacco smoking slowed down clearance of positive sputum and of lung tissue destruction (in smokers . % and . vs. . % and . % in nonsmokers, p b/ . ). drug-resistant mtb strains have been found to be isolated more often in smokers */ . vs. . % in non-smokers, p b/ . . resistance to streptomycin and isoniazid prevailed, reaching in heavy smokers . and . %, respectively. resistance to rifampicin increased . times. the concentration of rifampicin in the blood serum of heavy smokers decreased in . times. clinical data are in complete correlation with the findings of our experiments: % of experimental cultures developed resistance to streptomycin, isoniazid and less to rifampicin in the study of drug sensitivity under the effect of tobacco smoke condensate. thus, our findings show the development of drug resistance in mtb under the effect of components of tobacco smoke and also showed less effectiveness in therapy. kilani kb, ammari la, tiouiri ht , ben chaabène tbc. rabta hospital, infectious diseases, tunis, tunisia guerrero c a actinomycosis is a chronic disease characterized by abcess formation, tissue fibrosis and draining sinuses. it is caused by anaerobic bacteria belonging to the genus actinomyces. thoracic actinomycosis may involve the lungs, pleura, mediastinum or chest wall. the authors present a case of pulmonary actinomycosis complicating a cervicofacial site. a -year-old man with a history of cervicofacial actinomycosis treated by penicillin g years ago was admitted because of right-sided chest pain for months before presentation, cough and fever. physical examination shows a painless indurated mass in the neck with multiple fistula of the sternum. chest radiograph and ct scan revealed a mass in the upper lobe of the right lung with an infiltrate of the upper lobe of the left one. magnetic resonance imaging confirms the previous lesions, with extending process to the sternum and right collar bone. bronchoscopy was performed while patient was on antimicrobial therapy. culture of bronchoalveolar lavage fluid was negative. transbronchial biopsy was not conclusive. fungal serologies were negative for aspergillosis, histoplasmosis, blastomycosis. bacterial examination of purulent drainage from sternal wound shows inclusion bodies identified as actinomyces. he was treated then with penicillin iv for months, than switched to doxycycline. after months of treatment, he is asymptomatic with radiological improvement. kanellopoulou m a , skarmoutsou n a , iglezos i b , mylona e a , martsoukou m a , apostolopoulou f b , papafrangas e a . a laboratory of clinical microbiology, sismanoglio general district hospital of attica, athens, greece , b nd department of pneumology, sismanoglio general district hospital of attica, athens, greece introduction: achromobacter xylosoxidans is a rare human pathogen. it is an important cause of bacteremia in patients with cardiac diseases, malignancies and immunosuppression. it has been recently recognized as an emerging microorganism in cystic fibrosis (cf), whose its pathogenic role is unknown. aim: to investigate the sensitivity to eleven different antibiotics of a. xylosoxidans strains isolated from adults with cf, during . methods: the susceptibility was tested by kirby bauer and microdilution methods (wider i, fransisco soria melguizo, s.a.), according to nccls recomendations. results: the resistance to antibiotics was as follows : gentamicin, tobramycin, aztreonam %, amikacin %, ceftazidime %, ticarcillin %, carbapenems, cotrimoxazole %, colistin % and piperacillin %. conclusions: ( ) a. xylosoxidans isolated from cf patients appeared resistant to the most usually tested antibacterial agents. ( ) colistin which is used as aerolized antibiotic for cf patients seems to be effective in the half of the isolated strains. ( ) piperacillin was the most active antibiotic against a. xylosoxidans . morris ka, perry jd, jain s, gould fk. microbiology department, freeman hospital, newcastle upon tyne, uk alafosfalin, l-alanyl-l- -aminoethylphosphonic acid, is an antibacterial peptide mimetic which inhibits peptidoglycan biosynthesis. we report the in-vitro activity of this compound in combination with ceftazidime, cefsulodin, fosfomycin, piperacillin/tazobactam, aztreonam, ciprofloxacin and timentin. drug combinations were evaluated against burkholderia cepacia strains, and pseudomonas aeruginosa strains isolated from patients with cystic fibrosis. for this purpose a chequerboard technique was adopted using doubling dilutions of each antibiotic incorporated into a highly defined agar medium free of antagonists. the minimum inhibitory concentrations (mics) and fractional inhibitory concentrations (fics) of all the antibiotic combinations were determined which revealed the antibiotic interaction occurring. alafosfalin in combination with ceftazidime, meropenem, piperacillin/tazobactam and timentin demonstrated the highest percentages of synergy in both b. cepacia and p. aeruginosa . synergy was shown to occur in , , and % of b. cepacia strains respectively, and in , , and % of p. aeruginosa strains. these four combinations were re-tested with all isolates and the results were shown to be reproducible. alafosfalin shows potential as a treatment for cystic fibrosis patients colonised with p. aeruginosa and/or b. cepacia , when applied in combination with these agents. community-acquired pneumonia */does its aetiology matter? ps lintmaer i, popescu a, popescu c. institute of infectious diseases matei bals, unit , bucharest, romania the aetiology of a pneumonia is not one of the criteria used to determine pneumonia's severity. however, it is accepted that identified based Á/based therapy is less expensive (and possibly more effective). objectives: our study aims were to: ( ) to evaluate the role of aetiology identification in pneumonia; ( ) to evaluate the first-line therapy in pneumonia. methods: we conducted a retrospective study in an infectious diseases hospital on patients with pneumonia. we excluded all the cases with nosocomial pneumonia. primary end-point was the day clinical failure (deaths, icu admission), secondary end-points were the average time of fever and length of stay and the antimicrobial regimen changes. results: causative agent identification rate was . %. the evolution was different for patients with identified aetiology compared with other patients in terms of: -day failures, length-of-stay and changes of the antimicrobial regimen. the patients with inadequate first-line therapy had a more severe course of illness with a greater rate of day clinical failure, longer fever and length-of-stay. conclusions: pneumonia's treatment was better for the patients with identified causative agent. that is why we should include aetiology among the pneumonia severity criteria, especially at an 'after -day therapy' re-evaluation. results: pneumomococcal aom was detected in children ( . %) and s.pn. was the only pathogen in . %. the resistance rates of the organism to antibiotics were as follows: penicillin . % (micb/ mg/ml; intermediately resistant . %, mic . mg/ml . %, and mic!/ mg/ml; highly resistant . %), erythromycin . %, clindamycin . %, cotrimoxazole . % and chloramphenicol . %. all isolates were uniformly susceptible to rifambicin and vancomycin. the large majority of pneumococcal isolates ( . %) had the mphenotype and the remaining strains ( . %) the constitutive mls phenotype. a various of serogroups were detected; the serogroup was the most predominant one ( . %), followed by serogroups ( . %), ( . %) and ( . %). the non-typable s.pn. strains compromised the . % of the strains. conclusions: high prevalence of resistance to penicillin, macrolides and cotrimoxazole in pneumococcal aom of childhood was recognized. a strategy for preventing aom caused by drug-resistant pneumococci is mandatory to start. material and methods: a total number of strains were examined. the sensitivity test was performed by kirby bauer, microdilution method (pasco, difco) according to nccls guidelines and by e -test. results: a percentage of . % of s. pneumoniae strains revealed high level resistance to penicillin (mic]/ mg/ml), while the % showed intermediate resistance (mic . Á/ mg/ml). the resistance to erythromycin and cotrimoxazole was . % (mic ]/ mg/ml) and . % (mic]/ / mg/ml), respectively. all strains were sensitive to cefotaxime (mic . mg/ml), vancomycin (mic / . mg/ml), meropenem (mic / . mg/ml) and levofloxacin (mic / mg/ml). conclusions: ( ) the prevalence of high resistance s. pneumoniae to penicillin seems to be low in examined strains ( . %). ( ) intermediate resistance to penicillin of s. pneumoniae isolates was high as expected ( %). ( ) most of the strains were sensitive to erythromycin ( . %) and cotrimozaxole ( . %). ( ) s. pneumoniae isolates were completely ( %) sensitive to levofloxacin, vancomycin and meropenem. beghi g, aiolfi s, maghini l, patruno v, aiolfi e. s marta hospital, pulmonary rehabilitation unit, a.o., rivolta d'adda, italy aim: of this study was a retrospective ( Á/ ) evaluation of the more effective and practical antibiotic treatment in ae-copd patients (pts) admitted to our unit. methods: before introducing any antimocrobial drug, sputum specimens were collected for microbiological purposes, while blood analysis, to monitor adverse systemic effects, were performed at the beginning and the end of treatment. antibiotic treatment ranged from to days according to four regimens: regimen a ( pts) /oral therapy only: . % with amc g b.i.d.; . % with cip mg b.i.d.; . % with dox mg u.i.d.; . % with lev mg u.i.d.; . % with cla mg b.i.d.; . % miscellaneous. regimen b ( pts) /sequential therapy (e.v. for days /oral): . % with amc g b.i.d.; . % with cla mg b.i.d. regimen c ( pts) /e.v. therapy only: same drugs. regimen d ( pts) /an association of two antibiotics. results: of evaluated pts, only ( . %) required a second regimen of treatment because of failure of the previous one: . % of regimen a; . % of regimen b; . % of regimen c, and % of regimen d. mild adverse effects were detected only in four pts. our results confirm that oral antibiotic treatment is practical, safe, and effective, and can be considered as the first line regimen also in hospitalized patients with ae-copd. becher g a , gillissen a a , rothe m b . a st. george medical center, robert-koch-hospital, leipzig, germany , b filt, lung and chest diagnostics ltd., berlin, germany patients with severe form of chronic obstructive pulmonary disease (copd) are prone by frequent exacerbations. bacterial infections are judged to cause at least half of exacerbations. haemophilus influenzae and streptococcus pneumoniae are the most frequent isolates, gramnegative bacilli account for the severe cases, aggravating the inflammatory process in the airways eventually leading to respiratory insufficiency. the aim of this ongoing placebo controlled, parallel group, mono center study trial is to evaluate beneficial effect of cefixim to reduce bacterial load and pulmonary inflammation in patients (n / ) with acute bacterial exacerbation of severe copd. thus, patients received in randomized fashion either cefixim ( mg/day) or placebo ( days). on days , , and breath condensate is collected using 'ecoscreen' (jaeger germany) for ltb -, il- -, nitrite-and ph-analysis. in parallel sputum gathered for detection of bacterial strains, and for ltb -, and il- quantification purposes. these data are compared to clinical outcome parameters such as lung function tests, radiographic findings, serum inflammatory markers and length of hospital stay. the preliminary data obtained confirm successful antibiotic therapy with oral cefixim in bacterial related acute exacerbations of copd is a useful approach to reduce bacterial load, and concomitantly lower inflammatory indices of the central and peripheral airways leading to clinical improvement of the patients. soriano garcia f a , fenoll a b , fernandez-roblas r a , coronel p c , gimeno m c , rodenas e c , garcia m a , granizo jj d . a fundacion jimenez diaz, microbiology, madrid, spain , b instituto de salud carlos iii, centro nacional microbiologia, majadahonda, spain , c tedec meiji farma, scientific, alcala de henares, spain , d fundacion jimenez diaz, epidemiology, madrid, spain purpose: to describe the susceptibility of streptococcus pneumoniae against cefditoren and other antimicrobials by serotype a multicenter study in south europe was carried out. a total of strains were collected between september and march from adult patients (more than y.o.) with respiratory tract infection (respiratory tract samples and blood cultures). all the isolates were sent to a central laboratory (fundació n jiménez díaz, madrid, spain) where susceptibility test was performed by broth microdilution (sensititre) following nccls recommendations. serotype was determined by quellung reaction and dot assay in carlos iii institute in strains. results: a total of strains ( . %) were not typable. the most prevalent serotypes were ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %). two hundred and sixty-four strains were grouped in different serotypes. the proportion of susceptible strains by serotype to penicillin, erythromycin and levofloxacin were: serotype ( . , . , %); ( . , . , . %); ( . , . , . %); ( . , . , . %); ( . , . , . %); ( . , . , . %). the mic to cefditoren was / . (serotype ); . (serotype , and ) and mg/l (serotype and ). conclusions: the most prevalent serotype was . the susceptibility was higher in serotype than in serotypes and . community acquired pneumonia */a study among closed military community of young people ps martynova av, turkutyukov vb, vostrikova aa, andryukov bg. vladivostok state medical university, epidemiology, vladivostok, russian federation purpose: the etiology of pneumonia is still partly unknown. we should like to clear up an etiological role of respiratory pathogens in community-acquired pneumonia among youth. and we had chosen for it a model of a closed community both investigation of etiology of disease and for further investigation of mechanisms of transmission drug-resistant mechanisms. methods: we studied adults in age of Á/ from closed military collectives who presented to two public hospitals (one urban and one rural) with acute radiologically confirmed pneumonia during winter Á/ . we did blood and lung-aspirate cultures, mycobacterial cultures, serotype-specific pneumococcal antigen detection, and serology for viral and atypical agents. results: streptococcus pneumoniae is recognized as an important cause of community-acquired pneumonia, it probably accounts for % of cases of community-acquired pneumonia among youth. chlamydia pneumoniae and mycoplasma pneumoniae responsible for approximately % of cases. haemophilus influenzae caused . % sever cases of disease, % of all cases were due to moraxella catharralis . conclusion: pneumococcial infection accounted for % of the cases diagnosed. s. pneumoniae was the most common bacterial infective agent, with a low incidence of both m. pneumoniae and s. pneumoniae . other causative pathogens occurred only within groups of individuals with deficiency of immunological status. berezin en a , cardenuto md a , nobuko e b , guerra ml c , brandileone mc d . a santa casa, pediatrics, s. paulo, brazil , b santa casa, microbiology, s. paulo, brazil , c adolfo lutz, microbiology, s. paulo, brazil , d adolfo lutz, bacteriology, s. paulo, brazil to determine antimicrobial susceptibility of sp isolated from the upper respiratory tract, we collected np swab specimens from children, between months and years old. those children attended the outpatient clinic in s. paulo city, with diagnosis of bacterial infection requiring antibiotic therapy between march , to may , . penicillin susceptibility of isolates was determined by screening with oxacillin mcg disk and performing the minimal inhibitory concentration by the e -test. we performed also susceptibility test for amoxicillin and cefaclor. results: sp was recovered from np of children ( . %). the carriage of sp was more prevalent in children attending day care centers, children with young siblings at home, and children with tobacco users at home. the prevalence of penicillin non-susceptible strains was . % all of them with intermediate resistance. all the strains were susceptible to amoxicillin and . % were resistant to cefaclor. serotypes . b, f, n, , a and were the most common. these findings suggest that nasopharyngeal isolates of streptococcus pneumoniae from children with upper respiratory infections can be used to conduct surveillance for antimicrobial resistance in a defined geographic area. we were able to conclude also that penicillin intermediate resistent strains can be susceptible to amoxicillin. hinojosa rm a , saenz a a , collazo m a , echaniz g b . a universidad autonoma de nuevo leon, infectologia, monterrey, mexico , b instituto nacional de salud publica, epidemiologia, cuernavaca, mexico the emergence of penicillin-and multidrug-resistant pneumococcal strains has become a global concern, necessitating the identification of the epidemiological spread of such strains. material: ninety streptococcus pneumoniae clinical isolates were collected from march through march . typing was done by the capsular reaction with pooled, type, or group antisera. susceptibility testing to antimicrobials was done by the e -test and the disk diffusion method. results: only ( %) s. pneumoniae strains were classified by serotyping; the most frequent types were a/b, f, v, f and . the oxacillin screening test detected . % penicillin-resistant s. pneumoniae strains isolated from children and . % from adults. the susceptibility percentage of s. pneumoniae to ceftriaxone was % in both children and adults. s. pneumoniae isolates from children exhibit a susceptibility of % to azithromycin, while in adults % of the isolates were susceptible. for the rest of the antimicrobial agents, the susceptibility ranged from to %. s. pneumoniae had a lower susceptibility to ceftazidime and ciprofloxacin. conclusions: ceftriaxone and azithromycin had a good in-vitro activity against s. pneumoniae strains. but the percentage of penicillinresistant s. pneumoniae detected in this study is alarming, therefore we conclude that a continuous surveillance system is necessary in mexico. vertkine al, prokhorovitch ea, alexanyan la. a retrospective analyses of cases of ambulant pneumonia with fatal outcome was made. among the patients who died from ambulant pneumonia the prevailing age was over ( . %) and the prevailing sex was male ( . %). . % had pneumonia accompanied with some pathology: chronic lung disease ( . %), alcoholism ( . %), diabetes mellitus ( . %). . % of the patients had a big volume of lungs lesion */ . % of the patients suffered from bilobular pneumonia and . % */from trilobate pneumonia. in . % of the cases pneumonia was complicated with abscess formation and/or exudative pleurisy. we studied the antibiotics therapy used for the patients treatment. the change of antibiotics was made only in cases ( . %) whereas in the other cases no change of preparations was made though the signs of the therapy non-effectiveness were obvious. thus, the rational antibiotics therapy with the timely change of non-effective antibacterial drug is significantly important. while choosing the antibiotics, the patient's age, the accompanying diseases, the volume of the lungs lesion and complications which define pneumonia seriousness are to be taken into consideration. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to describe the management of lower respiratory tract infections (lrti) in healthy adults, by general practitioners (gp). methods: a questionnaire was sent to a representative national sample of gps. this questionnaire assessed their perception and management of lrti, the indication for antibiotics (ab) in a case of lrti in a healthy adult with no focal signs and no signs of severity, knowledge of the micro-organisms responsible for acute bronchitis and knowledge of the afssaps (french agency for the safety of health products) recommendations. results: three thousand seven hundred and thirty-eight gps, who reported seeing an average of patients per week, including . / . patients with lrti, returned the questionnaire. the main results are presented in the following for the majority of gps, the main objective of the visit is to determine the indication for antibiotics. according to gps, alp and whooping cough are rare, while atypical pneumonia is frequent. gps also declare that the diagnosis of alp is often easy right from the first visit, in contrast with that of atypical pneumonia. complementary investigations are not often requested. gps consider that they often delay prescription of antibiotics ( %) and declare that they tend to prescribe a macrolide as first-line treatment. finally, gps have a poor knowledge concerning the micro-organisms responsible for acute bronchitis and the majority of gps declare to be familiar with afssaps recommendations. perronne c a , rouveix b b , guillemot d c , zuck p d , reitz c e , tsatsaris a e . a hôpital raymond poincaré, service de maladies infectieuses, garches, france , b hôpital cochin, paris, france , c institut pasteur, paris, france , d hôpital de metz, metz, france , e laboratoires abbott, rungis, france objectives: to study the management of one case of lower respiratory tract infection (lrti) in adults by general practitioners (gps). methods: prospective study conducted on a representative national sample of gps. each gp had to include the first healthy adult patient seen during the -week data collection period, either on a home visit or in the office for recent cough and acute fever !/ . c. clinical data, the diagnostic perception and the therapeutic approach to the patient were collected by means of a standardised questionnaire, distributed by abbott laboratories. results: three thousand seven hundred and thirty-eight general practitioners included patients. conclusions: during lrti in adults, gps observe few focal signs, confirming the marked predominance of bronchitis compared to pneumonia. in view of the frequency of the signs, the diagnosis of pneumonia appears to be overestimated. one-third of clinical situations were diagnosed as 'bacterial superinfection of acute bronchitis', despite it is not a recognised diagnostic entity. antibiotic prescription was immediate in % of cases and delayed in % of cases. this last point shows that clinical practice differs from the gp's perception of their described prescribing practice shown in a simultaneous survey ( % of gps declared that they prescribed antibiotics immediately, while % delayed this prescription). results: thirty-three patients ( men, mean age b years, mean apache ii score . &bdqup;b . ) during the years Á/ were prospectively studied. thirteen patients ( %) had no identifiable risk factor for severe cap. an etiologic factor was revealed in patients ( %). in of them this was achieved with noninvasive methods. psb cultures were taken from eight patients and were positive in only . the offending organisms included: streptococcus pneumoniae in six cases, gnb as the sole pathogen in six cases, haemophilus influenzae (with s. pneumoniae or klebsialla pneumoniae ) in two cases, s. aureus in two and legionella pneumophila in one patient. initial antibiotic regimen a combination of marcodile '/ rd gen cephalosporin /aminoglycoside was successful in patients who all survived and had to be changed empirically or according to culture results in patients who had a mortality of %. the overall mortality rate was %. the identification of the causative factor did not seem to have any impact on survival. conclusion: severe cap in our icu was most often caused by s. pneumoniae and gbn. the high mortality of this entity seems to be influenced by the immediate use of the appropriate antibiotic combination and not by the identification of the causative organism. this underscores the need for knowledge of topical microbiology which helps in designing an effective empirical initial antibiotic regimen. mily mn a , golubev sa b , lugovoy vy a , voronov gg b . a vitebsk emergency hospital, pharmacotherapy unit, vitebsk, belarus , b basic and clinical pharmacology department, vitebsk state medical university, vitebsk, belarus purpose: the study aims were to assess the spectrum and predictors of the antibiotic use during pneumonia management in a regional emergency hospital in belarus. patients, treatment and physicians characteristics of cases ( Á/ ) were collected and possible associations were examined with using defined daily doses methodology (ddd) and american thoracic society (ats) guidelines. results: the mean treatment duration was . / . days, the total antibiotic ddd/ bed-days was . . the ddd/ bed-days of the most used antibiotics were: penicillins . ; aminoglycosides . ; macrolides . ; cephalosporins . ; tetracyclines . . in manova certain ats categories were associated with ddd/day, but not with frequency of definite antibiotic use. ddd/day was higher in case of multilobar infiltrates ( conclusion: our study indicated the low rate of macrolides and cephalosporins using, and the high one of aminoglycosides. antibiotic prescriptions were associated with disease severity and physician personality rather then with empirical choice rules recommended. acute exacerbation of copd: most frequent infecting agents and their suspectability to the different types of penicillins. analysis of medical documentation ps pertseva to, bogatska ke, gashynova ky. dsma, internal medicine , dniepropetrovsk, ukraine number of copd cases has been increased in ukraine. treatment of acute exacerbation (ae) of copd is not always successful because of inadequate antibiotic therapy. the aim of study was to reveal most frequent infecting agents and their susceptibility to the different types of penicillins in patients with ae of copd. medical documentation of patients with ae of copd (type i) was studied. data of sputum analysis and susceptibility of isolated agents to penicillin, ampicillin, oxacillin and amoxicillin/clavulone acid were evaluated. there were patients with haemophilus influencae/parainfluencae ( . %), klebsiella pneumoniae ( %), staphylococcus aureus ( . %), pseudomonas fluorescens , pseudomonas putida , serratia marcescens , serratia liquefaciens ( . % each), streptococcus agalactiae , acinetobacter baumanii ( . % each) in samples of sputum. in . % cases there was mixt infection. . % had no any bacterial agents. only % of agents were susceptible to penicillin, % */to ampicillin, % */to oxacillin. however, % of microorganisms were susceptible to amoxicillin combined with clavulone acid. this study has shown that most frequent infecting agents caused ae of copd were gram-negative microorganisms and s. aureus . according to antibiogram the prescription of amoxicillin/clavulone acid is most expedient in this case. pertseva to, bogatska ke, konopkina li, kireeva tv, gashynova ky. dsma, internal medicine department, dniepropetrovsk, ukraine we examined patients ( men, mean age . / . years) with acute exacerbation of cob (type i). the most frequently isolated agents were haemophilus influenzae and parainfluenzae */eight patients, gram-negative rods in , staphylococcus aureus in two and mixed in six and one patient had no bacterial agents isolated in their sputum. high susceptibility to azithromycin was found in all cases of gram-positive agents and in h. influenzae and parainfluenzae . other gram-negative agents were resistant to this drug in vitro. however, treatment with mg/day during days was clinically effective in . % of cases. only . % of patients had a further acute exacerbation of cob. there were peculiarities of the infecting agents causing acute exacerbation of cob in this study: klebsiella pneumoniae and s. aureus were found more frequently than in other studies. high efficacy of surnamed in the treatment of acute exacerbation of cob was established in . %. . % had partial positive clinical effect after this therapy. there were no patients with adverse events. efficacy and tolerance of amoxicillin mg/kg bid versus amoxicillin mg/kg tid in the treatment of acute otitis media (aom) in children / years ps borek m a , guggenbichler jp b . a biochemie gmbh, international medical department, kundl, austria , b department of pediatrics, university of erlangen, erlangen, germany five hundred and sixteen patients (mean age / . years) with clinical and otoscopic diagnosis of aom were included in a randomized, double blind, multicentre study, and were treated days either with amox mg/kg bid or amox mg/kg tid. assessments were made during therapy (day Á/ ), after end of therapy (eot, day Á/ ) and follow up (fu, day Á/ ). the primary efficacy endpoint was the clinical response at eot defined as success (cure/improvement) or failure. both regimens were well tolerated; one or more drug-related adverse events (aes) were reported in . % ( / ) of bid patients and in . % ( / ) of tid patients. the most frequently reported drugrelated aes in each group were gastrointestinal symptoms (bid . % vs. tid . %), which were mainly of mild or moderate severity. both regimens were clinically equivalent. the higher cure rates in the bid group suggest a possible higher benefit from bid therapy in children / years. children attending family day-care (fdc) should be less exposed to upper respiratory tract infections than those in group day-care (gdc) and therefore to antibiotic treatment; fewer should thus carry resistant bacteria. to test this hypothesis, np carriage of sp and hi with reduced susceptibility to penicillin (pdsp and hi bl'/, respectively) was investigated among children in fdc (maximum three children) and in gdc ( Á/ children) in the alpes maritimes (france) between november and march . a two stage cluster sample of children attending gdc or fdc was selected. np samples were cultured for sp and hi. penicillin susceptibility was tested by disk diffusion and e -test, and b-lactamase production by api-nh † tests (biomerieux, lyon). two hundred and thirty-five children in fdc and in gdc aged Á/ months were sampled. age and sex distribution were similar in both groups. sp was isolated in children in fdc ( %), and in ( . %) children in gdc (p b/ Á/ ). proportions of pdsp were . and . %, respectively (p / . ). hi was present in . % of children in gdc vs. . % in fdc (p b/ . ). proportions of hi bl'/ were . % vs. . %, respectively (p / . ). antibiotic exposure during the previous months concerned . % of children in gdc vs. . % in fdc (p / . ). there was no correlation between antibiotic use and carriage of pdsp or hi b'/ strains. sp and hi carriage rates are significantly lower among children in fdc than in gdc. advising alternative types of daycare for children attending gdc should reduce exposure and thus limit the spread of resistant bacteria. however, the proportion of pdsp and hi bl'/ is similar in both groups and comparable patterns of antibiotic use are observed. continued efforts must concentrate on parental education and enforcement of recommendations for management of pediatric upper respiratory tract infections. during a period between january and august , invasive haemophilus influenzae (hi) isolates had been collected at the children's hospital of tunis. we used haemophilus test medium to test antibiotic susceptibility. the mic of beta-lactams was measured by e -test. beta-lactamase production was determined by using the cefinase test and biotyping by apinh. presence of capsular antigen was determined by using hi typing anti sera. hi strains were isolated from meningitidis ( ), bacteremia ( ) and arthritis ( ). all strains were serotype b and . % of them belonged to biotypes i and ii. amoxicillin resistance with beta-lactamase producing mechanism occured in . %. mic of beta-lactamase producing strains was vs . mg/l in non-producing one. there is no betalactamasenegative amoxicillin resistant among these invasive isolates. antibiotic resistance concerned chloramphenicol: . %, trimethoprim-sulfamethoxazole: . %, tetracycline: . % and kanamycin: . %. invasive hi infections in tunisian children's were always associated with type b strains. introduction of a hib vaccine programme in tunisia is recommended. the aim of this study was to analyse the clinical picture and treatment of neurological manifestations of neuroborreliosis in children. the study included children ( Á/ years) with neuroborreliosis diagnosed on the basis of the clinical and serologic criteria. symptoms of facial palsy occurred in six children symptoms of iii Á/vi cranial nerves palsy in three children, meningitis in four and paresthesias in three. symptoms of v or viii nerve palsy, mental disturbances, radiculoneuritis or cerebellitis were found in singular cases. all children received ceftriaxone intravenously Á/ weeks. total recovery was obtained in children following the first course of therapy. recovery following the second course of therapy (amoxicillin) occurred in one child with mental disorders and one with vi nerve palsy. improvement was achieved after the second course in the patient with radiculitis, however, muscular atrophy persisted. irreversible, unilateral deafness was found in a child with viii nerve palsy in spite of three courses of therapy applied. infection with borrelia burgdorferi in children causes a wide spectrum of neurological manifestations. facial palsy was the most common sign in our study. applying ceftriaxone in the treatment of neuroborreliosis is characterised by a good effectiveness. double infection by c. pneumoniae and m. pneumoniae as a cause of cystic changes in the lungs ps streharova a, moravcikova d. department of infectious diseases, university of trnava, trnava, slovakia chlamydia pneumoniae and mycoplasma pneumoniae are human respiratory pathogens manifested in early childhood. immunological disbalance could trigger autoaggressive diseases. the authors describe the case of a -year-old girl with development of multiorgan failure and septic state, which followed multiple cystic changes in the lungs. the girl did not have a diagnosis of cystic fibrosis. the authors consider that cystic changes are a consequence of double infection by c. and m. pneumoniae. dzarlieva m, momeva l, temelkovska g, balevska p, pejkovska m. medical centre, neonatology, bitola, the former yugoslav republic, macedonia unicef skopje has supported a nationwide safe motherhood needs assessment in representative samples of hospitals. eighteen of maternity wards and facilities renovated and certified as 'babyfriendly'. all mature newborns with successful adaptation to extra uterine life and satisfactory vital parameters are h during the day with their mothers at rooming-in system. aim: with rooming-in system we reached decreasing of incidence of neonatal infections. material and methods: history records of newborns from our department. for the period of months, neonates have been borne. with suspection of infection there */ babies ( . %). newborns borne through meconium stained liquid */ ( . %). results: microbiological findings: from blood culture */staphylococcus coagulaza negative from swabs */staphylococcus aureus , escherichia coli , staphylococcus epidermidis. conclusion: in , from all babies who had risk factors for infection in newborns ( %) we had positive findings and in year (before rooming-in sistem), in ( . %). after that period (with rooming-in) newborns ( . %). with rooming-in system we reached decreasing in incidence of neonatal infections by breaking the chain of infection */only mothers take care of their babies with help of the staff. newborns are in their micro environment, the same they will have at their home. with this practice we have also reduction of nosocomial infections. a study on antibiotic susceptibility and resistance factor transmissibility among antibiotic resistant salmonellae isolated from children affected to diarrhea ps sharifzadeh a. azad university of shahrekord, microbiology, shahrekord, islamic republic of iran in spite of happened drug resistance, antibacterial therapy still is the best route of treatment of salmonellosis in man and animals. in order to detection of dominants serotypes of salmonellae in children and detection of antibiotic susceptibility and r-factor transmissibility among those isolated salmonellae. this study was conducted on diarrheic stool samples were collected from children affected by diarrhea in ayatollah kashany hospital of shahrekord, during spring of to autumn of . after isolation and identification of salmonellae, seven serotypes were detected. one of those was s. typhi and another six serotypes were s. paratyphi b . in order to detection of antibiotic different antibiotic disks were used in disk diffusion method. best results were taken from ceftizoxim, cephtriaxon, cephazolin and chloramphenichol. the r-factor were transferred from isolated salmonellae to escherichia coli k in all of cases of resistance to penicillin and ampicillin. pharyngeal colonization by streptococcus pneumoniae and group a bhs were evaluated in randomly selected school children aged Á/ years in riyadh, saudi arabia. fifty-six children ( . %) had positive culture for either organisms of the isolates from school children, ( %) were s. pneumoniae , of them ( %) were penicillin-sensitive, three ( %) were penicillin-resistant, and two ( . %) were resistant to two antimicrobials. forty isolates of bhs ( %) were group a bhs. all isolates were penicillin and erythromycin sensitive. the carrier rate among school children for penicillin-resistance s. pneumoniae and resistance to two antimicrobials were ( . %) and ( %), respectively. the carrier rate of group a bhs was ( . %). riyadh has a low rate of antibiotic-resistant s. pneumoniae and a similar rate of group a bhs carriers among school children as that seen in temperate areas. boukadida j a , boukadida n b , hannechi n a , said h b , erraii s a , elmhabrech h a . a chu f. hached, laboratoire de microbiologie, sousse, tunisia , b c s base, sousse, tunisia the acute pharyngitis is a very frequent pathology in which group a streptococcus is the most incriminated bacteria. however, other non a b-haemolytic streptococcus (sbna) could be responsible. the aim of this work is to determine the part of each non a b hemolytic streptococci (sbna) in acute pharyngitis and the related antibiotics susceptibility pattern. the study was realized in sousse-tunisia (north africa) during months from may . the origin materials of isolates are throat swab (transystem venturi, copan, bovezzo). the mean age of patients is years with extremes Á/ years. the samples are cultured on blood agar plates in a delay of h maximum. identification was done to samples that have over than b-hemolytic colonies, groupage with pastorex strep. sanofi pasteur france, susceptibility pattern according to nccls norms, mic is determined by e -test. the control strain is s. pneumoniae atcc . twentyone clinical isolates of sbna are distinguished from clinical isolates of b-hemolytic streptococci recovered from patients with acute pharyngitis without symptoms of viruses' infections (tearing, corysa, sneeze). all b-hemolytic streptococcus represents . % of all collected samples. sbna were . % of the isolates. sbna were strains group g streptococci, seven strains group c streptococci and three strains group f streptococci. susceptibility pattern of each sbna to antimicrobial agents is as follow: group g streptococci: peni g / %, amoxicillin / %, pristinamycin %, clindamycin and erythromycin / . %, tetracyclin / . %, telithromycin . % and levofloxacin / . %. group c streptococci: peni g / %, amoxicillin / %, pristinamycin %, clindamycin / . %, erythromycin / . %, tetracyclin / . %, tel-ithromycin / . %, levofloxacin / %. group f streptococci-peni g / %, amoxicillin / %, clindamycin / . %, erythro / . %, tetracyclin / . %, telithromycin / %, levofloxacin / . %. all sbna have mic to penicillin g under . mg/l. according to available data, penicillin g and amoxicillin still the reference treatment of acute bacterial pharyngitis in spite of the new antibiotics introduction. berezin en a , quevedo sg b , nicolla l b , viegas d c , eizenberg b d , pedrosa f b , santos ag a . a santa casa, pediatrics, s. paulo, brazil , b elly lilly, scientific, s. paulo, brazil , c fac. abc, pediatrics, santo andre, brazil , d university hospital, pediatrics, s. paulo, brazil a multicenter, open label, prospective, randomized trial in which patients Á/ years of age with proven gabhs pharyngitis were randomized to receive either -day course of the broad spectrum oral cephalosporin, cefaclor or a -day course of amoxicillin. patients were included if they have signs and symptoms of streptococcal tonsillopharyngitis and a rapid streptococcal rapid test positive . patients were evaluated at days Á/ , Á/ and Á/ posttherapy. pharyngeal cultures were conducted at baseline and at follow-up visit ( Á/ days). we considered for bacteriologic eradication analysis only patients with positive culture to gabhs. there were patients with a rapid streptococcal rapid test. clinical success were achieved in around % of the patients. for evaluate eradication of the initial pathogen we considered patients of the amoxil arm and patients of the cefaclor arm. thirty-four of patients of the amoxil arm ( . %) and of ( %) patients of the cefaclor arm were considered bacteriological cured at the second culture performed at day Á/ . ten days of a penicillin or amoxicillin therapy may not be the best therapeutic choice for all pediatric patients. in developing countries where rheumatic fever is still an important problem to evaluate the bacterial eradication achieved with the different antibiotics may be important. prophylactic affect of saccharomyces boulardii for antibiotic-associated diarrhea in a paediatric age group ps erdeve o, tiras u, camurdan mo, tanyer g, dallar y. ankara education and research hospital, pediatrics, ankara, turkey the process resulting in antibiotic associated diarrhea is the alteration of enteric flora due to bacteria. saccharomyces boulardii is a yeast, isolated from cover of a kind of hazelnut and its usage became widespread recently. there is no enough study about s. boulardii activity at pediatric ages. we aimed to define s. boulardii activity on azithromycin and sulbactam-ampiciline, and associated diarrhea at pediatric age group. the . % of cases only with antibiotic usage developed diarrhea, whereas rate was . % for probiotic using group (p b/ . ). all of the clostridium difficile toxin a defined cases were from sulbactam-ampiciline using group. the rate of diarrhea for sulbactam-ampiciline using group was . %, while it was . % for group used s. boulardii as probiotic beside sulbactam-ampiciline (p b/ . ). it was observed that probiotic usage decreases diarrhea rate four times (p b/ . ). when age groups considered, the rate of sulbactamampiciline associated diarrhea increased at Á/ ages and s. boulardii effect on preventing diarrhea was significant at Á/ ages (p b/ . ). antibiotic associated diarrhea is a common clinical problem at pediatric age group. s. boulardii is a hope giving probiotic especially for sulbactam-ampiciline associated diarrhea. grey e a bilikova e a , hafed bm a , kovacicova g a , chovancova d b , huttova m b , krcmery v a . a school of health, trnava university, trnava, slovakia , b postgraduate academy of medicine, neonatal clinic, bratislava, slovakia the purpose of the study: the aim of the study was to find out, whether artificial abortion of a mother has impact on neonatal infection of her future baby. the results obtained: therefore, we compared neonates with infection, who were born to mothers with past history of abortion ( Á/ artificial abortions within years), with the babies of mothers, who have not experienced abortion before. control group consisted of neonates hospitalized at the same clinic, at the same time period. according to the analysis of risk factors for neonatal infections, it was found, that prematurity ( Á/ weeks of gestation) ( . vs. . %, p . ) and low birth weight- Á/ g ( . vs. . %, p . ) were significantly more frequently observed in babies of mothers with past history of abortion. drug abuse (heroin) ( . vs. . %, p . ) and nicotine use ( . vs. . %, p . ), were more significantly related to neonatal infections in babies of mothers with past history of abortion. etiological analysis showed that only candida albicans ( . vs. %, p . ) was significantly related to neonatal infections in mothers with past history of abortion. the conclusion reached: in conclusion, artificial abortion has not only direct impact to the health of the mother, but also on her next pregnancy. bacteraemia and patient mortality in a peadiatric intensive care unit ps armenian sh, singh j, arrieta ac. children's hospital of orange county, infectious disease, orange, usa purpose: this study will identify the factors that significantly contribute to mortality in patients with bloodstream infections (bsi) at a pediatric intensive care unit (picu). results: medical records of patients who were admitted to the picu and had a documented bsi were reviewed. there were separate episodes of bsi's, with nine patients having multiple bsi's during their hospital stay. a casecontrol model was used. cases were bsi's with eventual mortality (n / ; . %) and controls were those who survived bsi (n / ; . %). patients who died were older ( . vs. . years; pb/ . ), more likely to have a nosocomial bsi ( . vs. . %; pb/ . ), longer hospitalization prior to bsi ( . vs. . days; pb/ . ), and have a polymicrobial bsi ( . vs. . %; pb/ . ). infection related mortality (irm)-defined as death within days of bsi */was significantly higher in those receiving inadequate antibiotic treatment at the time of diagnosis of bsi ( . vs. . %; p b/ . ), as well as in those with gram negative bacteremia and/or fungemia ( vs. %; p b/ . ). logistic regression was used to adjust for potential confounding variables. conclusions: we found that being older, multiple organisms, and a longer hospitalization prior to the bsi were significantly associated with overall patient mortality. irm was significantly higher for those with inadequate initial antibiotic coverage and in those with gram negative bacteremia and/or fungemia. somer a a , gü r d a , diri s a , yalcýn i a , salman n a , ongen b b , gü rler n b . a department of pediatric infectious diseases, istanbul university istanbul medical faculty, istanbul, turkey , b department of microbiology and clinical microbiology, istanbul university istanbul medical faculty, istanbul, turkey teicoplanin is a glycopeptide antibiotic active against a broad range of gram-positive pathogens including methicillin-resistant staphylococci and offers the advantages of once daily administration, choice of administration route, lack of requirement for routine therapeutic drug monitoring and lower propensity to cause nephrotoxiticy and anaphylactoid-like reactions. in this study the efficacy and safety of teicoplanin were evaluated retrospectively in children with serious bacterial infection. sixty-three children ( girls, boys) aged between month and years were treated with teicoplanin (three loading dosages of mg/kg at h intervals, followed by a maintenance dosage of mg/kg/day). the infections treated were pleural empyema (n / ), joint and bone infections (n / ), septicemia (n / ), skin and soft tissue infections (n / ), and lung abscess (n / ). the pathogens isolated were staphylococcus aureus (n / , of which were methicillin resistant), coagulase-negative staphylococci (n / ), s. pneumoniae (n / ), and group a hemolytic streptococci (n / ). the duration of therapy ranged from to days (median days). clinical success (cure plus improvement) was achieved in . % of cases. no side effects attributable to teicoplanin therapy were encountered. teicoplanin appears to be an effective and well-tolerated treatment for serious gram-positive infections in children. the risk of infection is present in all children with acute leukemia. two hundred and sixty episodes of febrile neutropenia were analyzed in children (aged . Á/ years) with aml */ cases and all */ cases over years during cytostatic therapy (protocols: mbfm */ aml and mbfm */ all). a degree of fn occurred in % of cases in aml, in % */in all. the sites of infection were: blood'/ central venous catheter ( %) and respiratory tract ( %). pathogens isolated from blood were: gram-positive */cns */ %, streptococcus spp. */ . %, gram-negative */enterobacteria */ %, pseudomonas aeruginosa */ %, candida spp. */ . %, aspergillus spp. */ . %, other */ . %. for the treatment of fn we used empirical antibiotics regimens. clinical response was noticed: in st line of therapy */ cephalosporins Á/ generation used */ %, carbapenems used */ % as nd Á/ rd lines */vancomycin */ %, amphotericin b */ %. thirtytwo percent of children with aml and . % children with all died because of sepsis. conclusion: carbapenems are more active in the st line of antibiotics therapy both for patients with aml and all. vancomycin is useful in the nd line for patients with all. amphotericin b and vancomycin are useful in combination in the nd line for patients with aml. pavlenishvili ivp. medical academy, pediatrics, tbilisi, georgia our drug of choice in cases of complicated neonatal sepsis i. pavlenishvili, g, iobashvili tbilisi, medical academy georgian society of pediatrics chemotherapy (gspc) with collaborations of drug and therapeutic committee (dtc) of childers hospital 'republic' finished the observation study about nosocomial infections of neonatal icu in above mentioned hospital. study begins from november . during this period we observe cases of neonatal sepsis, most of them were due gram-negative bacteria. thirty-four cases of neonatal sepsis were cause different stains of escherichia coli , proteus spp. and acinetobacter . we notice that during last years the role of acinetobacter in etiology of neonatal sepsis and nosocomial complications of neonatal sepsis in our hospital is gradually increased. as our observation reveals most optimal in the case of complicated neonatal sepsis was (according criteria of dts) use of ceftasidime. almost in all cases we used ceftasidime with success. we have only one case of mortality. since the macrolide resistance (mr) in streptococcus pyogenes has been increasing in europe, we studied the incidence and genetic basis of mr in s. pyogenes in russia. s. pyogenes isolated at baseline and during follow-up visits from children with acute pharyngitis receiving penicillin or midecamycin ( -membered macrolide) were studied. mr was evaluated by agar dilution (nccls), resistance mechanisms */by pcr. a total of s. pyogenes were obtained at the baseline, ( %) of which were erythromycin-resistant: strains ( %) were erm (a)-positive, one */mef (a)-positive, and one was negative for all primers used. all erm (a)-strains were inducibly resistant to clindamycin and represented seven pfge profiles with one profile found in / strains. in three midecamycin treated patients mr was selected during the therapy (one strain had erm (a), one */mef (a), one */ unknown resistance determinant). mef (a)-strain obtained during follow-up visit had different pfge pattern with mef (a) strain isolated at baseline, while both the baseline and follow-up strains with unknown mechanism of resistance had unique pfge pattern. these data showed moderate incidence of erythromycin-resistant s. pyogenes in smolensk. ribosomal methylation (erm (a)) was the most common mechanism and though the polyclonal nature of mr was established, most erm (a)-strains belonged to only a few clones. bacillus cereus infections in traumatology-orthopaedy department: retrospective study and re-evaluation of healthcare practices ps bacillus cereus was cultured for patients from traumatology department who had developed postoperative wound infections between august and march . all patients presented inferior members open fractures, frequently contaminated with telluric material and requiring external fixators. genomic study of clinical isolates by pulsed field gel electrophoresis and random analysis polymorphic dna, allowed us to eliminate an outbreak. furthermore, the reduced delay in which patients developed the infection ( days'//- ) led us to re-evaluate the procotols used in our institution. indeed, all patients had received amoxycillin '/ clavulanate iv g for antibioprophylaxis during anesthetical induction then relayed per os for hours. because of the production of a potent beta-lactamase by the bacteria, this association could not be efficient. furthermore, accordingly to the afnor en norm, we have tested clinical isolates' sensitivity to the principal antiseptics used for antisepsis and disinfection (iodophors, chloride derivated and biguanidines) and observed a major resistance of all strains tested. even if these postoperative wound infections are considered as nosocomial because of the delay in appearance, we actually think that bacillus cereus were initially present in telluric material. this fact led us to propose a systematic screening for bacillus at admission for this type of wound and to administrate quinolones such as ciprofloxacin for prophylaxis. internal thiols and reactive oxygen species in the candidacidal activity exerted by a n-terminal peptide of human lactoferrin ps the purpose of the study: the emergence of candida albicans strains resistant to current antifungals points to the need for new antifungal agents, e. g. antimicrobial peptides. the results obtained : we report that hlf( - ), a synthetic nterminal peptide of human lactoferrin, displays excellent killing effect against fluconazole-resistant c. albicans and that sub-optimal concentrations of this peptide combined with fluconazole act synergistically. previous investigations revealed that hlf( - ) required an energized mitochondrion, atp release by candida , and ligation of atp receptors for its killing effect. we now report that reactive oxygen species (ros) are involved in the killing effect of hlf( - ). since internal thiols protect cells from oxidative damage, our observation that hlf( - ) caused a % reduction of internal thiols in candida is of interest. as expected, n-acetyl-l-cysteine (nac), which is a precursor of glutathione and a ros scavenger, inhibited the killing effect of hlf( - ). diamide, which oxidizes internal thiols, was candidacidal and hlf( - ) and diamide acted synergistically in killing c. albicans and ros production. moreover, the hlf( - )-induced activation of mitochondria was inhibited by nac, indicating that internal thiols/ros affect mitochondrial activity. the conclusion reached : the candidacidal activity of hlf( - ) involves ros production and reduction of internal thiols. objective : an audit was conducted to explore the observation of increasing resistance to ciprofloxacin in enterobacteriacea isolated from specimens from such patients in the hematology unit. results : enterobacteriacea isolates in specimens from such patients processed over the years to were examined. number of specimens per year was */ , , , and respectively and the annual percent ciprofloxacin resistant enterobacteriacea from these was %, %, %, % and %. conclusion : conclusion drawn from the audit was that rapid rise in ciprofloxacin resistance may possibly be attributed to the use of this fluoroquinolone as a single agent in dose of / mg [cut off patient weight kg] twice a day in neutropenic patients till neutrophils exceed /c.mm. subsequent to the audit, prophylaxis protocol was modified to use of oral colistin -mega units/twice a day in combination with ciprofloxacin / mg twice a day. a prospective audit is proposed to test the benefits of this combination. rhinocerebral zygomycosis: diagnostic dilemma for emergency physician: can the associated morbidity and mortality in this rare but deadly disease be reduced? ps samavedam s, guleri as. western infirmary, clinical microbiology, glasgow, united kingdom rhinocerebral zygomycosis [rcz] is a rare, invasive, rapidly progressive opportunistic infection caused by ubiquitous fungi of the order mucorales. it usually occurs in diabetics or immunocompromised patients. the emergency physician will typically see patients with rcz in its earliest stages masquerading as a variety of other less serious diseases. the key markers like necrotic patch on hard palate, nasal septum or turbinate, marked facial pain, and cellulites with marked eye and neurological signs may present late in disease. we report a case of rcz caused by rhizopus arrhizus [oryzae] in an year old woman with poorly controlled diabetes. she presented with a right-sided facial droop of short origin and being generally unwell. ct scan was non-conclusive and delayed presentation of key markers of rcz permitted disease to rapidly progress. despite an intensive antifungal therapy with ambisome and insulin sliding scale, patient rapidly succumbed within days. fine needle aspiration cytology is less invasive, easier and equally effective alternative to pre op biopsy. the key to successful reduction in morbidity and mortality associated with this rapidly fatal disease is -increasing awareness of the disease, an early diagnosis, correction of underlying metabolic derangement, prompt intensive antifungal therapy with amphotericin b and radical surgical debridement of the necrotic tissue. an 'optimal dosage' of ambisome requires discussion. clinical audit in the haematology ward of a tertiary care hospital: study of degree of correlation between bacteraemia and oro-pharyngeal screens in immunocompromised patients over five years and role of antibiotic prophylaxis ps guleri as, butcher i. western infirmary, clinical microbiology, glasgow, united kingdom introduction : a clinical audit was carried out over -years [ ] [ ] [ ] [ ] [ ] in the immunocompromised patients including neutropenic patients and bone marrow [autograft] transplant recipients in hematology ward of gartnaval general hospital, glasgow, a tertiary care center. objective : it was aimed to establish the degree of correlation between bacterial isolates in oro-pharyngeal screen during bacteraemia episodes and role of antibiotic prophylaxis. methods purpose : to assess whether antiretroviral therapy (art) intensification, gm-csf use and remune initiation before stopping art lead to viremia containment, and long periods off art. methods : ten adults with chronic hiv disease, hiv- rna levels (vl) b/ . log copies/ml and median cd '/ -t cell count of /ml were enrolled. after art intensification with ddi ( months) '/ hydroxyurea [hu]( mo.) '/ gm-csf ( mo.) and a remune dose, art was stopped but remune continued. art was resumed if rebound vl did not decrease to b/ . log in months or if cd '/ counts decreased to b/ . results : vl rebounded in all patients after stopping art, and developed an acute retroviral syndrome (ars). cd '/-t cells decreased, and cd '/ '/ increased ( -fold). after a median stoppage of weeks, art was resumed in patients and vl decreased to b/ . log, cd '/ counts were regained, il- and il- levels rose. at the nd interruption, / patients had a rebound, and / had a nd ars, but peak vl and loss of cd '/ were lower (p / . ). after . weeks off art, patients resumed therapy. the breadth and magnitude of hiv-specific activity increased and thymus size grew. the patients were off art for a median of . out of weeks. two of them are off art for and weeks respectively ( vlb/ log). conclusions : this approach led to a high ars incidence, long periods off art, increases in hiv-specific responses, il- and il- levels, and thymus size. urinary tract infection in hospitalized population is a significant problem. this is a study of catherized patients urinary infection in corfu hospital. in Á/ , urine cultures were sent to our bacteriology laboratory. of them were collected from the catheters. clinical data of age and type of disease were analysed. the samples were cultured on mc conkey agar-urotube and vitek cards. the organisms identified with vitek automatic system. the sensitivity was tested with vitek and kirby-bauer method. results: no growth of organisms in . %. positive cultures %. . % of the bacteria were gram((/) rods ( . % e. coli , pseudomonas spp. %), % were gram('/) cocci (enterococcus spp.) and % was candida spp. the resistance of e. coli was: % to ampicillin , % to co-trimoxazol and % to quinolons. e. faecalis was resistant % to vancomycin . conclusions : ( ) the possible interference of acetaminophen in the amoxicillin/ clavulanic acid (a/c) or erythromycin (ery) efficacy in the treatment of acute otitis media (aom), and its possible role in the evolution to otitis media with effusion (ome), were determined in a gerbil model. a f streptococcus pneumoniae strain exhibiting a mic of a/c and ery of / . and . mg/l, respectively, was used. both antibiotics were tested at . and mg/kg. acetaminophen at mg/kg was administered min before each antibiotic dose. antibiotic concentrations in serum and middle ear exudate were determined. both antibiotics significantly reduced the number of culture-positive ears and colony counts, with serum concentrations over the mic of the microorganism for ]/ % of the dosing interval. antibiotic concentrations in middle ear exudate were almost identical in animals receiving and not receiving acetaminophen. clinical and microbiological efficacy was correlated with antibiotic concentrations in middle ear exudate ]/ . times the mic of the microorganism, for both antibiotics. both antibiotics demonstrated efficacy in the treatment of pneumococcal aom, with the same rate of ome. acetaminophen, concomitantly administered, did not interfere the efficacy of the two antibiotics tested and did not prevent the evolution of aom to ome. parra a a , ponte c a , cenjor c b , garcía-olmos m a , giménez mj c , aguilar l c , soriano f a . a fundación jiménez díaz, medical microbiology, madrid, spain , b fundación jiménez díaz, otorrinolaryngology, madrid, spain , c glaxosmithkline, medical department, madrid, spain a gerbil model of otitis media with effusion (ome) induced by haemophilus influenzae (amoxicillin/clavulanate-a/c-and erythromycin-ery-mics of / . and mg/l, respectively) was used to evaluate the efficacy of a/c ( / and / mg/kg) and ery ( and mg/kg). antibiotics were administered subcutaneously h post-middle ear inoculation, and continued t.i.d for h, with or without acetaminophen (ap), at mg/kg, administered min before each antibiotic dose. antibiotic concentrations in serum and middle ear (me) were measured by bioassay. me samples for colony counting were collected on day . a/c reduced (p / . ) positive me samples and colony counts versus untreated controls or ery: me positive cultures of % for controls, % for a/c , % for a/c , % for a/c '/ap, % for ery , % for ery and % for ery '/ap. this was due to a/c (but not ery) concentrations in me exceeding . times the mic despite the higher percentage of antibiotic penetration of ery versus a/c ( versus / %). animals receiving ap showed less polymorphonuclear cells and more bacteria in me than those receiving only antibiotics, suggesting that the anti-inflamatory drug diminish the phagocytes and therefore, the efficiency in bacterial clearance. amoxycillin treatment for acute otitis media caused by penicillinresistant streptococcus pneumoniae . a pharmacodynamic analysis pm parra a a , ponte c a , cenjor c b , garcía-calvo g a , giménez mj c , aguilar l c , soriano methods: a serotype f streptococcus pneumoniae strain exhibiting a mic of amoxycillin of mg/l was used in an experimental model performed in gerbils (meriones unguiculatus ) following previously described procedures. amoxycillin was tested at the following doses: . , . , . , . , . and mg/kg. amoxycillin concentrations in serum and middle ear exudate were determined after drug administration. results: doses of ]/ . mg/kg significantly reduced the number of culture-positive ears, colony counts and otorrhoea (p / . ) as compared with untreated controls or animals treated with doses lower than . mg/kg. doses of ]/ . mg/kg achieved antibiotic concentrations in the middle ear . Á/ . times higher than the mic of the infecting strain and serum concentrations over the mic for Á/ % of the dosing interval. conclusions: amoxycillin at doses achieving serum concentrations similar to those obtained in children after standard doses, obtained therapeutic and microbiological efficacy regardless the susceptibility of the infecting strain. better correlation was found between antibiotic efficacy and antibiotic concentrations in middle ear exudate than between efficacy and serum concentrations, which were suboptimal from the pharmacodynamic perspective. increasing prevalence of amoxycillin Á/clavulanate-resistance among e. coli strains in a hungarian university hospital pm veréb i, vígh a, urbán e, hajdú e, nagy e. department of clinical microbiology, university of szeged, szeged, hungary background: amoxicillin Á/clavulanate resistance (acr) is an emerging problem in escherichia coli as reported from different parts of europe. the aims of the present study were to evaluate statistically the prevalence of acr among e. coli isolates and to investigate the genetic background of the resistance. methods: all e. coli strains isolated between and were screened for acr by kirby Á/bauer disc diffusion method. the resistance to other beta-lactam Á/beta-lactamase inhibitor combinations and to different beta-lactam antibiotics were also tested. selected strains underwent determination of beta-lactamase activity. confirmatory tests for suspected extended spectrum beta-lactamase were performed. pcr testing for tem and shv genes were carried out on plasmids isolated from selected strains. results: in out of e. coli strains ( . %) were found to be resistant to amoxicillin clavulanate (amc). most of the resistant strains ( %) were obtained from the genitourinary tract and no acr isolate was found in blood cultures. in out of isolates ( %) proved to be acr and . % were isolated from blood cultures and . % from the genitourinary tract. thirty-five selected strains were further analysed. thirty-two were also resistant to (sam) and six were further resistant to tzp. quantitative beta-lactamase determination showed increased activity in strains which were partially susceptible to amc. the presence of esbl could be proved only in three acr isolates. this open parallel-group study compared the efficacy and tolerability of cef with cfix mg once daily in the treatment of community acquired uncomplicated uti. seventy-eight female patients were randomized to receive either oral cef or cfix for or days. the efficacy of treatment was evaluated by clinical response (by symptoms of uti dysuria frequency urgency suprapubian pain and by clinical signs) by bacteriologic response and health status measures at baseline and posttherapy. results: the clinical cure (complete resolution of symptoms and signs) rate for patients receiving cef was . % of the evaluable patients and . % of the patients receiving cfix. bacteriologic response (based on the results of urine cultures obtained posttherapy) the pathogen was eradicated in . % for cef . % for cfix. no drug related side effects have been reported in cef and side effects were experienced by . % of the patients receiving cfix. improvement in health status comparing visual scale scores baseline and poststudy to have detected a higher change in average score from to in cef, from to in cfix. wilcoxon improvement value was significant on the rd day of therapy in case of cef and on the th day of therapy in cfix group. in conclusion, the results of this study indicate that cef course is more effective than cfix in producing a favourable clinical outcome and achieving higher bacteriologic eradication rate, furthermore cef was better tolerated. cefepime is a fourth generation cephalosporine that has a broader spectrum of antibacterial activity than the third generation cepfalosporines and is more active in vitro against gram-positive aerobic bacteria. the purpose of this study was to measure cefepime concentrations in plasma, bile fluid and gall bladder tissue in patients undergoing cholecystectomy. thirty patients male, female, mean age: years had data acceptable for analysis and were included in this study. all patients received iv g of cefepime. several hours after administration and at different time intervals, during surgery, samples were obtained from plasma, bile fluid and gall bladder tissue concomitantly. antibiotic levels were measured by an agar diffusion method. the mean delta time was / min. the values for plasma, bile fluid and gall bladder tissue, were . / . , . / . and . / . mg/ml, respectively. the plasma/bile fluid ratio was . / . . there was a significant correlation between plasma and gall bladder tissue concentration (r / . , p / b/ . ). a correlation between bile fluid and plasma cefepime concentration was not observed. the minimum inhibitory concentration (mic) data from previous in vitro studies indicate that the cefepime concentration observed in plasma bile and tissue samples of this study would be adequate against typical biliary tract pathogens. furthermore, these cefepime concentrations correlated well with the favorable clinical outcome reported in previous clinical studies in biliary tract infections. there was also good correlation between delta time and plasma and tissue concentrations and if the dose were given closer to the time of surgery, cefepime concentration would be higher reducing the possibility of an infection. objectives: the use of antibiotics may lead to decreased colonization resistance and increased formation of resistant bacteria. present concept was developed to overcome these untoward effects. methods: b-lactamase of bacillus licheniformis was overproduced in bacillus subtilis . this targeted recombinant b-lactamase enzyme (trbl) was released in the small bowel from a controlled-release formulation. beagles (n / ) were treated bid with either mg/kg ampicillin (i.v.)'/placebo (p.o.), mg/kg ampicillin (i.v.)'/trbl (p.o.) or only placebo (i.v.'/p.o.). stool was collected at days and . samples were cultured for total and main groups of aerobic and anaerobic bacteria and yeast. temperature gradient gel electrophoresis (tgge) was used to separate the ribosomal rna genes. results: ampicillin'/placebo group had clearly decreased counts of both aerobic and anaerobic bacteria during the treatment, whereas those receiving trbl had only minor overall changes and some occasional changes by single species. intravenous ampicillin decreased the fecal similarity percentage to %. the similarity percentage during treatment with ampicillin'/trbl did not differ from that of placebo ( vs. %). conclusions: according to our results the trbl can maintain the large intestinal microflora almost unchanged. these results indicate that trbl is a promising novel approach for overcoming the ecological adverse effects on gut flora caused by b-lactam antibiotic agents. adamis since broad-spectrum â-lactams combined with amikacin are often applied for nosocomial infections, their pharmacokinetic interactions might be interesting. one gram of aztreonam and . g of amikacin were administered intravenously single and in combination in six healthy volunteers. blood samples were collected at regular time intervals and concentrations of antimicrobials were determined by a microbiological assay applying a strain developing resistance to single agent after serial passages. mean concentrations of amikacin in serum when administered alone and in combination with aztreonam were . and . , . and . , . and . , . and . , . and . and and . mg/ml immediately after and . , , , and h after infusion of antimicrobials. respective concentrations of aztreonam were . and . , . and . , . and . , . and . , . and . and . and . mg/ml. aucs for amikacin when administered alone and in combination with aztreonam were . / . and . / . mg h/l, respectively. respective auc for aztreonam were . / . and . / . mg h/l. it is concluded that the co-administration of aztreonam and amikacin results in earlier clearance of aztreonam and in higher levels of amikacin compared to the administration of each single antimicrobial. molecular modelling of b-lactams reveals the structural basis for their inhibition of penicillin-binding proteins, susceptibility to b-lactamases and oral bioavailability pm grail bm, gupta s, payne jw. school of biological sciences, university of wales, bangor, uk b-lactam antibiotics are peptide mimetics that act as suicide substrates for transpeptidase enzymes that cross link bacterial cellwall peptides. for the first time, the structural and electronic features needed for their recognition by transpeptidase have been fully described, using innovative molecular modelling techniques to compare the conformational forms adopted by cell-wall peptides and blactams. comparison of features in the backbone and c-terminal regions of conformers of active b-lactam antibiotics and model cellwall peptides, has allowed definition of the molecular recognition template required for substrate recognition by transpeptidase. these shared structural features allow both to act as substrates and to acylate the active-site serine. however, a significant difference in a critical backbone torsion between the two substrates, provides an explanation for the inability of the enzyme Á/antibiotic complex to undergo the deacylation step that causes inhibition of transpeptidase. on the other hand, b-lactamases appear to have evolved molecular mechanisms that facilitate the deacylation reaction through compensating for the altered structural orientations in b-lactams caused by the different backbone torsion. finally, analysis of the conformer repertoires of blactams for structural features required for substrate uptake by peptide transporters, provides insights into how their structures can be tailored for optimal oral absorption. antimicrobial susceptibility of proteus mirabilis clinical isolates producing extended spectrum beta-lactamases (esbls) pm objectives: the aim of the present study was to determine in vitro susceptibility to antimicrobials of proteus mirabilis isolated from urinary tract infections. methods: we studied the susceptibility profile of esbl positive p. mirabilis strains in three adopted children from india with age range from months to years. esbl was identified using the synergic effect of clavulanate with betalactams (ceftazidime and cefotaxime the in vivo efficacy of amoxicillin (amx) sub-therapeutic doses ( . mg/kg, t.i.d for h, achieving serum levels over the mic of only % of the dosing interval) and concomitant specific serotherapy (single intraperitoneal dose of / diluted hyperimmune serum (hs) obtained from mice immunized with the heat-inactivated strain) was assessed in a pneumococcal sepsis balb/c mouse model. mice (five mice/ treatment group) were intraperitoneally infected with . )/ cfu/ ml of a serotype b penicillin-resistant strain (mic of and mg/l for penicillin and amx, respectively). treatments started h after bacterial inoculation. study groups were: control (k; receiving nonimmune serum (nhs)), amx'/nhs, hs, and amx'/hs. survival rates (%) over time were: purpose of the study: the study was performed to determine the consumption of imipenem and resistance of gram-negative pathogens (pseudomonas aeruginosa , acinetobacter sp., klebsiella sp., escherichia coli , proteus mirabilis , serratia marcescens , enterobacter sp. ) to imipenem. gram-negative pathogens were isolated at the sestre milosrdnice university hospital from zagreb, croatia, in and . the imipenem sensitivity testing was performed by disk diffusion and e -test methods. the consumption of imipenem was expressed in ddd/ hospital days in the same periods. results obtained: imipenem resistance of acinetobacter sp. decreased significantly in the year (p / . ), especially in the first months (p / . ) when the lowest consumption of imipenem was recorded. imipenem resistance of other gram-negative pathogens did not decrease significantly. conclusion reached: comsumption of imipenem might lead to changes in resistance to imipenem among acinetobacter strains. vacheva-dobrevski rs, savov ez. military medical academy, clinical microbiology, sofia, bulgaria purpose: acinetobacter baumanii is becoming increasingly frequent nosocomial pathogen at our hospital, and beta-lactam resistant strains are on the increase, especially among icu isolates. to study the susceptibility of a. baumanii clinical isolates to beta-lactams and to determine the esbl-producing strains during , year. a total gram-negative nonfermenters (gnnf) isolates was investigated by semiautomated mini api system (bio merieux, france). eighty-four a. baumanii non-repeated isolates was studied for esbl-producing by double-disk synergy test (ddt) and atb-blse test (bio merieux, france). mics for beta-lactams were determined by e -test (ab biodisk, sweden). results: a. baumanii (n / ) showed a multidrug resistance. the isolates were resistant to cefotaxime ( %), cefoxitin ( %), ceftazidime ( %), amoxicillin/clavulanate ( %), piperacillin ( %), aztreonam ( %), imipenem ( %). the ( %) of investigated a. baumanii expressed esbl activity and originated more frequently from icu ( %). esbls producing strains were isolated from endotracheal aspirate ( %), surgery wounds ( %), blood culture ( . %). conclusions: in general resistance levels were higher in clinical isolates a. baumanii to beta-lactams. the ddt seems to be a practical method for esbl-screening; atb-blse method is more sensitive. our study display to be the first report of esbl-producing a. baumanii strains from our country. carbapenems seems to be the most active agents against a. baumanii . salmonella infantis , strain , was isolated from a newborn baby at wassila bourguiba maternity in tunis. it exhibited high resistance to penicillins, extended-spectrum cephalosporines (cefotaxime, ceftriaxone, ceftazidime, cefpirome) and aztreonam but remained susceptible to cefoxitine and imipenem. involvement and characterization of enzymatic mechanism in b-lactam resistance were investigated in strain . isoelectricfocusing revealed that this strain produced a b-lactamase of pi . this enzyme had a broad-substrate profile, hydrolyzing amoxicillin, ampicillin, ticarcillin, cephaloridine, cefuroxime, cefotaxime, ceftriaxone, cefpirome and ceftazidime. the highest specific activity was observed with ampicillin. cefotaxime was hydrolyzed the most efficiently of the extended-spectum cephalosporines. the pi extended-spectrum b-lactamase (esbl) was inhibited by clavulanic acid and sulbactam. no inhibition of the esbl was observed with mm edta. thus, no metal ion is involved in hydrolysis for this b-lactamase. resistance due to the production of the pi esbl was transferred with dna plasmid into escherichia coli . on the basis of substrate and inhibition profiles and isoelectric point, the pi esbl was not previously described in s. infantis in tunisia. the presence of such a resistance on a plasmid raises concer for rapid dissemination among bacteria and loss of effectiveness of blactams. poizot-martin i a , enel p b , benhaïm s c , vion-dury f c , dinh t c , drogoul mp c , gastaut ja c . a assistance publique hôpitaux de marseille, cisih sud, pr ja gastaut, marseille, france , b assistance publique hôpitaux de marseille, cellule santé publique dmi , marseille, france , c assistance publique hôpitaux de marseille, cisih sud, marseille, france objective: to assess liver biopsy (lb) practices in a cohort of co-infected hcv and hiv patients followed up in an hiv specialized medical unit. method: transversal study with questionnaire among patients in pre-therapeutic's evaluation with pcr'/ and without lb at months. results: among the patients, ( %) are lost of follow up, ( . %) have had lb, ( . %) have no lb. the characteristics of these patients are: median age / . / years; sex ratio / . , cdc-stage a / . % b / . % c / . %, undetectable viral load / . %, median cd / / , anti-retroviral therapy / . %, hcv-genotype / . %; a / . %; / . %. causes of non-made lb are: ( ) refusal from patients because of biopsy's fear / . %; ( ) contraindications because of hiv infection / . % (clinical events / . % which contraindicate anti-hcv treatment, grade iii thrombocytopenia / . % which contraindicate biopsy, non-adherence to previous hiv follow up / . %); ( ) other / . % (alcoholism / . %, psychiatric/depressive disorders / . %, decompensated cirrhosis / . %). drug use or methadone/buprenorphine treatment are not considered as contraindication. conclusion: one-third of patients are afraid of lb. alcoholism and psychiatric/depressive disorders are the principal contraindications to anti-hcv treatment. it seems important to improve information of patients about lb and to focus on alcohol and psychiatric/depressive disorders management in such population. kashiwagi kk a , furusyo nf a , nakashima hn a , kashiwagi sk b , hayashi jh a . a department of environmental medicine and infectious disease, kyushu university, fukuoka, japan , b national kyushu medical center, fukuoka, japan the purpose of the study: the aim of this prospective study was to explore the effect of htlv-i co-infection on the development of hcc among patients with chronic hcv viremia. a total of consecutive patients with chronic hcv viremia were studied and followed-up over a mean period of . years: ( . %) were infected with htlv-i infection and ( . %) were not. the results obtained the annual hcc development rate was . % in patients co-infected with hcv and htlv-i and . % in patients infected with hcv alone. hcc was significantly higher in ( . %) of the patients co-infected patients than in ( . %) of the patients infected with hcv alone (p b/ . , logrank test). in patients under the age of years, hcc development was significantly higher in seven ( . %) of patients co-infected with hcv and htlv-i than in eight ( . %) of patients with hcv alone (pb/ . , logrank test), whereas there was no significant difference in hcc development between patients over age with or without htlv-i infection ( ( . %) of and ( . %) of , respectively). the conclusion reached htlv-i infection accelerates the development of hcc in chronic hcv patients, especially among patients under the age of years. to analyze hbv genotype-related clinical differences among patients with chronic hbv infection, all patients were serially tested for serum alanine aminotransferase (alt) and hepatitis b e antigen (hbeag) and followed up for a mean . ( . ) year period. genotypes b and c were found in ( . %) and ( . %) of the patients, respectively. hbeag positivity and alt abnormality rates at the start of the observation period were significantly higher in genotype c patients ( . and . %) than in genotype b patients ( . and . %). the annual rate of spontaneous hbeag disappearance in genotype b patients was much higher than in genotype c patients ( . versus . %, respectively). patients with genotype c who were continuously hbeag negative from entry had significantly higher alt abnormality ( . %) than those with genotype b ( . %). interestingly, patients with genotype c who became hbeag negative by interferon treatment had high alt abnormality ( . %). all patients with alt abnormality were serum hbv dna positive. these findings indicate that hbv genotype c patients are more severe liver deterioration because of the delay of hbeag disappearance and continued hbv replication after hbeag disappearance. nossik nn a , nebolsin ve b , zheltukhina ga c , yevstigneeva rp c . a the d.i. ivanovsky institute of virology, viral reproduction, moscow, russian federation , b pparminterprisis co., chemistry, mocow, russian federation , c moscow state academy of fine chemical technology, piptide chemistry, moscow, russian federation objective: to study the effects of 'gamma'-l-glutamylhistamine (glu-ha) derivates on non-specific immunity ('alfa'-ifn, 'gamma'-ifn and nk cell activity) and antiviral activity on the experimental influenza and herpes virus infections in mice. the glu-ya and its derivate glu-ii were synthesized by peptide chemistry techniques. the glu-ha and glu-ii was administered i.p. . and . mg/kg before and after influenza virus (type a/aichi) and showed a protective effect even at the high infective dose ( ld ) */the rate of protection / Á/ / % in the positive/control group. they were not very effective in the protection of herpes simplex virus encephalitis in mice. the model of the physico-emotional stress in mice was used to investigate the ifn system and nk cell activity. the production of ifns and nk cell activity of splenocytes decreased in h after the stress and back to normal level in Á/ days. it was shown that glu-ha and glu-ii can protect or substantially prevent the decrease in nk cell activity and ifns synthesis in post-stress period (so normally did not induce the ifns' synthesis). conclusions: the glu-ha and glu-ii showed antiviral effect against influenza virus infection in mice. the immunomodulating activity and ability to normalize the ifn synthesis and nk cell activity depressed the post-stress period and probably play an essential role in the antiviral activity. no dose adjustment of an anti-influenza prodrug oseltamivir is required in patients with hepatic impairment pm oo c a , snell pr b , liu b a , martin d b , simkins t b , small i b , ward p b . a hoffmann-la roche inc., global development, nutley, usa , b roche products ltd., global development, welwyn garden city, uk background: oseltamivir (ose; ro - , tamiflu † ) is an oral ethyl ester prodrug of its active metabolite oseltamivir carboxylate (oc: ro - ), a potent and selective neuraminidase inhibitor of the influenza virus. the purpose of the study is to evaluate the need for ose dosage adjustment in hepatic impaired patients (hi). method: healthy volunteers (hv) versus hi (child-pugh score Á/ ) [matched on the basis of age ( / years), gender and weight ( / %)] were compared. each subject received mg ose. results: based on c max (ng/ml) and auc inf (ng h/ml) analysed using nominal times, ls mean ratios and % ci between hi and hv were similar. ose* values in hi were marginally elevated but not sufficiently to require dose adjustment. the aim of this study is to investigate the influence of molecular structure of macrocyclic pyridinophanes and their analogs on antiinfluenza and antiherpetic activity of these compounds. we used d-qsar approaches on the basis of simple representation of molecular structure. such representation for biologically active substances allows the description of the spatial structure of compounds with the complete stereochemical information. it determines spatial structures either promoting or interfering of the concrete biological activity. it is easy to realize the molecular design of compounds with the given level of activity with the help of the combinations of simplexes. statistic characteristics for qsar of partial least-squares models are satisfactory (r / . Á/ . ; cvr / . Á/ . ). the molecular fragments that increase the antiviral activity were defined and will be demonstrated. this information was used for design and directed synthesis of several novel antiviral agents with predicted high anti-influenza or antiherpetic activities. predicted activities were confirmed experimentally. d-qsar approaches are useful for development of antiviral compounds. this work was partially supported by intas foundation (grant intas - ). lozitsky vp. ukrainian mechnikov research anti-plague institute, chemotherapy, odessa, ukraine the purpose of this study was to research the anti-influenza activity of proteolytic inhibitor e-aca. it prevents the enhancement of proteolysis during the interaction of virions with cell membranes and decreases penetration of virions into cells. e-aca brings down proteolytic cleavage of ha-precursor to ha- and ha-polypeptides and reduces the infectious virus harvest. it shows the prophylactic and therapeutic action during the experimental influenza reducing the enhancement of alkaline proteases activity in lungs after infection. e-aca promotes the intensification of specific antibodies production and cell immunity, prevents vessels' permeability and hemorrhagic phenomena, decreases the destruction of bronchi's epithelium. it reduces the duration of intoxication, catarrhal instances and hyperthermia in sick children. e-aca improves the indexes of immunity, non-specific resistance and decreases the rate of bacterial complications. application of e-aca for treatment influenza and other arvi in children is recommended in ukraine on the base of results of our researches. the higher effects demonstrated as a result of combine usage of e-aca with specific ig, or deitiforin, or unithyol, or ribavirin. in our opinion, the study of effectiveness of e-aca combine application with inhibitors of influenza na is the perspective direction of anti-influenza researches development. we should we treat immediately all varicella patients with acyclovir if the patient is older than years pm during past years in institute for infectious and tropical diseases in belgrade, immunocompetent varicella patients were treated and cured. among them were older than years ( . %). x-rays were performed in all patients. diagnosis of pneumonia was made in patients ( . %), but in ( . %) patients older than years. varicella is a benign, self-limited disease, if it strikes early, i.e. preschool, school children and teenagers. at that time there is no need for specific therapy. but in neonates, immunocompetent adults and in all immunocompromised patients it can be difficult and life-threatening disease. in immunocompetent adult population pneumonia is a very serious, sometimes fatal complication. knowing the pathophysiology of primary varicella Á/zoster infection, specific therapy with acyclovir should be started immediately after making the diagnosis in patients older than years, without waiting for x-ray proof of pneumonia. brivudin compared to acyclovir for the treatment of herpes zoster: effects on acute disease and posttherapeutic pain pm objective: comparison of efficacy and safety of brivudin )/ mg and acyclovir )/ mg, both for days, in the treatment of herpes zoster. methods: randomised, double-blind study on immunocompetent patients ]/ years (brivudin: n / , acyclovir: n / ). a subgroup of patients ]/ years (brivudin: n / , acyclovir: n / ) was examined for the occurrence of posttherapeutic pain in a poststudy survey. posttherapeutic pain was defined as any zoster-associated pain, regardless of intensity, after the end of acute zoster. results: brivudin was superior to acyclovir in reducing time to last occurrence of new vesicles (rr(itt): . [ . Á/ . ], p / . ). the advantage of brivudin was more pronounced in patients ]/ years (rr(itt): . , [ . Á/ . ], p / . ). incidence of posttherapeutic pain was significantly lower with brivudin ( . %) than with acyclovir ( . %, p / . ). duration of pain was comparable in both treatment groups (rr: . , [ . Á/ . ], p / . ). potentially treatment-related adverse events occurred in . % of the brivudin recipients and in % of the acyclovir recipients. conclusions: brivudin mg once daily for days is superior to standard acyclovir in stopping viral replication in acute herpes zoster. in patients ]/ years, brivudin is more effective than acyclovir in reducing the risk of developing posttherapeutic pain. brivudin is as well tolerated as acyclovir. varadinova t a , genova p a , garcia-raso a b , terron a b , fiol j b , badenas f b . a laboratory of virology, sofia university, sofia, bulgaria , b chemistry, universitat de les balears, palma de mallorca, spain we have published that cu(ii) complexes of acyclovir (acv) are active against hsv infection (mbd, ) . here we present data on the activity of acv complexes of ni(ii), cd(ii), co(ii) and ag(i) against resistant to acv hsv strain r- in comparison with the effect against acv sensitive strain victoria. selectivity indexes (si) compared to that of acv were indicative for activity. the following data were obtained: (i) was times less selective inhibitor of strain r- than of strain victoria; (ii) under the action of [cd(acv)cl ], [ni(acv) (h o) ]cl ×/ acv and [ni(acv)(no )] ×/ h o was up to % higher than that in the control; (ii) was times less selective inhibitor than acv; (iv) si of was two times higher for strain r- and five times lower for strain victoria then that of acv. these data show that the selectivity of acv against resistant hsv strains can increase when acv is bond to a proper metal ion. methods: an antiviral activity against herpes simplex virus of the type i (hsv-i/leningrad/ / ) and variant hsv- (vvt/ / r) resistant to acyclovir was determined using commonly accepted method. viruses were grown on a continuous culture. maximal toxic dose was determined by the administration of compounds orally ( mg/kg) or intraabdominally ( mg/kg) to white mice that had mass Á/ g. condition of the animals was controlled during h. mice pneumonia model was used for the testing activity in vivo. results: derivatives tested have activity against hsv- and hsv- resistant to acyclovir. maximum protection of the cells up to % was reached at concentration of compounds Á/ mg/kg. tested compounds have low toxicity and animals did not die after intraabdominal and after per oral administration of the substances. using these compounds led to essential relief of diseases in animals. the number and square of virus specific areas of inflammation in lung was decreased to compare with control untreated group. tested compounds protected animals similar to acyclovir that was used as control. conclusion: derivatives of carboalkoxysulfanilic acids are active against hsv in vitro and in vivo and act on the acyclovir resistant variant viruses. markiewicz r a , szepietowski jc b . a jelfa s.a., medical department, jelenia gora, poland , b department of dermatology, university of medicine, wroclaw, poland background: denotivir is a -benzoamino- ?-chloro- -methyl- isothiazolecarboxanilide anti-inflammatory agent with antiviral and immunomodulatory activities. it possesses also mild antibacterial and antifungal action. the purpose of the study: the aim of this presentation is to give an overview of recent studies demonstrating denotivir efficacy in herpetic infections. the results obtained: in vitro studies revealed that denotivir in the doses below its cytotoxicity (about um) significantly inhibited (by Á/ %). herpes simplex virus (hsv)- and hsv- replication in fibroblast and kidney cell cultures. moreover, it was showed that denotivir in the dose of mg/ml markedly inactivated hsv- after min incubation in c. in giunea pigs research, % denotivir in % dmso appeared to be superior to % dmso alone and untreated groups in the therapy of animal skin infected by hsv- . there was no huge difference in eythema and oedema scorings between studied groups, however in the group treated with denotivir, in contrast to others, no vesicles developed. several clinical studies showed usefulness of denotivir in controlling herpetic infections in dermatology, ophthalmology and otolaryngology. in the majority of studies within few hours after the drug application itch and pain relief was noted and within Á/ days the vesicular lesions were dried up. the conclusion reached: in conclusion, denotivir is an effective antiherpetic agent. this study was conducted on cows affected with teat papillomatosis. in the first step, each cow was located on one of three groups. the first group contained four cows from to years that were treated with fig tree (ficus carica) latex. the second group contained four cows from . to years that were treated with a % solution of salicylic acid, and the third group contained four cows as control. in group one and two following treatment with fig tree latex and salicylic acid, superficial necrosis begun from day and all of the warts disappeared by day . in the control group, after day , there were no changes in number of lesions, but some of them were larger than first observation. on day , one of the marked warts disappeared and on day another wart was disappeared but six were present until day . comparison of effects of salicylic acid and fig latex showed similar effects in treatment of udder papillomatosis in cow. laboratory markers of skeletal muscle toxicity in hiv-infected patients: a cross-sectional case-control survey of frequency, potential correlation with antiretroviral therapy, clinical significance, and outcome pm manfredi r, motta r, patrono d, calza l, chiodo f, boni p. to assess skeletal muscle toxicity among Â/ hiv-infected outpatients (p), the p who had ]/ altered cpk assay ( !/ u/l) between may and november , were compared with p randomly selected among those who had ]/ laboratory exams in this -month interval, in a : case-control study. among the p with altered cpk levels only six were females, and received antiretrovirals. the overall frequency of altered cpk among all p who underwent ]/ laboratory workouts in months was . %. cpk alteration was transient in p, with values ranging from to (mean . / . ) u/l, but was recognized ]/ times in Á/ months in p ( %), of them showing concomitant high aldolase levels ( . Á/ . u/l). a myopathy or a rhabdomyolisis were recognized in four p only; a myositis was confirmed in one p by histopathology. in a multivariate logistic regression analysis, when excluding the unexpected prevalence of the male gender (p b/ . ), no significant difference emerged between p and controls as to age, risk for hiv infection, iv drug use, duration of hiv infection, prior anti-hiv therapy and its length, selected drug combinations, administered nucleoside analogues,hiv disease stage, mean cd '/ count and hiv viremia, signs and duration of lipodystrophy, increased glucose, triglyceride and cholesterol levels, and other therapies. muscle abnormalities, though frequently asymptomatic, are underestimated hiv disease complications, and the role of metabolic (i.e. mitochondrial) alterations, deserves investigation. poor efficacy of non-nucleoside reverse transcriptase inhibitor (nnrti)based salvage haart in hiv-infected patients heavily pre-treated with all other classes of antiretroviral compounds pm manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy poorly comparable literature series show conflicting results of nnrti-based rescue haart: Á/ % rate of virologic success. to assess the response to a Á/ -drug rescue haart including a nnrti, patients (p) treated with nucleoside analogues (na) and protease inhibitors (pi) for !/ and !/ months, respectively but naïve to nnrti, with a viremia !/ copies/ml, were prospectively followed during Á/ years, provided that they ensured a !/ % adherence. efavirenz was used in p, nevirapine in , and delavirdine in two. most p ( . %) had an early laboratory improvement, but mean peak viral load decrease was . log, and a significant reduction vs baseline (p b/ . ) lasted months only. a mean % increase of zenith cd count was obtained (p b/ . ), but only . % of p remained !/ cells/ml year after switch to nnrti. in a multivariate analysis, the concurrent introduction of novel pi(s) ( p) and/or different na(s) ( p) acted favorably until the th month of follow-up (p b/ . ), while genotypic mutations conferring nnrti cross-resistance, usually associated with a broad resistance profile, predicted failure in all p (p b/ . ), and the response did not vary according to duration and type of prior therapy, and selected nnrti. a deep salvage nnrtibased haart has a poor and transient virologic outcome also in nnrti-naïve p, while a more evident and sustained immunologic response is expected. p who can introduce novel pi/na and have no mutations impairing nnrti activity are entitled to a better outcome. fatal lactic acidosis without elevation of liver-enzymes during the treatment with stavudine, didanosine and efavirenz: a case report pm winzer r, langmann p, väth t, zilly m, klinker h. medizinische poliklinik der universität würzburg, schwerpunkt hepatologie/infektiologie, ürzburg, germany nucleoside reverse transcriptase inhibitors (nrtis) cause various side effects, many of which are thought to be due to their effects on mitochondria. a -year-old hiv positive (hiv rna: copies/ml, cd cell count: /ml), obese (body-mass-index: . ), therapy-naïve female patient, who after months of well tolerated and effective antiretroviral therapy (stavudine, didanosine, efavirenz), had slight gastrointestinal discomfort and suddenly developed a lactic acidosis (arterial-ph . [ . Á/ . she died days later despite intensive care (continuous venovenous haemodiafiltration, sodium-bicarbonate infusion, high doses of vitamins, respiration). the pathologic examination showed an enlarged liver ( g) with yellowish appearance and pasty consistency, which microscopically appeared as a massive macro-and microvesicular fatty degeneration, and only slight signs of terminal pancreatitis. this reported case gives evidence that a massive lactate acidosis may develop without previously disarranged laboratory parameters for liver or pancreatic function. a fatal outcome may evolve without further accompanying-illnesses. efficacy and tolerability of atorvastatin in the treatment of hypercholesterolemia in hiv-infected patients receiving haart pm calza l, manfredi r, chiodo f. division of infectious diseases, university of bologna, bologna, italy introduction: significant increases in plasma triglyceride and cholesterol levels have been reported in patients treated with haart, and prolonged metabolic imbalances could significantly act on the longterm prognosis and outcome of hiv-infected persons. patients and methods: fourteen hiv-infected patients on pi-based haart since at least months and presenting hypercholesterolemia ( !/ mg/dl) of at least -month duration and unresponsive to a hypolipidemic diet and physical exercise, have been treated with a single daily dose of atorvastatin ( mg) for months. results: one patient was ecluded from evaluation due to early dropout. ongoing antiretroviral treatment included ritonavir in four cases, indinavir in four, nelfinavir in three, and saquinavir hard-gel in two. at the close of -month follow-up of atorvastatin therapy, a decrease of total cholesterol level of . % versus respective baseline value was observed; eight out of patients reached normal values for cholesterol. mild gastroenteric symptoms were found in only one of the treated patients, while no skeletal muscle and liver toxicity has been observed. discussion: in our study, pharmacological treatment with atorvastatin proved certainly effective in the management of diet-resistant hypercholesterolemia, and was associated with a favourable tolerability and adherence profile. the effect of combination antiretroviral therapy regimens on hiv- proviral dna level in peripheral blood mononuclear cells (pbmcs) was examined in hiv- -positive patients, using endpoint dilution pcr and serially cloning and sequencing of the gag region of hiv- . the major clone was defined as the most numerous of analyzed clones, and observation periods ranged from to months (mean, . / . months). in five patients (one with primary-stage hiv- infection) receiving three antiretroviral drugs, hiv- rna levels reduced to undetectable (i.e. b/ copies/ml). hiv- proviral dna levels and the number of major clones reduced in four of these patients. hiv- rna levels reduced, but remained detectable, in five other patients. in the two remaining patients (both receiving two rather than three antiretroviral drugs) hiv- rna levels increased. these results suggested that the population of the major clones may be affected when hiv- rna levels reduce following combination regimens of antiretroviral therapy. saquinavir hard gel (shg) as a part of a spontaneous Á/ -month deintensification anti-hiv regimen following successful highly active antiretroviral therapy (haart) pm manfredi r, calza l, chiodo f. infectious diseases, university of bologna, bologna, italy the induction-maintenance concept was poorly studied in hiv'/ patients (p), and shg was never assessed after prolonged response to potent protease inhibitors (pi)-based haart. shg-naïve p who refused indinavir, ritonavir, or nelfinavir-based haart after achieving long-term viral suppression, and resorted to shg'/ nucleoside analogues (na), were followed prospectively. in . % of the p assessed for Á/ months, ]/ na was changed. prior haart was interrupted after . / . months, due to adverse events ( p), or p's request ( p), while a viremia b/ copies/ml was present since . / . months. a viremia of Á/ hiv-rna copies/ml was maintained in p ( . %), while a higher viral load occurred in p after . / . months, and was related to a pre-haart viremia !/ / ml, a more frequent !/ % recovery of cd count, mutations of codons Á/ , and failure to change na (p b/ . Á/ . ). a cd drop !/ %/ cells/ml was found after . / . months in only eight p, who also had virologic failure: immunologic deterioration was earlier and deeper when na were not changed (p b/ . ). all the p who introduced shg'/novel na after a successful !/ -month induction with a potent pi-based haart had a stable Á/ -month outcome. a suboptimal haart including the less effective but better tolerated shg may be effective for !/ year, especially when novel na are introduced, and specific mutations are absent. despite a lower potency, drugs with a good safety and compliance profile may be recovered for simplified regimens. objective: to evaluate efficacy of antiretroviral therapy (art) with two or three drugs in the nervous 'reservoir'. patients: thirteen acute neurological and art naive aids patients underwent a paired and simultaneous sample from plasma and cerebrospinal fluid (csf) for a quantitative detection of hiv- rna (amplicor roche) before art. all patients underwent a ct and/or mr of the brain to perform a diagnosis. all of them had an hiv related neurological acute inflammatory disease. after diagnosis all patients received art: / received two nrti and / received haart including two nrti and one protease inhibitor. all patients underwent a paired and simultaneous follow-up from plasma and csf during the nd month of treatment. results: in all patients baseline levels of hiv-rna were higher (p b/ . ) in the plasma (log . '/ . ) than in the csf (log . '/ . ). the / patients who received dual therapy had undetectable levels (cut-off copies/ml) of viral rna at the follow-up in csf, but not in plasma: three of these seven patients had a detectable plasma hiv- rna. all / patients with haart had undetectable hiv- rna both in plasma and in csf at the follow up. conclusions: dual nrti therapy is rapidly effective in csf (because of an high penetration of drugs through a more permeable blood Á/brain barrier and lower hiv rna baseline levels) but not in plasma. haart is rapidly and equally effective both in csf and plasma. there are no reports of fulminant and fatal hepatic failure after the start of highly active antiretroviral therapy (haart) in an hiv subject without chronic viral hepatitis. case report: a -year-old naive aids woman with clinical symptoms due by a pcp was observed. baseline alt was increased ( . m.n.v.) because of a mild hepatosteatosis and a silent cholelithiasis. serology for hbv, hdv and hcv was negative; igg anti-hav, anti-ebv, anti-cmv and anti-hsv were present. hiv- rna was . log , cd '/ count was /ml. during the pcp treatment with cotrimoxazole alt values increased ( !/ m.n.v.); nevertheless, she completed the treatment. liver enzymes returned to the pre-treatment values over several days. then she started haart with stavudine, lamivudine and efavirenz. after days the patient showed an efavirenz-related skin rush that resolved within days, without treatment discontinuation. fourteen days after the start of haart jaundice appeared. laboratory revealed severe alt increase ( !/ m.n.v.) and hyperbilirubinemia ( mg/dl) and she died because of an acute liver failure syndrome within few days. an haart efavirenzbased regimen can result highly hepatotoxic when given in presence of a hepatosteatosis, of a recent hepatotoxicity caused by a nonantiretroviral treatment and of a previous idiosyncratic reaction to efavirenz. our experience with bulgarian herbal extracts for improving the general condition of hiv-positive patients pm methods: we used a combination of bulgarian herbal extracts and treated six patients, divided in two groups: three with symptomatic and three with asymptomatic hiv-infection. the all three patients with asymptomatic hiv-infection were treated only with herbal extracts, another three patients with symptomatic hiv-infection were treated with combination of herbal extracts and anti-retroviral therapy. the general status of patients has been evaluated by both subjective and objective surveillance. the immunologic monitoring has been performed by absolute count of cd '/ lymphocytes. results: all patients have shown an obvious improvement in their general condition: high spirit and working capacity, good appetite and sleep, a restoration of body weight. the number of cd '/ lymphocytes has been lightly increased or constant. conclusion: the combination of bulgarian herbal extracts has shown significant positive effect on the general condition and improve the quality of life. antifungal activity of in vitro and in vivo combinations of voriconazole with -fluorocytosine and amphotericin b against candida and cryptococcus spp pm hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: the present study was designed to determine whether the activity of voriconazole (vor), a novel triazole, was reduced against candidal and cryptococcal infections by the addition of standard antifungal agents, amphotericin b (amb) and -fluorocytosine ( -fc). vor was tested in combination with standard antifungal agents both in vitro, using a checkerboard mic determination test, and in vivo in immune normal guinea pig models of fungal infections. the results indicate that the efficacy of vor against candida albicans and c. neoformans was not antagonised by amb or -fc in vitro. furthermore, in guinea pig models of systemic candidiasis and intracranial cryptococcosis, no antagonism was observed between the lower doses of vor and either amb or -fc on the basis of reductions in tissue fungal loads. at the highest doses of vor, both amb and -fc showed some antagonism, but the combinations were still effective in significantly reducing fungal tissue loads compared with vehicle-treated control animals. conclusion: these results suggest that vor may be used in combination with standard antifungal agents, and future studies to elucidate the clinical potential of vor combination therapies in the management of candida and cryptococcus infections are warranted. itraconazole in the treatment of pityriasis versicolor pm tiodorovic j, jovanovic d, binic i, nikolic lj. faculty of medicine, clinic of dermatovenerology, nis, serbia, yugoslavia a comparison of two short-term dose schedules with itraconazole was carried out in patients with pityriasis versicolor. the patients were divided in two groups. each group consisted of patients who completed the therapy and controls. the clinical diagnosis was confirmed mycologically, by direct microscopic examination. the first group received mg of itraconazole daily for days. the second group received mg daily for days. the patients were controlled clinically and mycologically and days after the initiation of treatment. erythema, scaling and pruritus was evaluated clinically. clinically and mycologically cured patients accepted as cured. the cure rate were % in the first group and % in the second group at day . the effects of these two groups are similar. none of the patients reported side-effects. fungal urinary infections: emerging species, antifungal susceptibility trends and antibody response pm badawi he a , kamel ai b , fam ns a , el-sayed me a , elian sae c . a theodor bilharz medical research institute (tbmri ), microbiology, giza, egypt , b theodor bilharz medical research institute (tbmri ), urosurgery, giza, egypt , c faculty of medicine, medical microbiology and immunology, cairo university, cairo, egypt objectives: to assess the role of candida species in patients with urinary tract infections (utis) with or without schistosomiasis and/or cancer bladder, to compare chromogenic; chromagar (cma), biggy agar; morphologic (corn meal, rice agar-tween ) media and biochemical candifast test for identification of candida species. susceptibility to antifungal agents using e -test and candifast and the performance of elisa test for detection of anticandida antibodies (igm and igg) in serum were evaluated. results: c. albicans was the most frequent ( . %) species responsible for fungal utis. however, non-albicans species, c. glabrata ( . %), c. tropicalis ( %) and c. krusei ( . %) were also isolated. rice agar-tween was found to be cheap, available and sufficient to make a final identification ( %). cma could not identify c. glabrata . biggy agar could not adequately differentiate candida species. candifast biochemical identification showed low sensitivity of . %. e -test on sabouraud dextrose agar (sda) is simple method for mics determination and could detect s-dd strains in case of azoles. conclusion: the emergence of non-albicans species such as c. glabrata , c. tropicalis and c. krusei have contributed to complicated utis. this necessitates accurate isolation and identification of candida to the species level. morphology on rice agar-tween and antifungal susceptibility using e -test on sda is a simle rapid scheme for routine identification of clinically important yeasts. purpose: classification of allergic fungal rhinosinusitis (afr) is based on the immunologic relationship of the host to the fungus. afr must be differentiated from other fungal rhinosinusitis infections, which include acute invasive, chronic invasive, fungal balls and saprophytic colonization. although many cases of fungal rhinosinusitis is caused by species of aspergillis , dermatiaceous moulds have become an emerging pathogen in immunocompetent individuals. results: our case study involved a year male suffering from facial pain, headache, postnasal drip and loss of smell. he was hiv negative and a nonsmoker. the following laboratory tests were performed: ige- . ( . Á/ . ) iu/ml iga- . ( . Á/ . ) g/l igm- . ( . Á/ . ) g/l allergen specific ige for alternaria */ . ( . Á/ . ) ku/l fbc-normal except eosinophils slightly raised . ( . Á/ . ))/ / l. ct scans indicated fungus proliferation, bone erosion and extension of disease into adjacent anatomic area. sinus tissue following debridement was sent for microscopy and culture. hyphae was microscopically observed and cultures yielded two dermatiaceous fungi, bipolaris spp and alternaria spp . conclusion: it is important to differentiate these two species from curvularia , helminthosporum , drechelria and exserohilum . knowledge of these dermatiaceous fungi is important in directing appropriate antifungal therapy and selecting the correct antigens for postsurgical immunotherapy after initial debridement and irrigation. antifungal activity of in vitro and in vivo combinations of voriconazole with -fluorocytosine and amphotericin b against aspergillus fumigatus pm hitchcock ca, andrews rj, lewis bgh, pye gw, oliver gp, troke pf. pfizer global research and development, department of discovery biology, sandwich, uk purpose: a key requisite for a new antifungal drug is to demonstrate that it is devoid of significant antagonism in combination with other agents. combinations of the new triazole, voriconazole (vor), and standard antifungal agents ( -fluorocytosine or amphotericin b; -fc or amb) were tested against aspergillus fumigatus in vitro and in guinea pig models of infections to confirm that antifungal activity was not antagonised by using combination therapies. vor was studied in combination with amb or -fc in vitro, using a checkerboard mic determination test, and in vivo, using immune normal and immunocompromised guinea pig models of systemic aspergillosis. results: the results indicate that the potency of vor was not antagonised by amb or -fc in vivo; indeed, at lower concentrations of vor, significant improvements in reducing fungal burden in both in vivo models were achieved by the addition of amb. in vitro, no antagonism was found between vor and amb, although -fc had a significant antagonistic effect on vor activity. conclusion: these results from in vitro and in vivo models of aspergillosis suggest that vor may be used in combination with standard antifungal agents and, therefore, justify further examinations of vor combination therapies in a clinical setting. in vitro activity of caspofungin compared to that of amphotericin b, fluconazole, and itraconazole against candida species pm arikan s, sancak b, hascelik g. department of microbiology and clinical microbiology, hacettepe university medical school, ankara, turkey purpose: to evaluate the in vitro activity of caspofungin against various candida spp. and particularly against isolates with decreased amphotericin b, fluconazole, and itraconazole susceptibilities. methods: susceptibility tests were done by nccls m a microdilution guidelines for clinical candida strains. the mics (mg/ml) were read at and h. results: caspofungin mics at h are shown in the table. mics at h were similar to h readings. expectedly, no evidence of crossresistance was detected between caspofungin and other drugs tested. caspofungin was similarly active against fluconazole-or itraconazolesusceptible and resistant isolates. conclusions: ( ) caspofungin is active in vitro against all candida spp. tested. ( ) caspofungin mics are slightly higher for c. parapsilosis compared to other species. ( ) its activity against fluconazole-and itraconazole-resistant isolates is noteworthy. ( ) validation of these data require clinical investigations. oropharyngeal microbiological samples of bmt patients were evaluated. weekly cultures (days (/ , and '/ ) revealed presence of fungi in patients ( . %): in four ( %) patients before bmt only, in ( %) after bmt only, and in ( . %) both before and after bmt. in one patient candida norvegensis was isolated from the throat, buccal and palatal surfaces. three c. albicans , two c. krusei , and three c. norvegensis from four patients were chosen to comper their antifungal sensitivities and extracellular virulence factors. using fungitest method we determined the sensitivities of these isolates to flucytosine, amphotericin b, miconazole, ketoconazole and fluconazole. the fluconazole sensitivities were also determined by the e -test. on the basis of the fungitest data the three c. norvegensis isolates were sensitive to flucytosine, amphotericin b, and also to ketoconazole. in case of fluconazole and miconazole they proved to be susceptible dependent upon dose. the mic fluconazole values determined by the e -test for the three c. norvegensis isolates were , ]/ and ]/ mg/ml, respectively. the oropharyngeal isolates of c. norvegensis produced high amounts of extracellular aspartic protease and phospholipase similarly to c. albicans strains. these enzymes may contribute to the pathogenesis of this new emerging candida species. in vitro activities of antifungal and antiseptic agents against rhodotorula sp pm preney l, théraud m, guiguen c, gangneux jp. laboratory parasitologie-mycologie, chu de rennes, france purpose of the study: rhodotorula species are common saprophyte yeasts widespread in nature. since the last years, they have been implicated in several severe infections, especially in immunocompromised patients, and various antifungal therapies were used. however, only limited data are available on the susceptibility of rhodotorula sp. to antifungal and antiseptic agents. material and methods: in this work, we evaluated the in vitro activities of eight antifungal agents against strains of rhodotorula ( strains of r. rubra and nine strains of r. glutinis using atb fungus system (biomerieux) and etest strips (ab-biodisk). beside, the effect of eight antiseptic agents was assessed on a suspension of r. rubra . the quantification of yeasts after exposure to antiseptic agents was performed by subculturings using a microtitration method in well plates. results and discussion: all strains tested were susceptible to amphotericin b, fc, and nystatin. twenty-nine out of strains were susceptible to ketoconazole, out of were intermediate to econazole. all strains were resistant to fluconazole (cmi!/ mg/ml) and itraconazole (cmi!/ mg/ml), and out of were resistant to miconazole, suggesting that antifungal therapy must be adapted when rhodotorula yeasts are implicated in invasive infection. beside, min exposure to sodium hypochlorite , chlorhexidine . % or ecodiol (isopropyl alcohol'/alkylamin) showed fungicidal activities. susceptibility testing of aspergillus fumigatus and emerging aspergillus pathogens by a modification of the nccls m -p method pm logotheti m a , kapsanaki-gotsi e b , velegraki a a , zagoura d b . a department of microbiology, mycology reference laboratory, medical school, university of athens, athens, greece , b biology department, section ecology and systematics, university of athens, athens, greece aspergillosis in high risk groups of patients is still associated with high mortality rate ( Á/ %). aspergillus fumigatus is the primary pathogen, while other opportunistic aspergillus species are emerging. amphotericin b (ab), itraconazole (it), voriconazole (vo) and terbinafine (te) minimum inhibitory concentrations (mic) were determined by modifying the nccls m -p microdilution method. stock drug solutions were prepared in rpmi , dimethyl sulfoxide (dmso), and polyethylene glycol (peg ). inocula, of the a. fumigatus group ( ), a. flavus group ( ) ( ) and the m -p quality control strains were prepared according to, and by modifying, the nccls guidelines. plates were incubated at and c and read at and h. peg effectively dissolved it and vo, while either dmso or peg dissolved te. low and c Á/ h ab, it, vo and te mics ( . Á/ . mg/l) were recorded. a. terreus ( ) and a. parasiticus ( ) were resistant to ab. certain clinical isolates demonstrate clinical and in vitro resistance. standardization of susceptibility testing would offer reliable assistance in selecting and monitoring antifungal therapy. otag f, aslan, g, ozturk c. microbiology department, faculty of medicine, mersin university, mersin, turkey rates of opportunistic fungal infections have risen markedly. because some of these species have potential resistance to antifungal agents, rapid presumptive species level identification is crucial in allowing for directed antifungal therapy. in this study, isolated yeasts from the clinical specimens were identified by atb id c (biomerieux, france). the number of identified yeasts were, respectively; ( %) candida albicans , six ( %) c. glabrata , five ( . %) c. tropicalis , three ( . %) c. parapsilosis , two ( . %) c. krusei , two ( . %) c. kefyr , one ( . %) c. guillermondii , one ( . %) c. dubliniensis . twenty-one of strains were investigated for antifungal sensitivities by atb fungus kit (biomerieux, france). the results are as follows: % sensitivity was detected to myconasol, % to flusitozin, nystatin and econasol, % to amphtericin b and ketokonazol. it is important to achieve empirik treatment of the opportunistic candida infections and the following of resistance to antifungals. shakhmatov da, strelchenco, ov. novosibirsk state medical academy, dermatovenerology, novosibirsk, russian federation at the present stage in russia with a background of a high case rate of syphilis, it becomes necessary to exclude biological false positive serological tests. because the serodiagnosis of syphilis has significant limitations, the direct detection of t. pallidum in suspect blood may serve as an alternate diagnostic strategy. polymerase chain reaction (pcr) has been the most widely used amplification method. the study of patients receiving examination related and treatment for syphilis in std clinic and persons directed from other hospitals where routine serologic examination revealed doubtful results. pcr reaction was carried out with nested primer pairs based on the dna sequence of the Á/ and Á/ kda gene of t. pallidum . pcr was utilized with whole blood. a complex of serological tests: fta-abs and tit was used as the &rdqup; gold standard''. as a result the sensitivity of pcr was . % and specificity was . %. selective comparison of pcr results with vdrl, the fta-abs and treponemal immobilisation test (tit) has shown concurrence . %. in conclusion, the preliminary results of pcr in whole blood in syphilis detection revealed its high sensitivity and specificity; possibility to obtain rapid results in unclear cases. chlamydia pneumoniae (cp) is an atypical pathogen whit intracellular location, whose eradication is very difficult. in the past years it has been objects of many studies that lead to the demonstration of a relationship between its presence and the development of widespread multifactorial pathologies such as atherosclerosis and asthma. the lack of its eradication can become an important clinical and social problem. the study objective is the comprehension of pathogen Á/host interaction mechanism, to characterize therapeutics protocols that cold lead to complete eradication of cp from organism. the research had been principally made on the studying the molecular mechanisms that are at the root of pathogen permanence inside host cell. using proliferation and apoptosis tests we underlined a different behaviour of infected cells towards control cells. in presence of p ( mg/ml), i.e. a peptide that can inhibit the proliferation and induce apoptosis in vitro inhibiting nf-kb, uninfected cells proliferation decreased of % in comparison whit the controls, while the one of infected decreased only of about %. moreover, using various apoptosis-inducers, the infected cells showing apoptosis were about % while the uninfected were about %. the caspace iii activity increased significantly in uninfected cells. in conclusion, cp could delay its elimination from the host inhibiting the apoptosis via nf-kb activation. hryniewiecki t a , gzyl a b , rawczynska-englert i a , a department of acquired valvular heart disease, national institute of cardiology, warsaw, poland , b department of sera and vaccines, national institute of hygiene, warsaw, poland infective endocarditis (ie) frequently causes problems in diagnosis, especially where blood cultures are negative and with fungal etiology (also as a fungal superinfection in bacterial ie). the purpose of the study: the purpose of the study was to evaluate the usefulness of broad-range fungal pcr in diagnosis of fungal superinfection of bacterial ie. twenty-five blood samples were taken for analysis from patients with infective endocarditis. ie was diagnosed according to duke criteria including positive blood cultures. suspicion of fungal superinfection was established on serological investigation in five patients, confirmed by blood culture in two patients. control group consisted of patients without infection. dna was isolated using the commercially available s.n.a.p. kit. amplification products were analyzed by gel electrophoresis stained with ethidium bromide. the results obtained: fungal dna was found in two patients with fungal superinfection of bacterial ie confirmed by culture. in the remaining patients with ie and controls no fungal dna was found. the conclusion reached: broad-range fungal pcr is a fast and inexpensive tool for the detection of fungal dna, but it is more prone to contamination than species-specific pcr. the method may be valuable in the identification of fungal superinfection of bacterial ie or diagnosis of fungal ie. rivanera d, lilli d, lozzi ma, piunno m, mancini c. microbiology, science and public health, rome, italy aim: the aim of this study was to evaluate the eia method for detection of antibody to ttv virus (ttv) and to investigate the anti-tt virus prevalence in patients with hepatitis b (hbv) virus, hepatitis c (hcv) virus, in group of 'high risk'subjects to hepatitis and in healthy subjects. the elisa methods (nuclear laser vienna lab) using ttv s and ns antigens: orf ( aa) and orf ( aa) was applied to detect anti-ttv; the serological screening was performed from samples to italian subjects. results: the positive rates of anti-ttv antibodies were . % in patients with hepatitis b Á/c and . % in 'high risk' hepatitis patients. the anti-ttv was also found in . % in healthy people. conclusions: the anti-ttv were detected in all groups studied, however, its positive rate was similar in patients with hepatitis b Á/c and in 'high risk' hepatitis respect to heathly people. our results shown that tt virus is frequent in italy both in patients infected by others transmitted viruses and in general population. the positivity found in healthy adults included in our studies suggests that the virus might be transmitted non-parenterally. the study of pattern of antibody to ttv may be an infectious marker of ttv similar to that of anti-hcv. a stress test on a miniaturized identification system designed for neisseria and haemophilus pm rich m a , bannatyne rm a , memish za b . a king fahad national guard hospital, division of microbiology, riyadh, saudi arabia , b king fahad national guard hospital, infection prevention and control, riyadh, saudi arabia we report an incident that occurred in our laboratory when the bbl crystal identification system for neisseria and haemophilus was used to identify a haemophilus-like-organism. the numerical profile generated was not in the system database. conventional biochemical tests subsequently revealed an identification of brucella melitensis , a common isolate in our area. as a result of this revelation we subjected this system to a mini 'stress-test' with a collection of isolates of b. melitensis . two numerical profiles were obtained, and , neither of which are listed in the system database. brucella species have been misidentified as moraxella species, moraxella phenylpyruvica , and as haemophilus influenzae biotype iv in various identification systems. two cases of laboratory-acquired brucellosis have been attributed to misidentification. to its credit the bbl crystal identification system for neisseria and haemophilus neither generates a profile number with a misidentified organism nor assigns a confidence level. instead it properly directs the user to resort to conventional methods to secure an identification. if further studies on additional brucella isolates and strains from different geographical sources confirm the unique biochemical profiles identified here, it may be worthwhile to incorporate these into the database where they would be of considerable assistance in areas where brucellosis is widespread. cloning and characterization of aflmp in aspergillus flavus pm chong tk, woo pcy, leung asp, yuen ky. the university of hong kong, microbiology, hong kong, hong kong purpose of the study: to clone and characterize an antigenic protein for serodiagnosis of infection caused by aspergillus flavus which is the commonest aspergillus species causing aspergilloma (ao) and invasive aspergillosis (ia) in asia. result obtained: we cloned the aflmp gene, which encodes the first antigenic cell wall protein in a. flavus . aflmp codes for a protein, aflmp p, of amino acid residues, with sequence features that are present in mp p and afmp p, the antigenic cell wall mannoprotein in penicillium marneffei and aspergillus fumigatus that we described previously. it contains a serine-and threonine-rich region for o glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. specific anti-aflmp p antibody was generated with recombinant aflmp p protein purified from escherichia coli to allow further characterization of aflmp p. indirect immunofluorescent staining indicated that aflmp p is present in the cell walls of the hyphae and conidia of a. flavus . furthermore, it was observed that patients with ao and ia due to a. flavus develop a specific antibody response against aflmp p. conclusion reached: this suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with ao or ia, and the protein may represent a good cell surface target for host humoral immunitiy. grape purpose: to investigate the basis for increasing resistance to trimethoprim and sulphamethoxazole. methods: pcr screening for integrons of clinical urinary tract isolates was performed. isolates were tested for resistance to antibiotics. integrons in isolates were sequenced. results: integrons of class were found in isolates and class integrons were found in . eight isolates in the study were resistant to five antibiotics or more and not shown to carry any integron. nineteen of isolates resistant to trimethoprim did not carry integrons. only one of these isolates was shown to carry sul and is thus probably also carrying an integron. none of the isolates were shown to carry dfr , one of five trimethoprim resistance genes known to exist outside integrons. three isolates were resistant to sulphonamides but were not shown to carry neither sul nor sul . only dfr and aad gene cassettes were found in the sequenced integrons. conclusions: resistance to trimethoprim in of trimethoprim resistant isolates is mediated by genes not detectable, as in the case with three sulphonamide resistant isolates. sequenced integrons that contain dfr genes do not carry any gene cassettes mediating resistance to modern antibiotics. unusual diagnosis tool for an unusual presentation of alveolar echinococcosis: report of two cases of local progression after an animal bite pm bardonnet k, bart jm, loiseau j, gérard a, estavoyer jm, heyd b, badet jm, dubiez a, piarroux r, bresson-hadni s. who collaborating centre for prevention and treatment of human echinococcosis, university of franche-comté, besançon, france introduction: the classical human contamination route for alveolar echinococcosis (ae) is ingestion of eggs. two exceptional human cases are reported with extensive local evolution of ae after a bite. case no. : between and , a patient underwent surgery seven times for a muscle growing tumour which developed after a bite. the diagnosis of muscle ae was assessed on histopathological examination. in , serological tests were in accordance with echinococcus sp infection. case no. : in , a man presented 'cat-scratch fever' with a right supraclavicular tumefaction following a cat bite. between and , five recurrences occurred. different surgical explorations indicated multiple abscesses of the cervical muscles. in , serological tests were in favour of echinococcus sp infection and the pathologist described a parasitic wall suggesting hydatidosis, but specific pcr from histological samples prompted the diagnosis of ae. conclusion: in these exceptional observations, the liver which is the most usual location of ae was lesion-free. the chronic inflammatory ae lesions have developed in the local lymphatic chain area of the bite site. to perform diagnosis in these very unusual forms of ae, it is necessary to add unusual tests such as specific pcr to classical tests. antibiotic resistance in foodborne salmonella is an emerging public health concern. integrons are now recognized as the main genetic vehicles of antibiotic resistance in gram-negative bacteria, including in salmonella . the purpose of the present study was to investigate the presence of class i integrons in resistant isolates of several serotypes of salmonella isolated from poultry products and to determine their association with multidrug-resistance phenotypes. a total of isolates of salmonella belonging to seven different serotypes were tested. the most frequent multiresistant phenotype, found alone or together with other resistances, was to streptomycin and tetracycline. all but seven were resistant to three or more antimicrobial agents, including quinolones and amoxicillin. pcr analysis with the ?cs and ?cs primers detected the presence of class i integrons of . kb in one isolate, with the multiresistant phenotype: amoxicillin, chloramphenicol, streptomycin, trimethoprim-sulphametoxazol and tetracycline. our findings suggest that the uncontrolled use of the antimicrobial agents in food animals may have contributed to the development of the pattern of resistance observed in salmonella isolates. also the presence of integrons in low prevalent human salmonella serotypes but associated with food animals underscores the public health problem of antibiotic resistance acquisition and spread. prevalence and antimicrobial resistance of campylobacter jejuni and c. coli isolated from broilers and pigs in france pm avrain l a , humbert f b , sanders p c , kempf i a . a afssa, umb, ploufragan, france , b afssa, hqpap, ploufragan, france , c afssa, lermvd, fougères, france in , caeca from standard, export or free-range broilers and in , fecal samples from pigs, were collected in french slaughterhouses. prevalence of campylobacter jejuni and c. coli strains was . % in standard, . % in export and % in free-range broilers. in standard and export productions, the most often isolated species was c. jejuni , whereas c. coli was predominant in free-range production. . % samples collected from pigs contained c. coli . the sensitivity of strains to ampicillin, nalidixic acid, enrofloxacin (broilers) or ciprofloxacin (pigs), tetracycline, erythromycin and gentamicin was tested by an agar dilution method. in broilers, the percentages of resistant strains were, respectively , , , , . and % for c. jejuni and , , , , and % for c. coli . in pigs the percentages of resistant c. coli were, respectively , , , , and %. in broiler production, significant differences between distributions of species or percentages of resistant strains were observed according to type of production or administrated antimicrobials. the enzyme dhps (dihydropteroate synthase) participates in the folate synthesis pathway, and is well recognized as the target for sulphonamides. the enzyme preceding dhps in this pathway, pppk (dihydropterin pyrophosphokinase), is another interesting candidate drug target. the metabolic role of pppk is to provide one of the substrates for dhps. earlier studies have suggested that pppk and dhps enzymes need to have physical contact with each other for full enzyme activity. studies of potential interactions between the enzymes have been initiated. so far, indication of a weak interaction has been detected in gelfiltration experiments and the two-hybrid system. to confirm these results, we are currently developing a method to study substrate channeling, as interfering with such interactions could lead to impaired growth and thus be used as inhibitory drugs. we have also cloned and sequenced the operons coding for the enzymes in the folate biosynthesis from different clinical isolates of streptococcus pyogenes . comparisons revealed some isolates with a mosaic structure in the operon, suggesting that horizontal transfer of genetic material has occurred. multi-resistance gene cluster on a plasmid in a clinical isolate of e. faecium pm werner g, hildebrandt b, klare i, witte w. department of nosocomial infections, robert koch institute, wernigerode branch, germany purpose: strain uw was isolated from an urine sample of a patient with a permanent catheter. the purpose of our study was to identify and localize the resistance determinants in this isolate. results: isolate uw was resistant to the following antibiotics: penicillin, ampicillin, gentamicin (high-level), streptomycin (highlevel), erythromycin, clindamycin, vancomycin, teicoplanin, ciprofloxacin, moxifloxacin, nourseothricin, rifampicin, and fusidic acid (lowlevel, mic / mg/l); but showed susceptibilities to oxytetracycline, phosphomycin, chloramphenicol, trimethoprim/sulfamethoxazol, linezolid, and quinupristin/dalfopristin. hybridization, pcr and sequencing experiments localized a cluster consisting of several resistance genes in a composite element on a plasmid. the cluster included genes and transposons tn (vana) Á/tn (ermb) Á/tn (aade Á/ sat Á/apha- ). the plasmid itself was not transferable in filter-matings into a fusidic acid high-level resistant enterococcus faecium recipient while selecting either for erythromycin or vancomycin resistances. however, after transposing a tn -related determinant into uw , determinants became mobilizable with the help of the conjugative transposon. transconjugants were, besides others, high-level resistant to fusidic acid, but susceptible to penicillin and ampicillin. pfge of transconjugants demonstrated a pattern almost identical to the recipient but clearly different from the donor. conclusion: resistance genes in e. faecium could be arranged in a cluster and are mobile via mobilizable/transferable plasmids. lilli d, rivanera d, barbacini ig, lozzi ma, mancini c. department of science and public health, university la sapienza, microbiology, rome, italy aim: hepatitis g virus (hgv), a new rna virus that is parenterally trasmitted has frequentley been found in patients with chronic hepatitis c infection but its role in chronic liver desease is unknown. the aim of this study was to determine the prevalence of hgv infection in patients infected with hcv. ninety-eight patients infected with hcv were evaluated for the presence of hgv rna. the hcv genotypes distribution was genotype b, genotype a, genotype a and four genotype c/ d. hcv rna and hgv rna were detected by rt-nested pcr. results: infection with hepatitis g virus was detected in ( . %) patients and ( . %) were hgv rna negative. none of our patients with genotypes a and c/ d results hgv rna positive. prevalence of hgv infection was % in patients infected with hcv genotype b and . % with genotype a. conclusions: infection with hgv occurred frequently ( . %) in this sample of patients with chronic hepatitis c. we observed a height prevalence of hcv/hgv coinfection in patients infected with hcv genotype a. this association with hcv genotype a was indipendent of the source of infection, infact some of our patients have not history of intravenous drug use. characterization of extended-spectrum beta-lactamase (esbl)mediated resistance in salmonella spp. from durban, south africa pm moodley p a , essack s b , gajee k a , sturm w a . a department of medical microbiology, nelson r. mandela school of medicine, school of infection, university of natal, durban, south africa , b school of pharmacy and pharmacology, university of durban westville, durban, south africa background: gastroenteritis is a common condition among the paediatric population presenting to king edward viii hospital in durban, south africa. from july , we noticed that the susceptibility of the salmonella spp. isolated from stool samples among these children were resistant to multiple antibiotics. aim: to characterize the phenotype of the resistance mechanisms involved. methods: minimum inhibitory concentrations (mics) of ampicillin, azithromycin, ciprofloxacin, cefepime, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefoxitin, chloramphenicol, cotrimoxazole and gentamicin were determined by means of the agar dilution method. isolates were subjected to the e -test for extended-spectrum betalactamase (esbl) production. isoelectric focusing was performed as a preliminary step in enzyme characterization. results and conclusion: thirty isolates of multiresistant salmonella spp. were obtained. antibiogram typing revealed six different resistance phenotypes. all isolates depicted ceftazidime/ceftazidime Á/clavulanate ratio of !/ and were considered putative esbl-producers. isolates expressed Á/ beta-lactamases each with pi values ranging between and . indicative of tem-, shv-and/or ctx-m-related esbls. nine isolates expressed two beta-lactamases each and two isolates expressed three beta-lactamases each. there was evidence of the simultaneous expression of both tem-and shv-derived esbls as well as the simultaneous expression of multiple tem-or shv-derived esbls in single isolates, a phenomenon reported in esbl-positive klebsiella pneumoniae isolated at the same hospital. neutrophils exhibit reduced chemiluminescence response to serum opsonized klebsiella pneumoniae producing extended spectrum b-lactamases (esbl) pm objective: to investigate the ability of esbl and non-esblproducing klebsiella pneumoniae isolates treated with human serum to induce a chemiluminescence response in neutrophils. methods: oxidative burst induced by the interaction of esbl (n / ) and non-esbl-producing (n / ) klebsiella pneumoniae isolates with neutrophils from healthy individuals was monitored by measuring the chemiluminescence response (cl). pooled sera from healthy individuals served as source of complement for pretreatment of the bacteria. cl responses triggered by serum treated zymosan served as positive control. the serum opsonized klebsiella strains were arbitrarily graded as high (h) and low (l) inducers of cl when the cl response induced by the bacteria was cl b/ and !/ %, respectively, of that induced by opsonized zymosan. results: out of non-esbl-producing klebsiella isolates, . % induced high cl response in neutrophils whereas only % of esbl-producing klebsiella isolates did so (pb/ . ). conclusions: strains harboring the esbl plasmid were more virulent than non-esbl-producing strains by virtue of their higher tendency to escape serum-dependent recognition by neutrophils. osiris */an automated system for susceptibility testing in agar diffusion technique pm chegrani f, kolbert m, shah pm. universitätsklinik frankfurt, zentrum der inneren medizin med iii schwerpunkt infektiologie, frankfurt am main, germany objective: osiris measured zone sizes were compared to manually measured inhibition zones using round (rp) and mm mueller hinton square agarplates (sqp). variations of / mm in zone size measurements were defined as tolerable. 'very major errors' (vme) were defined as classification of a resistant organism as sensitive by osiris. thirty thousand two hundred and ninety-eight single measurements testing antibiotics on staphylococci and enterobacteriaceae were done according to the din recommendations. results: vancomycin, rifampicin, gentamicin gave the best results on rp with a concordance of , and %. vancomycin, rifampicin, teicoplanin performed best with , , % on sqp testing staphylococci . worst results on rp gave cefuroxim ( . %) and fosfomycin ( . %), on sqp fosfomycin ( . %), ofloxacin ( . %). for enterobacteriaceae amikacin ( %), gentamicin ( %), ciprofloxacin ( %) performed best on rp; worst nalidixinacid ( . %), piperacillin ( . %). concordance on sqp amikacin ( %), cefotaxim ( %), gentamicin ( %), nitrofurantoin ( . %), cotrimoxazol ( %). very major errors were seen in b/ % of all test performed. interpretation: osiris is a rapid and reliable system for susceptibility testing with round and square agarplates and has an excellent expert system. altindis m a , aktepe oc a , kocagoz t b . a kocatepe university school of medicine, microbiology, afyon, turkey , b diomed inc. tr, istanbul, turkey dio-bacit, in a two section plate, that contains % sheep blood agar on one side and sheep blood agar with bacitracin ( mg/ml) was compared for its efficiency in identification of group a beta hemolytic streptococcus (gabs) with other two different growth plates, one containing % sheep blood agar with bacitracin (b) and the other containing b-sxt. we used latex-agglutination for this comparision. throat specimens obtained from cases were inoculated to dio-bacit plates, first to one side with % sheep blood agar and to the other side with b. after an overnight inoculation at c, colonies with beta hemolysis an % sheep blood agar but no growth with b, were inoculated to % sheep blood agar again and antibiogram identification discs containing . u b and . and . mg sxt (oxoid, uk) were placed onto the plate and incubated overnight at c. after that, colonies with beta hemolysis were defined as b-sensitive while colonies resistant to sxt were defined to be gabs. all colonies are serologically classified by latex-agglutination (oxoid, uk). seventy-one ( . %) inoculations revealed growth of gabs at dio-bacit plates. after inoculating these colonies to % sheep blood agar, of them were found to be sensitive to b, while were found to be sensitive to b but resistant to sxt and of them were defined as gabs by latex test. when compared with latex-agglutination test, we found dio-bacit method's sensitivity and spesificity to be and . %, respectively. method: agar diffusion technique as recommended by din was used to determine izs (read using aura and manually) for staphylococci and enterobacteriaceae . variations in automated measured zone sizes of / mm to the manual readings were considered to be within acceptable range. results: six thousand and fifty-two zone sizes were determined for staphylococci and for enterobacteriaceae . mha displayed tendency to smaller zone sizes in automated readings than isa, as well in staphylococci and enterobacteriaceae . on the other side automated readings presented on isa more precise results than mha. overall less major discrepancies ( b/ mm) were found on isa. izs were generally smaller on mha. the tables below show differences in manually and automated measured zone sizes on different media and species. we discovered seven more cases of resistance in the case of metronidazole. we did not have such experience with clarithromycin. conclusion: our results show that e -test is comparable to ad for clarithromycin, but for metronidazole our findings confirm nccls recommendantion. classical ad is time consuming for every day use in the laboratory. the use of screening agar plate with mg/ml of metronidazole to detect possible resistance could be the solution. rokosz a a , sawicka-grzelak a a , meszaros j b , luczak m a . a department of medical microbiology, the university medical school, warsaw, poland , b department of bacteriology, state institute of hygiene, warsaw, poland purpose: to identify esbl-positive strains and to compare two methods applied for the detection of extended-spectrum beta-lactamases (esbls). methods: two hundred and sixty strains of gram-negative rods were cultured from clinical specimens from hospitalized patients. identification of strains was performed in the automatic atb system (biomerieux, france). these strains were identified as esbl-positive on the basis of the double-disc synergy test (ddst according to jarlier et al., ) results. all strains were also determined using a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: cpd (cefpodoxime) and cd (cefpodoxime/clavulanic acid) (oxoid, england). results: consistent results of two methods (ddst and dd) were obtained in the case of from among of examined strains ( . %). consistent results concerned out of strains of enteric rods ( . %) and only five from among other strains (mostly nonfermenting rods). conclusions: the novel method of esbl-producers detection (dd) is more objective and easier for interpretation than the double-disc synergy test (ddst). diagnostic disc test should be used as the basic one or to confirm the results of ddst in difficult cases. assessment of e -test for determining penicillin resistance in pneumococci pm sener b, yeniþehirli g, ercis s, hasçelik g. department of clinical microbiology, hacettepe university medical faculty, ankara, turkey there is a greater need for susceptibility testing methods that distinguish between susceptible and resistant pneumococci. an alternative method could be the e -test, which is compared with the reference agar dilution method in this study. penicillin susceptibility of a total of pneumococci was determined by e -test and agar dilution methods. streptococcus pneumoniae atcc and enterococcus faecalis atcc were used as controls. the results were given in the effect of anoxic conditions on the minimum inhibitory concentration of metronidazole in helicobacter pylori pm de la obra sanz p, lomas e, roman jl, alarcon t, lopez-brea m. the objective of this study was to determine the effect of incubation under anoxic conditions on the metronidazole resistance of helicobacter pylori . methods: a total of clinical isolates were used in this study. mics were determined by an agar dilution method using mueller-hinton agar plus % lysed horse blood. three plates series contained twofold dilutions of metronidazole from to . mg/l were prepared. the first one was incubated under microaerophilic conditions (oxoid) for days; second and third series were incubated anaerobically (anaerobic system, oxoid) for and h, respectively, and were then transferred to the microaerophilic enviroment up to complete days of incubation. results: with microaerophilic incubation, of strains were resistant (mic and mic were and , respectively). with h anaerobic preincubation, four of strains were resistant (mic and mic were . and , respectively). with h anaerobic preincubation, of strains was resistant (mic and mic were . and , respectively). conclusions: anaerobic preincubations causes an increase in sensitivity to metronidazole, the extent of which was dependent on the length of the anaerobic period. methods: the susceptibility to antibiotics was performed by microdilution method according to nccls guidelines. the production of b-lactamase was tested by nitrocefin sticks (oxoid). results: the mics /mics (mg/ml) appeared, respectively: ampicillin / , amoxicillin/clavulanic . / . , cefaclor / , ceftriazone . / . , erythromycin . / . , azithromycin / . / . , clarithromycin . / . , ciprofloxacin . / . , imipenem / . / . , tetracycline / . / / . , trimethoprim/sulfamethoxazole . / . . b-lactamase was detected in . % of the strains. conclusions: ( ) m. catarrhalis isolates were uniformly susceptible to all tested antimicrobials except ampicillin. ( ) the production of blactamase was responsible for ampicillin resistance ( . %). ( ) m. catarrhalis strains had almost the same behavior 'in vitro' to the tested microlides (erythromycin, azithromycin, clarythromycin). rokosz a, sawicka-grzelak a, luczak m. department of medical microbiology, the university medical school, warsaw, poland purpose: to isolate, identify and determine the drug-susceptibility of fungal strains cultured from fecal samples routinely submitted for detection of clostridium difficile and its toxins in cases of antibioticassociated diarrhea (aad). methods: one hundred fecal samples from hospitalized patients were examined (may Á/october ). c. difficile toxins a/b were detected directly in stools with c. difficile tox a/b ii test (techlab † , usa). fecal specimens were inoculated on ccca and candida id (biomerieux, france) media. c. difficile and fungi were identified with standard microbiological procedures. susceptibility of fungal strains to anti-fungal agents was determined (atb fungus, biomerieux, france). results: c. difficile toxins were detected in and c. difficile strains were isolated from of examined specimens. sixty-two fungal strains of genera were cultured from stool samples ( c. albicans isolates). massive fungal growths were observed on primary plates in all cases. fifty-five fungal strains were susceptible to nystatin, -to fluorocytosine, -to amphotericin b, -to ketoconazole, -to miconazole and -to econazole. conclusions: in some cases of antibiotic-associated diarrhea fungal strains are responsible for symptoms of this disease. certain persons having aad should be treated with anti-fungal agents. results: a total of isolates ( . %) were penicillin nonsusceptible (intermediate and resistant) and ( . %) were erythromycin non-susceptible. non-susceptibility to both antibiotics was found in ( . %). conclusions: if penicillin administration eliminates all penicillin susceptible strains, the prevalence of penicillin non-susceptible strains will increase as well as the erythromycin non-susceptible ones. this means that the proportion of erythromycin non-susceptible strains should increase from . to . %. at the same time, if erythromycin eliminate all susceptible strains to this antibiotic, the prevalence of penicillin non-susceptible strains would increase from the initial . to . %. these data can explain the co-selection results observed in different surveillance studies. antimicrobial susceptibility and capsular types/groups of streptococcus pneumoniae isolates causing pneumococcal diseases in bulgaria pm setchanova l a , gergova r a , ioneva m b , sredkova v c . a department of microbiology, medical university, sofia, bulgaria , b department of microbiology, ii city hospital-sofia, sofia, bulgaria , c department of microbiology, faculty of medicine, pleven, bulgaria a prospective study of pneumococcal infections was performed in cooperation with five clinical microbiology laboratories in bulgaria. mics values to antimicrobials and serotype/serogroup distribution were determined for strains of streptococcus pneumoniae . pneumococci were isolated from patients with systemic or respiratory infections. the incidence of penicillin g-intermediate and penicillin gresistant isolate was . and . %, respectively. the rates of resistance to other antimicrobials were: cefotaxime/ceftriaxone */ . %; erythromycin */ . %; clindamycin */ . %; tetracycline */ %; chloramphenicol */ . %; trimethoprim/sulfamothoxazole */ %; ciprofloxacin */ . %; rifampin */ . %. the s. pneumoniae isolates belonged to capsular types/groups. the most common serotypes/serogroups in bulgaria are , , , , , , we aimed to determine the pneumococcal antibiotic resistance rates and the serotypes of those resistant isolates in our hospital. the mic values of isolates (year Á/ ) were determined by agar dilution method. serotyping was performed by using pooled antisera of the pneumo-test. the results were as follows (n / ). objectives: to asses the antimicrobial susceptibility of clinical isolates of pseudomonas aeruginosa obtained from to and to monitor trends in antimicrobial resistance. methods: mics were determined by microdilution testing according to nccls. the antibiotics tested were: ceftazidime (caz), aztreonam (atm), imipenem (imp), gentamicin (cn), tobramycin (tb), amikacin (ak) and ciprofloxacin (cip). results: a total of isolates were included. urine was the most common site of isolation for outpatients isolates ( . %) while for hospitalized patients respiratory samples were the most frequent ( . %). susceptibility to antibiotics was: % caz, . % atm, % imp, . % cn, . % tb, . % ak and . % cip. comparison of susceptibility data through Á/ showed that the increase in the resistance rate was significative for caz ( . vs . %), atm ( . vs %), cn ( vs %), tb ( . vs . %), ak ( . vs . %) and cip ( . vs %). significant differences were found under the following circumstances: isolates from intensive care units and from inpatients were significatively more resistant to caz, atm and imp. isolates from respiratory samples were more resistant to caz and atm and isolates from urine samples were more resistant to cip. conclusions: although the antimicrobial susceptibility level has been decreasing p. aeruginosa isolates still show good susceptibility percentages for all antibiotics tested. antimicrobial susceptibility testing of clinical isolates of bordetella pertussis : report on isolates from rouen, france pm lemee l a , nouvellon m a , caron f b , lemeland jf a . a chu rouen, bacteriologie, rouen, france , b chu rouen, maladies infectieuses et tropicales, rouen, france reports of an increased clinical incidence of pertussis and the development of resistance by bordetella pertussis to erythromycin prompted the collection and antimicrobial susceptibility testing of recent clinical isolates from patients, who were hospitalized in rouen between and . mics of nine antimicrobial agents (erythromycin, josamycin, spiramycin, roxithromycin, ketolide hmr , cotrimoxazole, ciprofloxacin, rifampicin and amoxicillin) were measured by agar dilution method on mueller-hinton agar containing % horse blood. mbcs of erythromycin and rifampicin were also determined against four isolates of b. pertussis . all isolates were fully susceptible to the nine antimicrobial agents tested. mics (mcg/ml) were . for erythromycin, ketolide hmr and ciprofloxacin, . for josamycin, . for spiramycin, roxithromycin and rifampicin, . / . for cotrimoxazole, and for amoxicillin. mbcs (mcg/ml) were . Á/ . for eyrthromycin and . Á/ for rifampicin. in conclusion, our isolates of b. pertussis remain extremely susceptible to all antimicrobial agents tested, especially macrolides. no resistance was detected. finally, if erythromycin remains the molecule of choice, other macrolides (c and c ) also confirm their good in-vitro activity. in addition, the good in-vitro potency of rifampicin, together with its great diffusion within the respiratory tract, suggests that rifampicin has potential clinical efficacy in pertussis too. the emergence of streptococcus pneumoniae (sp) with diminished susceptibility to penicillin g (psdp) suggests the use of other antibiotics such as newer fluoroquinolones (fq). the resistance phenotypes of consecutive pneumococcal strains isolated from patients of four hospitals (observatoire régional des pneumocoques du nord-pas de calais) were studied: strains were susceptible to penicillin g and were psdp. reference strains provided from the centre national de réfeacute;rence des pneumocoques were added to the study. the activity of pefloxacin, ciprofloxacin, norfloxacin, sparfloxacin, levofloxacin and moxifloxacin was studied. reserpine was used to detect the efflux phenotype. methods used were performed according to the recommendations of the comité de l'antibiogramme de la société française de microbiologie. for each strain, the resistance phenotype to fq was deduced by comparison of mics or diameters obtained with those obtained with the reference strains of known phenotypes. fq resistance phenotypes were not correlated to blactam agent susceptibilities. wild type phenotype was observed among . and . % of the susceptible and psdp strains, respectively. a 'wild efflux' mechanism, deduced by addition of reserpine to norfloxacin, represented the predominant phenotype. it was detected among sp susceptible to penicillin g ( . %) as well as among psdp ( . %). the aim of this study was to determine macrolide resistance phenotypes of sp isolated in three french departments (alpes maritimes, doubs, nord) from nasopharyngeal aspirates of children aged months to years attending a dcc. a random sample of children attending randomly selected dccs was obtained during three periods (spring, autumn and winter ) in each department ( children attending dccs and children sampled). analysis of macrolide susceptibility of sp strains was performed using the ca-sfm method. out of strains, . % had decreased susceptibility to penicillin (spdp) and . % were resistant to erythromycin. the triple disk diffusion method (erythromycin (e), clindamycin (cl) and spiramycin) was used to determine resistance phenotypes. macrolide resistance is a well known phenomenon in france and is confirmed by our study. these results show that the constitutive phenotype is predominant as in other parts of europe and the frequency of efflux mechanism is lower than that observed in the usa and canada. developing antibiotic resistance surveillance of helicobacter pylori in england and wales pm elviss nc, owen rj. central public health laboratory, laboratory of enteric pathogens, london, uk purpose: helicobacter pylori antibiotic resistance is a key contributing factor in Â/ % of infected patients failing drug treatment. our aim was to survey rates of primary in-vitro resistance at different locations, and links to disease severity. antral gastric biopsies/cultures were received from phls in chelmsford, mid-essex ( isolates Á/ ); london ( isolates Á/ ); and bangor, north wales ( isolates Á/ ). susceptibilities to metronidazole (mtz), clarithromycin (cla), tetracycline (tet) and amoxicillin (amx) were tested by disc diffusion and also by e -test for cla and mtz. results: overall resistance rates ( isolates) were % for mtz and % for cla. all were susceptible to amx and tet. dual resistance rate was %. breakdown by location showed some marked differences. mtz resistance was highest in london ( %) compared to % in chelmsford and % in bangor. by contrast cla rates were % for london, and about % for bangor and chelmsford. in london, the majority of mtz resistant isolates were from non-uk borne individuals ( % non-uk vs % uk). comparison of duodenal ulcer-associated isolates with those from non-ulcer patients indicated similar rates of mtz resistance ( %). conclusion: resistance rates may vary significantly between locations depending on the local population with non-uk birth being a key risk factor for primary resistance with a mtz resistant strain. local resistance rates should be taken into account in test and treat strategies. potrykus j, benetkiewicz m, wegrzyn g. department of molecular biology, university of gdansk, gdansk, poland purpose of the study : because of their ability to extrude a wide range of compounds, multidrug efflux pumps have recently become an important issue in combating bacterial infections. acrab-tolc is the major efflux system of escherichia coli . we investigated the effect of acra on plasmid-borne and intrinsic chloramphenicol, tetracycline and ampicillin resistance. results and conclusions: recently, we reported a chloramphenicol sensitivity of e. coli mutant expressing cat , the chloramphenicol resistance gene. the strain was shown to bear a nonsense mutation in the acra gene. our studies indicate that this mutation is, at least in part, responsible for the observed chloramphenicol sensitivity phenotype. the mutation seems also to influence the strain's susceptibility to ampicillin and teta (c )-mediated (plasmid-borne) tetracycline resistance. although the teta(c) protein retained its biological function, there was a considerable growth impairment of the mutant strain when cultured in tetracycline containing medium. deletion of the acrab locus prevented any growth in the presence of tetracycline. upon the addition of ampicillin, the mutant underwent lysis more rapidly than the control strain. such was also observed in acrab deletion derivatives of other e. coli strains. we are trying to elucidate the role of the acra gene product in the phenomena described above. existence of efflux pumps in wild type isolates of drug-resistance bacteria pm raja ray rr. medical microbiology and parasitology, calcutta university, kolkata, india efflux pumps possessed by the bacterial cells of different kinds of bacteria had presented as a newer mode of drug resistance in many organisms. the capacity of bacterial cells to cause outward flow of noxious agents was known, however, for a considerable time with respect to tetracycline. recently, interest in the efflux pump system has brought to light some previously ill-understood mechanisms of drug resistance, involving noxious agents, toxins or poisons. we have found high level of resistance in pseudomonads towards cetrimide and other germicides for which no definite chromosomal/plasmid-mediated genes/mechanisms could be identified. likewise, occurrence of nonantibiotic sensitive vibrios, staphylococci and pseudomonads in the background of their high level of resistance to most of the common antibiotics suggest a mechanism of interference with the efflux pump, which accounts for such sensitivity in such cases. involvement of multiple resistance of marine isolates of v. parahaemolyticus to numerous clinically used antibiotics to which they have never been exposed also suggests a possible role of efflux pumps in determining such resistance */that these can simultaneously develop against multiple marine toxins/poisons and other noxious agents. interaction between oxacillin and glycopeptides in a teicoplanin-resistant mutant of staphylococcus epidermidis with reduced susceptibility to vancomycin pm greco aa, ben hassen a. laboratory service of national bone-marrow transplant center, tunis, tunisia we selected a laboratory-generated mutant of staphylococcus epidermidis capable of growing in the presence of mg/l of teicoplanin ( b tm ), from a methicillin-resistant (mic!/ mg/l), teicoplanin-sensitive (mic mg/l) and vancomycin-sensitive (mic mg/l) clinical isolate of s. epidermidis ( b so). in a previous work, we studied the different phenotypic characteristics acquired by the teicoplanin-resistant mutant b tm ( th interdisciplinary meeting on anti-infectious chemotherapy, december , poster sessions, /p ). in this work, we examined the interaction between oxacillin and glycopeptides against this teicoplanin-resistant mutant of s. epidermidis with reduced susceptibility to vancomycin. to study the combined antibiotic activity of oxacillin and glycopeptides, we used different methods: a modified disk diffusion test, the e -test, time-kill assays and population analysis profiles. the synergistic activity of glycopeptides in combination with oxacillin against the teicoplaninresistant mutant b tm was demonstrated with a bactericidal effect. no synergy was seen against the parental strain b so. moreover, the synergy between glycopeptides and oxacillin occurred with suppression of the subpopulation with the highest level of glycopeptides resistance. we concluded that combination of glycopeptides and oxacillin may be a possible alternative in the treatment of infections caused by methicillin-resistant, teicoplanin-resistant s. epidermidis . compositional changes in microcosm biofilms induced by application of minocycline: a preliminary study pm the aim of the study was to observe the effect of application of minocycline upon microcosm dental plaques. the plaques were cultivated in a constant departmenth film fermentor (cdff), which produces biofilms under conditions mimicking those present in vivo. the composition of the biofilms was determined by viable counting on selective and non-selective media. the proportion of antibiotic resistant genera within the biofilm was determined by viable counts utilising media containing minocycline ( mg/ml). before commencing antibiotic pulsing, the biofilms had a total viable anaerobic count of . )/ cfu per biofilm, with negligible ( cfu/biofilm) minocycline-resistant bacteria. however, h after introduction of the antibiotic, the total count had been reduced to . )/ cfu/biofilm whilst the number of minocycline-resistant bacteria had risen to . )/ cfu/biofilm. at the final sampling time point ( h) the total viable anaerobic count was . )/ cfu/biofilm whilst the number of minocycline-resistant bacteria was . )/ cfu/biofilm. hence, there is a very low basal level of inherent resistance to minocycline within microcosm dental plaques, but this increases considerably once the biofilms are exposed to minocycline. mechanism of resistance to aminoglycosides (amg) e. coli isolated from children with community-acquired urinary tract infections (cau-tis) pm methods: during the Á/ years nine centers took part in the study. the mics of antimicrobials were determined by the agar dilution method as described in the nccls guidelines. results: a total of consecutive urine isolates from children aged month to years with cauti were collected. the most frequently isolated species from children with cauti was e. coli ( . %), followed by klebsiella spp. ( . %) and proteus spp. ( . %). results of the in vitro susceptibility testing of e. coli to amg are shown in table below. resistance of the strains was conditioned on production of amg-modifying enzymes. there has been found following phenotypes among resistance strains: gentamicin Á/ tobramycin Á/netilmicin ( . % */aac( )-v and . % */aac( )-iv enzymes) and gentamicin Á/tobramycin ( . %, due to ant( ƒ) enzyme). conclusion: amikacin is most active amg against e. coli . resistance to gentamicin and netilmicin was mainly determined by production of aac( )-v enzyme. effect of b-lactamase inhibitors (b-l-i) on the evolution of resistance (r) to b-lactams (b-l) in gram the b-lactams antimicrobials still are the most frequently used. among the bacteria responsible of high resistance to b-lactams are gramnegative rods; its most frequent mechanism is the production of b-lactamase. the use of b-l-i has reversed partially this mechanism of resistance. we expect changes in the frequencies of r using b-lƒci after more than years. since , the venezuelan group of bacterial resistance, with health institution in the country; analyse and publish data on bacterial resistance of isolates from patients with bacterial infection coming from hospitals. it was used diffusion disk, according nccls. the software program whonet (world health organization net) was used. we follow the trends of r of gram-negative rods to b-l alone and with b-l-i during the decade Á/ . statistical analyses were made by evaluating the differences among percentages of resistance between the two series (p / . ). results and discussion: the difference in r between b-l and b-l/b-l-i are: ( ) piperacillin, piperacillin/tazobactam: between and % of r for most isolated, except for escherichia coli ( %) and serratia spp. ( %); ( ) ampicilin, ampicilin/sulbactam: between and %; ( ) cefoperazone, cefoperazone/sulbactam: between and %. how is expected gramnegative rods resistance to b-lactams with a b-l-i is lower than the b-lactam alone; furthermore the difference between both series, grows higher with time. these results are relevant and they were not expected, since b-l-i have been shown to be b-lactamase inductors. trends in the resistance (r) to b-lactams and others antimicrobials in p. aeruginosa in venezuelan medical centres. nosocomial (nos) and communitarian resistance pm in order to approach the infection produced by resistant bacteria, it is convenient to consider the hospital and the community as two separate ecosystems. the hospital ecosystem has special relevance in the infection and r of gramnegative aerobic bacilli. today, they are the main responsible of nos infection, with special reference to pseudomonas aeruginosa . infection by resistant bacteria is a world wide problem, specially related to nos. since , the venezuelan group of bacterial resistance, with health institution in the country; identify, analyse and publish data on bacterial r to antimicrobials: b-lactams, quinolones and aminoglicosides of isolates from patients with bacterial infection coming from hospitals and the community. it was used diffusion disk, according nccls. the software program whonet (world health organiza-tion net) was used. statistical significance (p / . ) was determined by application of the x technique. we show significant differences in the r of p. aeruginosa nos and communitarian (highest differences: piperacilina / %, tobra / %). we also established significant differences between the r arising in public hospitals and private hospitals (highest differences: ceftazidime / %, amika / %). we show the tendency in decreasing of frequency of r since ; this is more evident in private hospitals (b-lactam and aminoglicosides). new antimicrobials and new mechanism of action, and in the future the new technology will solve today's problem. however, the most important tools we have today are prevention and antimicrobials, and we must make them suitable. susceptibility to antibiotics of enterobacter cloacae and citrobacter freundii from drinking water pm quintera sm, sousa jc, peixe l. department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal the increased use of antimicrobials in farming, together with the practice of raw sewage discharge into receiving waters, has resulted in a significant increase in the number of antibiotic resistant bacteria present in aquatic environment. our objective was to determine the antimicrobial susceptibility, with focus on b-lactam resistance, among enterobacteriaceae strains isolated from raw drinking water samples. several isolates (n / ) of enterobacter cloacae and citrobacter freundii obtained from drinking waters were screened for antibiotic susceptibility patterns, using the agar diffusion technique, according to nccls's procedures. only % of e. cloacae strains, as well as % of c. freundii strains show resistance to amoxicillin and amoxicillin/ clavulanic acid. a reduced incidence of resistance to several others antibiotics was also observed. the obtained results suggest that strains isolated from raw drinking water have greater susceptibility to antimicrobial agents than pathogenic strains from hospital or outpatients infections. the 'natural' antimicrobial resistance phenotypes, usually described for c. freundii and e. cloacae , only seem to apply to strains isolated from human infections. notwithstanding the high susceptibility of the tested isolates to b-lactams, the role of environmental bacteria as a reservoir of resistance genes justify its periodical monitoring as a valid index for resistance spreading. a snapshot of the soil. using bacterial communities for tracing the evolution of metal-resistance pm quintera sm a , sousa jc a , peixe l a , monteiro nm b . a department of microbiology, faculty of pharmacy, university of oporto, oporto, portugal , b department of zoology and anthropology, faculty of sciences, university of oporto, oporto, portugal it is well known that pathogenic bacteria, specially those resistant to antimicrobial agents and heavy metals poses public health risks of great concern, and its detection, namely in soils is generally related to pollution. in this study, the heavy metal resistance patterns of the microflora isolated from polluted (dump area) and unpolluted soil environments were examined. the plate growth covering percentage in the soil samples was determined using mueller-hinton plates supplemented with different heavy metal (al '/, cd '/, cu '/, pb '/, hg '/ and zn '/) concentrations. parallelly, using icp-aes, it was possible to ascertain the real heavy metal concentration for each soil sample. we found that the percentage of plate growth covering from the used samples was closely linked to the level of chemical pollution measured for each location. moreover, using anova, we found significant differences between locations. the dump site showed the highest tolerance to all the tested metals (newman Á/keuls test). this pattern of results was consistent when using the data from the icp-aes. furthermore, it was possible to observe that pseudomonas spp., with a relatively high mic for the studied metals, might become a relevant model for both public health issues and eco-toxicological studies. biochemical characteristics of environmental isolates of listeria monocytogenes pm moshtaghi h a , garg sr b , mandokhot uv b . a shahrekord university, food hygiene, shahrekord, islamic republic of iran , b haryana agricultural university, food hygiene, hisar, india purpose: the investigations were carried out to study the biochemical reactions of listeria monocytogenes isolated from different sources in the environment. results: a total of isolates of l. monocytogenes were obtained from samples of agricultural soil, faecal matter of animals and sewage. all the isolates were gram-positive, small rods, catalase positive, oxidase negative, motile with tumbling motility in hanging drop at Á/ c, aerobic, facultative anaerobic, fermentative and produced acid from glucose. all the isolates of l. monocytogenes were beta haemolytic and positive for camp reaction with staphylococcus aureus . all the isolates were negative for phenyl alanine deaminase, ornithine decarboxylase, lysine decarboxylase, malonate utilization and beta galactosidase tests. these were also negative for acid production from arabinose, d-xylose, mannitol, soluble starch and sucrose but acid was produced in rhamnose, salicin, and trehalose. hydrogen sulfide production was recorded in tripticase soy broth with lead acetate paper strips but negative with triple sugar iron agar. all the isolates were found to hydrolyse aesculin. out of isolates of l. monocytogenes only two produced acid from lactose. in serotyping all the isolates were serotype b. conclusion: we can conclude that l. monocytogenes serotype b at least in fermentation of lactose shows different reactions. methods: the samples were pre-enriched in bhi broth with and without vancomycin ( mg/l) and then plated onto m-enterococcus agar with and without antibiotics: vancomycin ( mg/l), gentamicin ( mg/l), kanamycin ( mg/l), and streptomycin ( mg/l). representative colonies of each morphology were isolated and identified as enterococcus sp as previous described. pcr was used to identify e. faecium and e. faecalis and to characterise vancomycin resistant genotype. api strep was also used in the identification. susceptibility testing to antibiotics was performed by an agar dilution method (nccls). results: three hundred and fifty-three enterococci were isolated from of a total of faecal samples ( %, n / / ). the majority of enterococci were identified as e. faecium , e. faecalis and enterococcus sp. resistance to almost all antibiotics studied was observed: vancomycin */ . %; teicoplanin */ . ; ampicillin */ . %; tetracyclin */ . ; erythromycin */ . %; ciprofloxacin */ . %; chloramphenicol */ . %; gentamicin */ . %; streptomycin */ . %; kanamycin */ . %; linezolid */ %. the vancomycin resistant enterococci presented a vana genotype. conclusion: resistance to several common antibiotics used in therapy was observed among enterococci isolated from healthy human from community. many of these isolates presented multi-resistance. of concern is the presence of vana genotype among these populations that may constitute a reservoir of vancomycin resistant genes. antimicrobial resistance in tetracycline-resistant oral bacteria pm mercury release from dental amalgam may select for mercuryresistant oral bacteria. mercury resistance is often associated with multiple antibiotic resistances. the aims of this study were to determine whether tetracycline-resistant oral bacteria from children with and without amalgam fillings were also resistant to: (a) mercury; and (b) multiple antibiotics. tetracycline-resistant organisms were isolated on iso-sensitest/blood agar containing tetracycline ( mg/ml). the mic of hgcl and several antibiotics were determined using agar dilution (bsac). one hundred and three organisms were isolated from patients without amalgam. ninety-one were streptococcus species, seven neisseria species, three veillonella dispar and two rothia species. fifty-seven percent exhibited resistance to at least one antibiotic, % were mercury-resistant, % were penicillin-resistant, % were ampicillin-resistant and % erythromycin-resistant. fiftytwo organisms were isolated from patients with amalgam. forty-five were streptococcus species, five neisseria species, one v. dispar and one staphylococcus aureus . sixty-three percent exhibited resistance to at least one antibiotic, % were mercury-resistant, % penicillinresistant, % were ampicillin-resistant and % showed erythromycin-resistance. statistically, the results showed that in tetracyclineresistant organisms, the presence of dental amalgam did not affect the level of resistance to mercury or to the antibiotics tested. conway-wallace hl a , mullany p a , bedi r b , wilson m a . a eastman dental institute, university college london, microbiology, london, uk , b eastman dental institute, university college london, transcultural oral health, london, uk the purpose of this study: to determine the prevalence of antibioticresistant oral bacteria in children who had not received antibiotics during the months prior to sampling. plaque samples were obtained from children aged Á/ years and plated onto media containing: penicillin, ampicillin, tetracycline, erythromycin and vancomycin. resistant isolates were enumerated, sub-cultured and frozen for subsequent identification. the process was repeated and months later. the results obtained: bacteria resistant to each of the antibiotics were present in all of the children at each sampling time (except in the case of ampicillin and penicillin at months). the proportion of antibiotic-resistant bacteria in the oral microflora ranged from ]/ . (erythromycin) to / . % (ampicillin). the proportions of bacteria resistant to a particular antibiotic remained reasonably constant over the -month sampling period. in only two cases (penicillin and ampicillin) was there a statistically significant change in the proportions of resistant bacteria at different time periods. the conclusion reached: the results of the study have revealed that bacteria resistant to a wide range of antibiotics may be isolated from children who have not been administered these agents during the months prior to sampling. furthermore, in many cases the proportion of bacteria resistant to a particular antibiotic remains constant over a -month period. antimicrobial use in the intensive care unit: results of a pharmacoepidemiological study in italy pm periti p. e.i.f.t. srl, firenze, italy a retrospective survey of antimicrobial chemotherapy use in intensive care units in italy was carried out in using a computerized questionnaire under the auspices of the journal of chemotherapy. of the icus contacted, . % replied, being mainly general or post-surgical and pediatric units having a mean of beds, nine doctors and nurses. the antimicrobial agents used in these wards were almost always polychemotherapy with prevalent use of beta-lactams, aminoglycosides and glycopeptides or as empirical treatment during the first h after hospital admission. the continual use of medium-high dose combinational antimicrobial chemotherapy was justified by microbiological testing, which revealed that more than one-third of bacterial pathogens were resistant. approximately, % of gram-positive bacteria were methicillin-resistant, whereas about % of gram-negative strains were resistant to at least one of the tested antibiotics. forty percent of the responding icus furnished microbiological testing data, of which three quarters indicated the incidence of chemoresistance of the isolated strains. fungal infections were less frequent than bacterial, the most commonly isolated agent being candida spp. in conclusion, the sample of icus examined showed adequate and reasonable use of antimicrobial agents, with heavy reliance on medium-high dose combination therapy due to the elevated incidence of resistant isolates found. plasma concentrations (p), urinary excretion (u) and bactericidal activity of gatifloxacin (gat) mg versus ciprofloxacin (cip) mg in healthy volunteers after a single oral dose pm boy d a , kinzig-schippers m b , sö rgel f b , well naber kg a . a hospital st. elisabeth, urologic clinic, straubing, germany , b institute for biomedical and pharmaceutical research, ibmp, nürnberg-heroldsberg, germany twelve volunteers received a single oral dose of mg gat versus mg cip to assess p up to h, u (by hplc), and urinary bactericidal titers (ubt) in eight intervals up to h. the mean pmax of gat/cip was . / . mg. the ucum (mean) for gat/cip was . / . %. the ubts, i.e. the highest twofold dilution of urine still bactericidal, were determined for nine uropathogens and one reference strain */mics (mg/ml) (microdilution) for gat/cip: escherichia coli atcc ( . / . ); e. coli ( . / . ); klebsiella pneumoniae ( . / . ); proteus mirabilis ( . / . ); pseudomonas aeruginosa ( / . ); s. saprophyticus ( . / . ); two strains of s. aureus ( . / . ); two strains of e. faecalis ( . / and / ). the median ubts measured within the first h for gatifloxacin were between : and : for the five gram-negative strains (incl. p. aeruginosa ) and between : and : for the five gram-positive strains. the median ubts for ciprofloxacin were between : and : for the gramnegative strains (incl. p. aeruginosa ) and between : . and : for the five gram-positive strains. for the ubts up to h, gat was significantly superior to ciprofloxacin in all gram-positive strains, not different in the two e. coli strains, and inferior in the klebsiella , proteus and pseudomonas strains. for the ubts at Á/ h, gat was generally superior to cip, but showed no difference in the proteus and pseudomonas strains. gat showed overall comparable urinary bactericidal activity as cip. this is in agreement with a clinical study performed previously. malaria is one of the most prevalent endemic infectious disease affecting humans. in bichat hospital cases of malaria acute illness were reported during . among them, patients were hospitalised and intravenously treated by quinine. this retrospective study consisted of comparing the therapeutic drug monitoring (tdm) of quinine distinguishing, respectively and patients cured in infectious medical department (imd) and intensive care unit (icu) where a standardised quinine regimen was established ( and % malaria attacks, respectively). in icu, the treatment consisted of an infused loading dose mg/kg/ h of quinine diluted in % glucose followed by mg/kg/day. plasma quinine maximal concentrations were assessed after selective liquid Á/liquid extraction and spectrofluorometry detection. statistical analysis was performed using t -test. results showed that patients had comparable weight ( . / . and . / . kg) but quinine doses and plasma concentrations were significantly different in icu and imd, respectively ( . / . versus . / . mg/kg/day, pb/ . and . / . versus . / . mg/l, p b/ . ). in icu and imd, respectively: and % were in the therapeutic range ( Á/ mg/l) with and % below the requested therapeutic concentration ( mg/l) and and % above the limit of toxicity ( mg/l) conveying the importance of tdm in intravenous quinine treatment to avoid infra-therapeutic or toxic concentrations. simultaneous central nervous system distribution using microdialysis and pharmacokinetic Á/pharmacodynamic modelling of the electroencephalogram effect of norfloxacin in rats pm chenel m, marchand s, dupuis a, bouquet s, couet w. university of pharmacy, pharmacology, poitiers, france purpose: to investigate the epileptogenic activity of norfloxacin by a pharmacokinetic Á/pharmacodynamic (pk Á/pd) modelling approach and to assess the contribution of distributional processes across the blood Á/brain barrier (bbb) to the delayed effect. methods: rats (n / ) received an iv bolus dose of norfloxacin ( mg/kg). convulsant effect was quantified by electroencephalogram (eeg) recording during h post-dose. arterial blood samples were collected for drug assays in plasma. unbound norfloxacin concentrations were monitored in brain extracellular fluid (ecf) using microdialysis with in vivo calibration of the probes by retrodialysis with ciprofloxacin. results: the eeg effect reached its maximum between and min post-dose. a pk/pd effect compartment model was successfully fitted to these data. the relationship between effect and concentration at the effect site was best described by a spline function. norfloxacin concentrations in brain ecf were relatively low compared to plasma levels (ecf/plasma areas under curve (auc) ratio equal to . / . %), but central distribution was rapid. therefore, the effect versus brain ecf concentrations curves still exhibited a marked hysterisis. conclusion: the delay observed between plasma concentrations and norfloxacin convulsant effect cannot be explained by a slow distribution of norfloxacin across the bbb. pagoulatou a a , kanellakopoulou k b , vafiadou m a , kostakopoulos th c , kastriotis i b , giamarellou h c . a department of anesthesia, sismanoglio general hospital, greece , b th department of internal medicine, athens medical school, athens, greece , c department of urology, athens medical school, athens, greece csf kinetics of van and fu were studied in patients who underwent short urological surgery under spinal anesthesia. patients were excluded if they were already receiving an antibiotic or were suffering from renal and hepatic dysfunction. van was administered at g over h infusion. serum and csf samples were collected post-dose and the mean serum levels were as follows: min Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (six patients) and Á/ h: . mg/ml (seven patients). fu was administered at mg dose over h infusion. serum and csf samples were taken post-dose and the mean serum concentrations were found as follows: Á/ min: . mg/ml (six patients), min Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (five patients), Á/ h: . mg/ml (six patients), Á/ h: . mg/ml (five patients). in csf, both van and fu were undetectable. it is concluded that in the absence of meningeal inflammation van and fu do not penetrate (with the applied microbiological assay) the csf barrier. comparison of the pharmacology of intravenous and orally given moxifloxacin in an in-vitro model pm wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: the intravenous form (iv) of mg moxifloxacin (mox), one of the newer fluoroquinolones, has been recently approved by the fda. during the iv treatment higher peak serum concentrations are achieved in comparison to the oral administration (po) of the same dose. the antibacterial activity of fluoroquinolones is concentration dependent. we therefore simulated human pharmacokinetics of single po and iv dosages of mg mox in an in-vitro model using six different gram-negative and -positive pathogens to elucidate the different effect of these two dosing schedules. results: the comparison of the pharmacological parameter auc/ mic shows an increase ( table ) that could predict an enhanced antibacterial effect. however, the analysis of the killing curves with the following parameters, ka.max (maximal killing activity) and aac (area above the killing curve between and h), reveals no major difference between the po and iv dosage. conclusion: the serum concentration after oral administration is already sufficiently high to show the optimal bactericidal effect of mox that can only be slightly increased by higher peak concentrations and higher auc/mic ratios. thus the concentration dependence is not linear but ends already at concentrations achievable by oral dosing and documents that auc/mic calculations cannot easily be translated into dosing schedules. background : bacteria growing in vivo multiply much more slowly than in vitro. whether the bactericidal activity of quinolones may be affected by an increase in generation time (g) was studied in batch cultures. methods: by limiting the nutrient supply, generation times were lengthened from approximately . to . h up to . h. alternatively, the quinolones were added to the bacterial cultures during the lag-, exponential-and stationary phase. recent clinical isolates of escherichia coli were exposed to multiples of the mics of ciprofloxacin or norfloxacin. the 'killing rates' were calculated in analogy to the growth rate. results: the bactericidal activity of the quinolones tested against e. coli was minimally influenced by the reduced generation time. ciprofloxacin concentrations of ]/ )/mic eliminated the test strains within / h from the test system if added during the lag or exponential growth phase; four times higher concentrations were needed to reduce cfus by % within h, if added during the stationary phase. norfloxacin was significantly less active. conclusion: in contrast to norfloxacin, the bactericidal activity of ciprofloxacin is minimally affected by the generation time or growth phase of the bacteria. wiegand i, pfeil e, wiedemann b. university of bonn, pharmaceutical microbiology, bonn, germany purpose: moxifloxacin (mox) is one of the newer fluoroquinolones, now available for parenteral application. the pharmacology of an intravenous once-daily dose (od) of mg mox was determined with five gram-negative and -positive pathogens (streptococcus pyogenes , streptococcus pneumoniae , moraxella catarrhalis , escherichia coli , and klebsiella pneumoniae ). a twice-daily dose (bid) of mg mox was simulated with the gram-positive species in order to increase the bactericidal effect. results: to determine the efficacy, killing curves were analyzed, and following parameters were calculated: ka.max: maximal killing activity [log cfu]; ka. conclusion: an intravenous once-daily dose of mox is active against all tested pathogens. the gram-negative species are rapidly killed (ka. h similar to ka.max). there is no pronounced initial effect on the two gram-positive species but a general slow reduction in the viable cell count (ka.max is reached after h). the efficacy of mox (measured as aac and ka.max) on s. pyogenes and s. pneumoniae is to some extent increased after the second dose. however, the analysis of the killing curves reveals no major difference between od and bid. even the od nearly gives the maximal bacterical activity of mox against gram-positive pathogens. objectives: to evaluate the dose proportionality of amoxicillin and to compare the respective pk/pd parameters of two dosage regimens. methods: the dose proportionality of amoxicillin was evaluated using linear regression of mean auc -inf and c max data of different bioequivalence studies (n / volunteers) performed with formulations containing various amounts of amoxicillin alone or in the combination with clavulanic acid. the volunteers received a single oral dose in the range of Á/ mg. amoxicillin plasma concentrations were determined by hplc/uv or lc/ms/ms methods. time above mic (tmic) expressed in% of dosing interval was calculated with three target mic values ( . , . and . mg/l) for mg hourly and g -hourly dosage regimens. results: the absorption of amoxicillin (auc -inf ) showed a linear dependence with a correlation coefficient of . . the correlation coefficient of the linear regression for the cmax dependence on the actual dose was . . the respective tmic for both dosage regimens were very similar, with largely overlapping confidence intervals, supporting a pd breakpoint of mg/l for the g -hourly regimen (tmic ]/ mg/l: . %, % ci . , . %). conclusion: this analysis shows the dose proportionality of amoxicillin over the dosage range of Á/ mg and supports the pharmacodynamic rationale for a g bid dosage regimen. piperacillin/tazobactam concentration profile after high dose administration pattern in nosocomial pneumonias due to mecanical ventilation pm pedeboscq s a , gruson d b , bassoua v a , hilbert g b , pometan jp a . a st. andré hospital, pharmacy, bordeaux, france , b pellegrin hospital, reanimation, bordeaux, france the piperacillin (p)/tazobactam (t) antibacterial spectrum covers the largest part of bacteria responsible for pneumonias due to mechanical ventilation. but, due to important bacterial inoculum and pharmacokinetic parameter modifications in intensive care patients, high doses of beta-lactamines seem to be necessary to obtain antibiotic concentrations above suspected bacteria's mic (minimal inhibitory concentration) . this led us to compare, in patients with pneumonia due to mechanical ventilation, two intermittent administration patterns: g three times a day (usual pattern) versus g four times a day (high dose pattern). this study is carried out in collaboration with intensive care unit, bacteriological department and pharmacy where antibiotic concentrations are determined. twenty-three takings of blood are executed within a h period, in addition to two bronchial secretion samples. concerning p seric concentrations, the high dose pattern seems to be more adapted because of relatively high residual concentrations ( !/ mg/ml). three hours after each injection, t seric concentrations are lower than the mg/ml activity threshold. first and second day residual bronchial concentrations of p seem to be sufficient although t concentrations are below activity threshold. these results are to be correlated with the mic determined by the bacteriological department, and only this correlation will make us able to conclude the better efficacy of the high dose pattern in intensive care patients. anti-inflammatory drugs interference in absorption and tissue penetration of amoxycillin pm del fiol fs a , menon sz b , caramez th b , celotto tf b , lopes ras b . a university of sorocaba, pharmacology, sorocaba, brazil , b school of pharmacy, university of sorocaba, sorocaba, brazil antibiotics and anti-inflammatories are frequently associated in clinical practice. there is some concern about the quantity of antibiotic that reaches the infection sites, which may be reduced in the presence of an anti-inflammatory drug. the purpose of the present study was to analyse how steroids (dexamethasone (dexa)) and aines (celecoxib (cele)) influence on the penetration of amoxicillin to inflamed tissues. thirty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs to form granulomatous tissue. one week later the animals were divided into three groups. one group received only amox ( mg/kg), another received amox ( mg/kg) plus cele ( . mg/kg) and the last received amox ( mg/kg) plus dexa ( . mg/kg). one hour later the animals were sacrificed and the concentration of amoxicillin in the serum and tissue investigated. there was no difference among the groups in the quantity absorbed (amox / . / . mg/ml; amox'/cele / . / . mg/ml and amox'/dexa / . / . mg/ml). there was a reduction in the tissue concentration of amoxicillin (p b/ . tukey-kramer) for the group that received the drug with dexamethasone. for the other groups, there was no difference in the tissue concentration of amoxicillin. the results indicated that in inflamed tissue, a significant reduction of antibiotic penetration was induced by sinultaneous dexamethasone therapy. prediction of the optimal amoxicillin dose regimen based on coupling of pharmacokinetic data and bactericidal activity pm background: given its short half-life, amoxicillin (amx) should be administered at least three times a day to patients with acute exacerbations of chronic bronchitis, in order to achieve serum concentrations well above the mic of the responsible pathogen. however, several authors have recommended twice-daily administration of a higher dose for a shorter period. we assessed the relationship between amx sputum concentrations and antibacterial activity following two treatment schedules in healthy volunteers. subjects and methods: twelve healthy volunteers were randomized to receive amx for days at a dose of either g bd or mg bd. serum and sputum were collected every day, h after the morning administration, and again days after the last dose. amx concentrations were determined by hplc with fluorometric detection. sputum killing activity was determined against haemophilus influenzae , streptococcus pneumoniae and moraxella catarrhalis . results: mean serum concentrations measured h after the morning administration were . ( mg bd) and . mg/l ( g bd), and were above the mics of the three microrganisms. in contrast, sputum concentrations were always below . mg/l. in terms of sputum killing activity, g bd was more effective than mg bd against s. pneumoniae and m. catarrhalis , whereas no sputum samples were active on h. influenzae . conclusion: the optimal amoxicillin treatment schedule cannot be established on the basis of serum pharmacokinetics only. galmiche h, louchahi k, tod m, drugeon h, giroud jp, rouveix b. service de pharmacologie clinique, hopital cochin, paris, crepit , hopital avicenne, bobigny, service de microbiologie, hopital laennec, nantes, france background: cysteine-based mucolytics are commonly used in combination with antibiotics to treat patients with acute exacerbations of chronic bronchitis (aecb). they are also used to allow in vitro mic determination in sputum specimens. we conducted an in vitro and ex-vivo compatibility study designed to detect a possible interaction between mucolytics and antibiotics. methods: serial samples of bronchial secretions were collected from aecb patients and from healthy volunteers who received g of amoxicillin twice a day for days. two mucolytics were used to fluidify sputum specimens: , -dihydroxy- , -dithiolbutan (digest-eur † ) and acetylcysteine ( % solution). amoxicillin was assayed using a chromatographic system with fluorometric detection. each sample was also tested in a microbiological assay. results: amoxicillin could not be detected in the presence of the mucolytic agents. conclusions: this mucolytic Á/amoxicillin interaction may be explained by amoxicillin fixation to fluidified mucoproteins, and should be taken into account when assessing antibiotic efficacy in vivo. del fiol fs a , ferro c b , albuquerques et b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil physicians frequently recommend that macrolides should be administered with milk to decrease the discomfort they cause. thus the objective of this study was to verify the interference of milk in the absorption and distribution of erythromycin (eryt); clarithromycin (clar); roxithromycin (roxi) and azithromycin (azit). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their backs for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs eryt, clar, roxi and azit with and without milk ( . ml/kg [ca'/'/] / . mg/ml). the animals were sacrificed and the serum and tissue concentration of the drugs was investigated. there was no reduction (pb/ . tukey-kramer) in the serum and tissue concentration in the presence of milk for azit and clar. there was a % reduction for roxi in the serum concentration in the presence of milk ( . / . and . / . mg/ml), but no alteration in the tissue concentration. there was a % reduction for eryt (p b/ . ), in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and a % reduction in the tissue concentration. the milk decreased the effectiveness of treatments with erythromycin and roxithromycin and the bioavailabilities of this macrolides were affected by co-administration with milk. del fiol fs a , souza gp b , duzzi mr b . a university of sorocaba, pharmacology, sorocaba, brazil , b uniso, school of pharmacology, sorocaba, brazil the degree to which tetracyclines are absorbed differs greatly. this absorption is impaired by the concurrent ingestion of divalent and trivalent cations. thus the objective of this study was to investigate the interference of milk in the absorption and distribution of tetracycline (tetr), oxytetracycline (oxyt), minocycline (mino) and doxycycline (doxy). forty female rats (rattus norvegicus ) were used with surgically implanted pvc sponges on their back for granulomatous tissue formation. one week later the animals were divided into groups that received the drugs: tetr; oxyt; mino; and doxy with and without milk ( . ml/kg [ca'/'/] / . mg/ml). the animals were sacrificed and the drug concentrations in the serum and tissue were determined. there was no reduction (pb/ . tukey-kramer) in the serum and tissue concentrations in the presence of milk for mino. there was a % reduction (p b/ . ) for doxy in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. for oxyt, there was a reduction of % (pb/ . ) in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. the tetr results show a . % reduction (p b/ . ) in the serum concentration in the presence of milk ( . / . and . / . mg/ml) and % in the tissue concentration. milk decreased tetracycline bioavailability and effectiveness. isotopic studies with oxine labelled platelet. platelet kinetics in thrombocytopenic malaria patients pm introduction : thrombocytopenia is a common feature in human malaria ( ). excessive splenic platelet pooling has been suggested to play a role in uncomplicated cases of malaria, but a moderately shortened platelet life span during the period with decreasing parasitaemia seems the most plausible cause of the frequently observed thrombocytopenia ( , ). consumption coagulopathy, eventually manifested as disseminated intravascular coagulation, has been described in malaria ( ). in uncomplicated malaria, however disseminated intravascular coagulation is rarely found ( ). results: in malaria patients the sequestration was not different to normal. platelet half-life was reduced in patients with p. falciparum malaria to Á/ h (normal Â/ days). in one patient with p. vivax malaria platelet half live was . h. conclusion: no significant differences in the sequestration of platelets when compared to healthy individuals could be detected by in-labelled platelet scintigraphy. especially, no enhanced splenic sequestration, as previously expected, was the cause of the thrombocytopenia. therefore, other mechanisms than sequestration are responsible for the dramatically reduced life span of the platelets during acute malaria. zaharanka ag, rozhdestvensky da. vitebsk state medical university, vitebsk, belarus aim: the studying of chromatingeterogenic test (ct) results in sperm of subjects taking doxycycline (d) and some macrolides (erythromycine (e), jozamycine (j), and azytromycine (a)) in moderate therapeutic doses. methods: forty healthy volunteers ( Á/ years) were studied. daily dose of d was . ; e was administered in dose . four times per day days; j */ . before meals twice daily days; a */ . before meals once daily days. ct for evaluation of dna condition in human spermatozoids was performed before treatment (twice), on the th and th days of treatment, as well as after and months after treatment course completing. results: ct data analysis revealed that the mean amount of defective spermato-zoids before treatment was . '/ . %. by the th day of d treatment the index of de-natured dna was . '/ . % (pb/ . ), by the th day */ . '/ . % (pb/ . ). one and months after the d treatment course the amount of generative cells with denatured dna was . '/ . and . '/ . %, respectively (p b/ . in both case). under e treatment the amount of defective spermatozoids changed as . '/ . ( th day), . '/ . ( th day), . '/ . (after month), and . '/ . % (after months) (pb/ . in any case). under a using the ct results at the same control points were . '/ . , . '/ . , . '/ . , . '/ . % (pb/ . in any case); and under j treatment */ . '/ . , . '/ . , . '/ . , . '/ . %, respectively (p !/ . in any case). conclusions: the data obtained permit to conclude that d demonstrates the high level of toxicity to male generative cells. this effect preserves during months after the course of d treatment. objective: to study the effect of aggressive isolation and decontamination measures to control an outbreak of multi-resistant acinetobacter baumanii (mr-ab) in an icu. the outbreak: the index case was transferred from a mediterranean hospital, directly into an open-plan -bedded icu, with severe injuries to his head and thorax. he died shortly after admission. sputum, bronchoalveolar lavage fluid, blood cultures and a chest drain swab grew mr-ab, resistant to ampicillin, co-amoxiclav, aztreonam, amikacin, ceftazidime, cefotaxime, cefuroxime, ciprofloxacin, gentamicin, meropenem, piptazobactam, tobramycin and sensitive only to colistin. within days, mr-ab was isolated from two further icu patients. all isolates demonstrated identical antimicrobial susceptibility profiles. the icu was closed to admissions and thoroughly cleaned. all patients were isolated and their contacts screened. the icu was reopened, however, mr-ab was isolated from a fourth patient. this patient was isolated, the icu closed, for a second time, thoroughly cleaned, and all contacts isolated until discharge. all subsequent patients screened were negative for mr-ab conclusion: this illustrates the importance of aggressive isolation measures and thorough supervised cleaning in control of an outbreak, and the need to screen patients for resistant bacteria before admission to the intensive care unit in a general hospital. extended spectrum beta-lactamase-positive bacteria isolated in neonatal intensive care unit pm sandorcinova z a , siegfried l a , kmetova m a , viragova s b . a institute of medical microbiology, faculty of medicine, university of p.j. safarik, kosice, slovakia , b hospital, kosice-saca, neonatal intensive care unit, kosice, slovakia extended-spectrum beta-lactamases hydrolyse all penicilins, cephalosporins, including third-generation cephalosporins and aztreonam. esbl are predominantly produced by klebsiella spp. but may be presented in other enterobacteriaceae, too. the aim of present study was to investigate the occurrence of esbl-producing bacteria isolated from patients hospitalized at the neonatal intensive care units (icu). fifty escherichia coli and klebsiella spp. were isolated from rectum of patients hospitalized at the neonatal icu. the mics of antimicrobial agents were determined by the standard agar plate dilution method according to the nccls guidelines. for screening of esbl production we investigated strains showing reduced susceptibility (mic equal and/or more than ml/l) to at least one of third generation cephalosporins. esbl production was detected by double disk synergy test (ddst), e -test for esbl, and pcr employing specific primers for the presence of blashv and blatem genes. by using ddst and e -test, among e. coli isolates, an expression of esbl was detected in the three by the former method, while among klebsiella spp. isolates, a production of esbl was found in the two by the latter method. in esbl-positive e. coli strains the presence of blatem genes fragments was detected while in esbl-positive klebsiella spp. genes encoding for shv-type beta-lactamases were found. isolation of staphylococci from wound swabs and their susceptibility to antibiotics pm markov mij, shopovski e, despotovski v, angelevski a, nikolovski b. military hospital, microbiology, skopje, the former yugoslav republic, macedonia purpose: to determine percent of staphylococci from wound swabs and to establish their susceptibility to antibiotics. material and method: the wound swabs have been evaluated with standards microbiological techniques. bacteria have been identified with strips from the 'atb expression' system. the susceptibility testing has been performed with strips with dilution technique, read by the same system. results: during the last years ( Á/ ), a total of wound swabs have been evaluated in the military hospital in skopje. positive bacterial finding have been determined in ( %) swabs with isolated bacterial species, from which ( . ) were staphylococci: staphylococcus aureus ( . %) isolations ( methycillin resistant s. aureus ); s. epidermidis ( . %); s. haemolyticus ( . %); s. hominis ( . %); s. chromogenes and s. lugdunensis nine ( . %); s. intermedius , s. lentus , s. sciuri and s. warneri all with seven ( . %) isolations. the susceptibility of s. aureus was to: penicillin %, ampicillin %, amoksicillin/clavulonic acid %, ceftazidime %, gentamicin %, tetracycline's %, erythromycin %, lincomycin %, ciprofloksacin %, cotrimoxazole %, vancomycin %, fusidic acid %, cefixime %, and azitromicin %. conclusion: in our study most frequently isolated bacteria from the wound swabs were staphylococci, especially s. aureus . susceptibility, except for the penicillin ( %), was high to other antibiotics. the study went on from january to december . at maribor teaching hospital, staphylococcus aureus isolates were collected. in , ( %) and in , ( . %) were mrsa. mrsa were recovered from routine clinical material and from surveillance swabs (nose, throat, skin). for isolation, conventional culture media were used and for surveillance swabs mrsa-screening plate (manitol salt agar with % oxacillin) and trypticase soy broth with . % nacl were added. s. aureus was identified by catalase, dna-se and tube-coagulase test. antibiotic susceptibility was determined by the disk-diffusion method according to ncci guidelines. all mrsa isolates were tested for sensitivity to the following antimicrobials: gentamicin, netilmicin, ciprofloxacin, erythromycin, chloramphenicol, tetracycline, cotrimoxazol, vancomycin and clindamycin. it was found that all mrsa isolates were sensitive to vancomycin and partially or totally resistant to the rest. there were no important differences between the years and . our mrsa isolates were completely ( %) susceptible to vancomycin, but resistant to the other antimicrobials in use to some extent. although the monitoring of mrsa susceptibility to antimicrobials once a year did not show any important change in antimicrobial resistance, the periodical monitoring of mrsa susceptibility to antimicrobials and revaluation of current treatment regimens of mrsa infections is necessary. staphylococcus aureus strains with reduced susceptibility to vancomycin among clinical isolates in university hospital in warsaw pm the visa and especially h-visa are very difficult to be found in the routine laboratory. in our investigations we examined of s. aureus strains isolated and stored in our laboratory for several last years ( Á/ ) . most strains were isolated in , and some in . for all staphylococcal strains mrsa as well as mssa the mics of vancomycin were performed by the standard dilution method. among strains isolated in the last year three strains were recognized as visa (mic values were mg/l). the frequency of visa was . %. in the aim of founding the h-visa strains the population analysis was used. for this analysis all strains growing on the concentration mg/l of vancomycin from the inoculum were chosen. it was strains, but only of them were recognized as h-visa. the frequency of h-visa among investigated strains was about %. most but not all of the h-visa and all visa strains were methicillin resistant. in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci isolated from patients of surgical intensive care unit pm kucukates e, karayel n, kansiz e. institute of cardiology, university of istanbul, istanbul, turkey objectives: oxacillin-resistant staphylococci have emerged as a major infection control problem in our hospital. the aim of this study was to evaluate the in vitro activity of vancomycin, teicoplanin and oxacillin against staphylococci. material and methods: this study was performed between january and december , at university of istanbul, institute of cardiology. the antimicrobial susceptibilities of staphylococci isolates for vancomycin, teicoplanin and oxacillin have been investigated by e -test according to nccls guidelines. results: fifty-five ( . %) of clinical isolates were staphylococcus aureus . one hundred and twenty-four ( . %) of clinical isolates were coagulase negative staphylococci (cns). none of staphylococci isolates were resistant to vancomycin. but three of cns isolates were intermediate and six of cns isolates were resistant to teicoplanin. twenty-eight ( . %) of s. aureus were resistant to oxacillin. ninety ( . %) of cns isolates were also resistant to methicillin. conclusions: nosocomial staphylococcal infections, especially in intensive care units increase day by day. staphylococcal infections are a major problem in many hospitals. according to our experiences the rate of oxacillin resistant staphylococci isolates in our hospital has also increased. methicillin-resistant staphylococcus aureus (mrsa) is a clinically significant pathogen because mrsa is resistant to many kinds of antibiotics and causes nosocomial infections around the world. the antiseptics are used for prevention of infections by mrsa. antisepticresistant mrsa strains have been isolated from clinical specimens. antiseptic resistance genes confer resistance to many kinds of drugs structurally and the resistance mechanism is the energy-dependent drug efflux system. in addition, the fluoroquinolone (fq)-resistance gene, nora , confers also resistance to many kinds of antiseptics. we studied the relation of the susceptibility to antiseptics and fqs of mrsa strains isolated in japan. a total of strains of mrsa were isolated from hospitals in japan from to . acriflavin (af), acrinol, benzethonium chloride, benzalkonium chloride and chlorhexidine digluconate were used as the antiseptics. norfloxacin and sparfloxacin were also used in this experiment. the mic was determined by agar double-dilution method as recommended by the nccls. about % of mrsa showed resistance to af (mic: !/ ug/ml). no strain was resistant to a specific antiseptic. fq-susceptible strains were susceptible to all antiseptics. this study showed that antiseptic-resistant mrsa are widely spread at hospitals in japan. the drug of choice in treatment of serious infections caused by mrsa was still vancomycin, however sometimes failures were observed, especially in monotherapy. some conflicting are present in literature about an effect of combined action of vancomycin and betalactams. in the present work, the common effect of vancomycin and methicillin against chosen staphylococcus aureus strains was examined. the investigated strains were characterized as visa, h-visa and clones obtained from h-visa in population analysis. two methods were performed: e -tests with methicillin and vancomycin placed on the media supplemented with the second antibiotic and the chessboard micro-analysis with increasing concentrations of both antibiotics. the fic indexes were calculated for different combinations of concentrations. on the basis of the fic indexes it was shown that the simultaneous action of vancomycin and methicillin was synergistic in all examined strains visa and h-visa, but only in appropriate concentrations. in different combinations the observed effect was addition or indifference. antagonism was never observed. the synergistic effect was not observed in the case of standard s. aureus strain sensitive to methicillin. supplementation of media with % of nacl substantially decreased the observed effect. incidence of antibiotic resistance in staphylococcus aureus strains in hungary with special reference to mrsa pm ghidán Á , maró di c, csukás z, kamotsay k, szabó d, ostorházi e, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary between january and december , a total of staphylococcus aureus strains isolated from patients admitted to the clinics of the semmelweis university were examined for antibiotic sensitivity with the disc diffusion test. resistance to individual antimicrobials were as follows: penicillin %, oxacillin %, erythromycin %, ciprofloxacin %, amikacin %, netilmicin %, tobramycin %, gentamicin %, clindamycin %, mupirocin %, tetracyclines %, chloramphenicol %, teicoplanin % and vancomycin %. all mrsa were b-lactamase producer. they showed coresistance to erythromycin ( %), ciproflxacin ( %), amikacin ( %), netilmicin ( %) and mupirocin ( %). multiple resistant mrsa strains to mupirocin'/tetracyclines'/chloramphenicol amounted to . %. triple resistance to oxacillin'/ciprofloxacin'/netilmicin was %. the detection of meca gene by pcr in randomly chosen mrsa qualified with mg oxacillin disc resulted in only % meca positivity indicating that the traditional disc diffusion test overestimates the frequency of mrsa strains particularly in such an environment where the usage of penicillins and cephalosporins is so liberal as in hungary. consequently, the selective pressure for blactam-resistance and b-lactamase induction exists everywhere. this conclusion is coherent with the relatively low frequency of multiple resistant mrsa strains and urge the need of a routinely available genetic method to apply for mrsa detection. objectives: the main objectives of this study were to monitor antibiotic resistance, identify new/emerging resistance mechanisms at an early stage, prevent their dissemination, early detection and prevent the outbreaks. methods: our laboratory used antibiotic disc sensitivity testing methodology (nccls ) . zone sizes were measured objectively using a biomic automated radius zone reader. results: throughout years (january till december ) we have surveyed organisms collected from outpatient departments ( , . %), radio-oncology department ( , . %), medical department ( , . %), obg department ( , . %), surgical-oncology non icu department ( , . %), icu department ( , . %). from ( %) strains of enterobacteriaceae ( . %) were resistant to th generation fluoroquinolone and ( . %) strains were esbl positive. from ( %) strains of staphylococcus aureus were only five ( . %) strains resistant to methicilin (mrsa). we collected ( %) strains of enterococci, whereabout only two ( . %) were resistant to glycopeptides (vre). from ( %) strains of pesudomonas aeruginosa , ( . %) were resistant to aminoglycosides. conclusions: national restrictive antibiotic policy hand in hand with local hospital antibiotic policy and regular rotation of antibiotics used in prevention and treatment on all departments is leading in our case in positive situation in antibiotic resistance in comparing with other slovakian and european centers. antimicrobial resistance of nosocomial strains of staphylococcus aureus pm dekhnich av, stratchounski ls, edelstain ia, narezkina ad. institute of antimicrobial chemotherapy, smolensk, russian federation purpose: to determine the antimicrobial resistance of staphylococcus aureus causing nosocomial infections in smolensk regional hospital. results: a total of s. aureus strains isolated during Á/ were studied. antimicrobials tested included oxacillin (oxa), erythromycin (ery), clindamycin (cli), gentamicin (gen), vancomycin (van), linezolid (lnz), tetracycline (tet), chloramphenicol (chl), rifampicin (rif), fusidic acid (fus), trimethoprim/sulfamethoxazole (ts), ciprofloxacin (cip), mupirocin (mup), quinupristin/dalfopristin (qd). susceptibility testing and its interpretation were performed by agar dilution according to nccls guidelines where applicable. results are presented in the conclusions: the most active antimicrobials were vancomycin, linezolid, quinupristin/dalfopristin, fusidic acid, mupirocin, followed by co-trimoxazole, rifampicin. beta-lactams, macrolides, lincosamydes, tetracyclines and chloramphenicol should not be used for the treatment of nosocomial s. aureus infections. we investigated all staphylococcal infections within years among neonates hospitalized for infection in the neonatal icu in a tertiary neonatal referral center. univariate analysis, to assess risk factors for neonates infected with staphylococcus aureus ( ) vs. without s. aureus ( ) was performed. from the total number of cases, in cases s. aureus was isolated from various samples; in cases from blood cultures, in cases from urine, in cases from eye swabs and in cases from gastric content (no significant differences in comparison with control group). colonization with s. aureus , was a predictor of infection: nasal swabs, throat swabs, ear swabs, skin swabs and umbilical swabs were significantly more commonly observed in neonates infected with s. aureus , than with other infections. etiological analysis showed that co-pathogens escherichia coli and viridans streptococci were significantly more frequently associated with neonatal infection caused by s. aureus , in comparison to other organisms. according to localization of infection site, conjunctivitis and thrush stomatitis was the commonest s. aureus neonatal infections. outcome was similar to other infections and without any significant differences between both groups. mortality was similar to other infections, probably because: initial therapy in our centre contains an antistaphylococcaly active agent (cefuroxime or cefotaxime plus aminoglycosides). morozova ot, semina na. laboratory of hospital infections, central research institute of epidemiology, moscow, russian federation purpose is to study the role of enterococcus spp. in the aetiology of nosocomial infections among the patients of the childrens clinical hospital and susceptibility of these strains to antibiotics. methods: the strains of enterococci were isolated from patients with hospital infections in Á/ . results: the aetiological structure of enterococcus -infections showed the predominance of skin and soft tissue infections ( . %), urinary tract infections ( . %), bloodstream infection ( . %), pneumoniae ( . %), infection of central nervous system, gastrointestinal tract, eye, surgical wound infections were of rare incidence ( Á/ . %). various nosological forms of infections were caused more often by e. faecalis than e. faecium ( . , . %). the antibiotic resistance to ampicillin and other beta-lactams occured in % of e. faecium isolates, but all strains of e. faecalis were susceptible to these drugs. high-level gentamicin resistance demonstrated e. faecalis isolates */ . %, e. faecium */ %; and high-level streptomycin resistance showed e. faecalis */ . %, e. faecium */ . %. all the e. faecalis were active against fluoroquinolones, but e. faecium were resistant in . %. there were no vancomycin resistant enterococci. conclusion: e. faecalis predominated in the aetiological structure of nosocomial infections due to enterococcus spp. antibiotic resistance patterns for two species of enterococci were different, all the strans were susceptible to vancomycin. evaluation of antimicrobial resistance of enterococcus spp. experience of years ( Á/ ) pm lopez-barba j, jesus de la calle i, solino-ocañ a i, rodríguez-iglesias m, perez-ramos s. microbiology laboratory, puerto real university hospital, cádiz, spain objective: determination of quantitative changes in antimicrobial resistance of enterococcus isolated from clinically significant not urinary samples of patients remitted to the laboratory of microbiology during a years period ( Á/ ) . material: the period of the study was comprised between and . the samples has been processed for the isolation of enterococcus following conventional methods. were isolated enterococci strains. the identification and susceptibility to antibiotics have been performed in automated system microscan(c) dade-behring(c) through panels combo cgp. the data were processed by the statistical system statgraphics plus . . results: of the enterococcus , have been identified e. faecalis and e. faecium . the resistance is shown in table . conclusions: the resistance to va and tei of e. faecalis remains through last years ( Á/ %). the high resistance to erythromycin and tetracilin ( !/ %) and the resistance ( Á/ %) to quinolones, antibiotics all of them used in community-acquired infections justify the susceptibility testing to the clinical strains isolated of this group of microorganism. in e. faecium the antimicrobial resistances was high and increasingly to imipenem, meropenem, erythromycin and quinolones. characteristics of strains e. faecium colonizing the neutropenic patients pm abbassi ms, achour w, gréco a, ben hassen a. laboratory of bone marrow transplant center, tunis, tunisia digestive colonization by enterococcus faecium in the neutropenic patients under gut decontamination is important. seventeen multiresistant strains of e. faecium isolated from stools of seven neutropenic patients were the target of an epidemiological analysis through the determination of the mics of amoxicillin, gentamicin, vancomycin, the transferability gentamicin resistance to the recipient strain e. faecalis jh - by filter-mating assay, analysis of plasmid profiles of e. faecium -strain and of transconjugants and the amplification by pcr of the gene aac( ?)-aph( ??) coding for the bifunctional enzyme by using primer m who gives a fragment of kb. among the seventeen strains, eleven had the same antibiotype a , had a gentamicin mic!/ mg/l. the mic of the amoxicillin was of mg/l. all the strains were sensitive to vancomycin. ten strains harbored a plasmid of kb transfered at a frequency of . Á/ , also found in gentamicin-resistant transconjugants. however, strains belong to nine distinguished plasmids profiles. all high-level gentamicin resistant-strains had a positive pcr amplification of the aac ( ?)-aph ( ƒ) gene. the features of the studied strains establish their endogen origin, specific for every patient, sharing only high-level resistance to gentamicin. gut decontamination treatment with gentamicin enhance either the spread and the preservation of easy-transferable plasmid carrying genetic transposable element. frequency and antibiotic resistance of bacteria isolated from patients suffering infectious complications following the implantation of prosthetic devices pm kristó f k, rozgonyi f. institute of medical microbiology, semmelweis university, budapest, hungary for patients with indwelling joint prosthesis, early recognition and prompt therapy for infection in any location may be critical to reduce the risk of seeding the joint implant heamatogenously. a year period ( ) a total of swabs of aspiration from patients with infectious complications following the implantation of prosthetic devices were cultured. cultivation and identification of the strains were performed by conventional methods and by vitek system (biomȇrieux) and susceptibility testing by disc diffusion. potentional pathogens were recovered in cases ( . %). gram positive cocci, in particular staphylococcus spp. proved to be the most commonly isolated bacteria. coagulase-negative staphylococcus (cns) was isolated more frequently ( %), followed by s. aureus ( %), enterococcus faecalis and e. faecium ( %), gram-negatives ( %), anaerob isolates ( . %). resistance to individual antimicrobials of s. aureus and cns were as follows: methicillin and %, clindamycin . and . %, fluoroquinolones and %. mupirocin resistant strains of s. aureus were not found, while . % were among the cns strains. our results could be essential for the rational selection of treatment at our orthopedic wards. results: in the analysed period the percentage of isolated enterococcus sp. strains among all non-repetitive clinical isolates in , and was . , . and %, respectively. cultured strains were identified as e. faecalis, e. faecium, e. gallinarum and e. avium . the most prevalent was e. faecium strains, isolated with a frequency of , and %, respectively. vancomycin-resistant strains were all identified as e. faecium and in they comprised % of all isolates of this species. conclusions: ( ) the frequency of isolation of enterococcus sp. in blood cultures of haematological patients remained relatively stable in Á/ . ( ) the predominant enterococcal species isolated from these patients was e. faecium . ( ) in we recorded for the first time an emergence of vancomycin-resistant e. faecium , which comprised % of all isolates of this species. kalai s, ben hassen a, achour w, greco a. laboratory of microbiology, national bone marrow transplantation center, tunis, tunisia from april to june , non-repeated strains of pseudomonas aeruginosa were isolated from immunocompromised patients. thirty-six percent of strains were isolated from abscess, % from blood culture and % from urine. susceptibility to antibiotic was studied by the routine disk diffusion method (ca-sfm). mics were determined using agar dilution to antibiotics ( b-lactams, four aminosides and two fluoroquinolones). serotyping of the different trains was performed using antisera to the international antigenic typing systems serotypes. the study showed % of resistance to c cochin port royal hospital, service de gynecologie-obstetrique site st vincent-de-paul, paris, france , d cochin port royal hospital, cclin, paris, france aims: to determinate risk markers of an outbreak of postpartum endometritis due to group a streptococcus . design: a case-control study using data collected with a structured form. setting: the cases of postpartum endometritis were diagnosed in the department of obstetric of the paris hospital network during days (december Á/january ). the group of controls consisted of women delivered in the same department during the same period. participants: cases (n / ) and controls (n / ). findings: cases had smoked more often during pregnancy ( vs. %; p / . ) and received more often immunosuppressive treatment than controls ( vs. %; p / . ). instrumental delivery has been needed more often for cases than controls ( vs. %; p / . ). cases had been hospitalized after delivery in a ward z of the department more often than controls ( vs. %; p / . ). they had been examined after delivery more often by a midwife x ( vs. %; p / . ) and a nurse y has provided care to cases and not to controls ( vs. %; p / . ). conclusion: smoking, receiving immunosuppressive treatment during pregnancy, and instrumental delivery were significantly associated with postpartum endometritis (pb/ %). a midwife and a nurse might be involved in the transmission of the infection. petoukhova i a , sokolova e a , dmitrieva n a , nummaev b b . a laboratory microbiology, cancer research center of russia, moscow, russian federation , b department of oncogynecology, cancer research centre, moscow, russian federation the aim of the study was to assess efficacy of perioperative ap. total pts were included. two hundred and one pts with cervical cancer (cc) undergone extensive hysterectomy, pts with cancer of vulva (cv)-extensive vulvoectomy, pts with ovarian cancer (oc) Á/ extensive/combined operations. fifty-one pts (group ) received ap with amoxicillin/clavulanate (am/cl) . g iv min prior to operation, then . g iv thrice per day for Á/ days. fifty pts (group ) received cefotaxime (ctx) g iv min prior to operation, then g four times per day for Á/ days'/metronidazole (mtz) mg two times per day for the same period. one hundred and forty pts were retrospective control (they received ii Á/iii generation cephalosporins or linkosamides only after operation). the rate of swi in pts with cc, cv and oc was . , , %, respectively; dwi */ . , and %, respectively; uti */ , , . %, respectively. the ap with am/cl was more effective compared to ctx'/mtz (swi */ vs %, respectively, pb/ . , dwi */ . vs %, respectively, p b/ . ). the rate of postoperative uti was equivalent in two groups ( vs %, p /n.s.). thus, ap with am/cl is preferrable option in extensive operations in og pts. fifty-eight patients were randomized into two groups. group , a treatment consisting of patients and group , consisting of patients as a control. the patients were at the age of / . and had had different surgical interventions with general anaesthesia from to h and accompanying copd ( %). artificial pulmonary ventilation was used in cases. in the early post-surgery period the patients of group were administered inhalation therapy, including ipratopium of bromide in the combination with fenoterol (atrovent, berodual) and ambrocsol (lazolvan) through a nebulizer. the inhalation therapy was not administered to the patients of group . under the influence of the inhalation therapy pulmonary ventilation and respiratory metabolism was restored faster in all the resuscitation patients (in group */by the end of the first h, in group */on the rd Á/ th day). the percent rate of pef was . '/ . and . '/ . , respectively. artificial pulmonary ventilation ended in . and . h, respectively. the time of the patients' stay in the resuscitation department was . days in group and . days in group . by the end of the st week pneumonia developed in one patient from group and in eight patients in group . aerosol therapy application accelerates medication delivery to the respiratory tracts, increases the local activity and effects good prophylaxis for surgical hospital pneumonia. variation in ethiology of early and late onset ventilator associated pneumonia pm nikolopoulos j, daganou m, michailidou m, karabela e, kavada k, retsou s, antoniadou a, rasidakis a. department of respiratory abstracts s failure and icu, sotiria general and chest disease hospital, athens, greece purpose: to compare the distribution of causative microorganisms, their susceptibility to antibiotics and outcome of 'early' and 'late' vap in a greek icu. methods and results: retrospective study of mechanical ventilated (mv) patients (pts) with early and late vaps during a -month period. diagnosis of vap was made by clinical, radiographic criteria and quantitative cultures of bronchial secretions. vap was diagnosed in pts ( %) out of consecutive admissions in icu. all pts before the development of vap received antibiotics. three episodes of vap ( . %) were developed before the th day of mv (early vap) and were caused: ( ) by multi-resistant acinetobacter; and ( ) by antibiotic-susceptible pseudomonas aeroginosa . in this group one pt died from septic shock related to vap and two pts survived. fourteen pts ( . %) developed vap after the th day of mv (late vap). five cases were caused by multi-resistant p. aeroginosa , two cases by mrsa, two cases by multi-resistant acinetobacter, two cases by susceptible to antibiotics klebsiella pneumoniae , and three were polymicrobial and caused by multi-resistant microorganisms (mrsa and gnb). four pts died ( . %) from septic shock related to vap, five pts ( %) died because of another cause and five pts ( %) survived. conclusions: early and late onset episodes of vap were caused by 'potentially drug-resistant bacteria'. p. aeroginosa as a cause of early vap was susceptible. mortality attributed to early and late vap was similar. antibiotic prophylaxis in oncological and major reconstructive orthopaedic surgery pm de biase p, ciampalini l, astone a, capanna r. azienda ospedaliera careggi, oncological and reconstructive centre, aoc, florence, italy during last year patients scheduled for oncological surgery or major reconstructive procedures were randomised to either vancomycin or teicoplanin prophylaxis. prophylaxis was performed with either vancomycin g i.v. twice daily or teicoplanin once daily i.v. two hundred patients were included. four patients did not agree the study protocol and were excluded. we treated patients with teicoplanin and patients with vancomycin. out of the patients were operated for oncological disease, while the remaining underwent major orthopaedic procedures. we experienced cases of red man syndrome, and five cases of moderate hypotension. five patients had postoperative complications: two deep venous thrombosis, one pulmonary embolism, two postoperative haematoma. in five patients we observed a wound dehiscence; two of these patients showed clinical sign of ssi and microbiological examinations were positive for mrsa. one patient recovered from infection with medical therapy, while the other patient showed a local tumour recurrence and was amputated at the thigh. at last surgery infection was still present clinical and at microbiological examination. in conclusion we had an infection rate of . % which is comparable to the infection rate of a 'clean' surgery in patients with normal risk of infection. teicoplanin showed lower toxicity, has a longer half-life and has a simpler way of infusion and it is our current choice in high risk surgery. injuries with contaminated sharp articles in health care workers in general hospital celje, slovenia pm lesnicar g, sibanc b. department of infectious diseases, medical center celje, celje, slovenia in a prospective study carried out from january till june , we registered subcutaneous injuries with sharp objects, mostly in nurses and cleaning service workers. in % of cases the incident occurred outside the hospital, in persons who were not medical workers. in cases the injury causing object was a needle that had been used in known patients, of which were hepatitis b positive. fifty-five ( . %) of the injured health workers had been previously vaccinated against hepatitis b; the protective antibodies to hepatitis b in the blood were found in / ( . %) health workers only, while the tests for antibodies to hepatitis c and hiv were negative in all cases. following the incident, the majority of the injured persons, i.e. , were vaccinated against hepatitis b, while persons ( . %) also received passive prophylaxis with human immunoglobulin against hepatitis b. none of the injured persons have developed the disease or showed evidence of sero-conversion. in the year the general as well as specific preventive measures practised in our hospital became more rigorous. thus, approximately % of our health workers at risk have already been vaccinated against hepatitis b. to improve measures preventing dissemination of multidrug-resistant bacteria (mrb), a cross-sectional survey ( ) was conducted to analyse healthcare workers' (hcws) isolation precaution knowledge for mrb infection at investigation and outpatient departments excluding four declaring not to be involved in care to mrb patients (emergency, obstetrics, nuclear medicine, and bacteriology). two hundred and eight hcws answered ( % of the paramedical staff, % of the physicians). thirty-three percent of them reported they do not know frequently or always the patient mrb status. they ( %) wish mrb status to be mentioned on the test form or on the advice request letter. mrb patient visit or test was appropriately timed in % answers. gowns ( %) or masks ( %) use were not systematically reported. other hcws ( %) reported better isolation precaution knowledge than physicians ( %) and nurses ( %) than investigation assistants ( %). physicians declared lower compliance with use of gowns, gloves or draw-sheets than other hcws. they had also lower education in isolation precautions and were less interested in education programs. this study suggests the necessity to improve mbr infection information. physicians and investigation assistants seem to be insufficiently aware of hospital infection control. therefore, education strategies targeted at physicians and investigation assistants working at outpatient and investigation departments should be developed. outbreak of clostridium difficile -associated diarrhoea in infectious disease department: risk factors and hygiene measures assessment pm henoun loukili n, martin m, remy v, hansmann y, christmann d. hôpitaux universitaires de strasbourg, maladies infectieuses et tropicales, strasbourg, france (shock)'/ * (bedridden status)'/ * (age !/ years)'/ * (previous antibiotic treatment) and points (women) / * (shock)'/ * (bedridden status)'/ * (age !/ years)'/ * (immunosuppression). the vast majority of patients ( and % of males and females, respectively) could be classified in the subgroups with lower scores (six points or less) which had a very limited risk of death ( Á/ . and Á/ . % for men and women, respectively), whereas for patients in the highest score subgroup ( points or more), the risk was % for men and % for women. conclusion: risk stratification of patients with ap is possible from simple clinical variables available on admission. procalcitonin (pct) and c-reactive protein (crp) for differentiation of systemic inflammatory response syndrome (sirs), sepsis and severe sepsis pm lupse m a , ursu l b , slavcovici a a , carstina d a . a clinical department, teaching hospital of infectious diseases, cluj-napoca, romania , b laboratory department, teaching hospital of infectious diseases, cluj-napoca, romania objectives: to evaluate the value of pct in the differentiation of patients with sirs, sepsis, severe sepsis and bacteremia in comparison to crp. design: prospective study including patients who meet criteria for sirs, sepsis or severe sepsis (consensus conference of the accp/ sccm) admitted over -month period. patients and method: a total of patients were included: eight with sirs, with sepsis and with severe sepsis. sixteen from patients had bacteremia. pct and crp were evaluated in the first h after admission: pct by brahms pct-q test and crp by turbidimetric assay. the sensitivity, specificity, predictive value of different cutoff points for crp and pct were determined. results: with a cut off point of . ng/ml for pct and . mg/dl for crp sensitivity and specificity for sepsis were %, respectively % (ppv . , npv . ) and %, respectively % (ppv . , npv ). a cutoff point of ng/ml for pct accurately predict severe sepsis (sensitivity %, specificity %, ppv ). a pct level of at least ng/ml was a good predictor for bacteremia (sensitivity %, specificity %, ppv . and npv ). conclusion: pct is a good discriminating marker to characterize the level of inflammation caused by infection and can predict bacteremia . giamarellou h a , aoun a b , klastersky j b , anagnostopoulos n c , galani l c , grecka p c , panaretou e c , papageorgiou e c , repoussis p c , syrseloudis pct has been considered as a useful diagnostic marker in neutropenic patients with bacteremia and/or severe sepsis (giamarellos-bourboulis ej et al. clin infect dis ; : ) . in an attempt to define its value in the diagnosis of localized infections in neutropenic hosts, daily determinations of pct and of c-reactive protein (crp) were performed before and after the onset of fever in subjects male and female aged . / . years with various haematologic malignancies (aml , nhl , mds , all ) developing neutropenia ( b/ pmns/mm ) after chemotherapy. thirty-three patients were presented with fever of unknown origin (fuo) and with localized bacterial infections (lbi; pneumonia , acute pyelonephritis , soft tissue infections , acute pharyngitis ). pct was determined by an immunoluminometric assay and crp by nephelometry. it is concluded that febrile neutropenia followed by a localized bacterial infections is accompanied by significantly higher levels of pct than in case of fuo ( . / . vs . / . ng/ml). similar differences are not observed with crp, which lacks the appropriate specificity. our study included patients who were categorized as having proven ( ), probable ( ), or possible ( ) systemic fungosis according to eortc criteria; showed no sign of infection, and were used as controls. blood samples were received on the st, rd, and th day from the onset of signs of a fungal infection, and then twice a week. pct levels were determined by an immunochemioluminent assay, and candida and aspergillus antigen levels by elisa. in only five patients pct indicated early signs of infection, albeit at barely detectable limits. six patients, however, showed significantly increasing titres preceding time of death. positive antigens titres were observed only in patients who had proven or probable systemic fungosis. only half of the control group had negative antigen titres; a high rate of false negatives was also observed. both pct and antigens titres increased in parallel in / patients with unfavorable outcome. pct and antigens titres cannot reliably indicate early diagnosis of systemic fungal infections although may be used as a prognostic tool of severity. lactulose, a factor that decreases endotoxaemia, in obstructive jaundice? pm koutelidakis im a , papaziogas v a , makris i a , giamarellos-bourboulis ej b , giamarellou h b , papaziogas t a . a thessaloniki med school, nd surgical clinic, thessaloniki, greece , b th department internal medicine, athens medical school, athens, greece bacterial translocation is a process implicated in the pathogenesis of spontaneous peritonitis. in order to evaluate the impact of lactulose administration on systemic endotoxaemia, obstructive jaundice was induced in rabbits by common bile duct ligation. animals were divided into two groups, group a of five rabbits not receiving lactulose and group b of six rabbits, which received . ml/kg of lactulose orally by an oral catheter. blood was collected daily, before and after operation for a total duration of four days. samples were applied for culture and for determination of endotoxins (lps) by the lal qcl- assay. concentrations of lps (mean /sd) of group a were . / . , . / . , . / . and . / . eu/ml on the st, nd, rd and th day, respectively. respective concentrations of lps (mean /sd) of group b were . / . , . / . , . / . and . / . . all blood cultures were sterile in both groups. differences activity of linezolid against nosocomial strains of staphylococcus aureus in russia: results of multicentre study pm were included in the study. antimicrobial susceptibility testing was performed by agar dilution method in accordance with the nccls recommendations. all tested strains including mrsa strains ( . % of all strains) were found to be susceptible to linezolid with the mic ranged from . to mg/l. both mic and mic were mg/l. conclusions: linezolid had excellent in vitro activity that was not affected by resistance to other classes of antimicrobials susceptibility to antiseptics of mrsa isolated in japan during Á % to piperacillin, % to ceftazidime, % to cefepime, % to imipenem, % to amikacin and % to ciprofloxacin. fiftythree percent of p. aeruginosa strains were multiresistant ( strains) and were isolated in patients. wild phenotype to b-lactams was observed in % of strains. the most frequent b-lactams resistance phenotypes were: cephalosporinase over production ( %) and penicillinase ( %). imipenem, ceftazidime and piperacillin-tazobactam were the most active b-lactams (mic of . and mg/l, respectively) these results showed high rates of antibiotic resistance and predominance of o serotype in multiresistant strains compared to the o serotype in europe. infectious complications sustained by stenotrophomonas (xanthomonas ) maltophilia in hiv at present, very limited informations are available about s. maltophilia infections in the setting of hiv disease. patients and methods: a retrospective survey of clinical and microbiological records of hiv-infected patients referring to out tertiary care centre between and was performed, in order to identify all episodes of s. maltophilia infections, and analyze its epidemiological, clinical, and microbiological variables. results: sixty-one episodes of s. maltophilia infection were observed in patients: sepsis/bacteraemia in cases ( . %), lower airways infection in five, urinary tract infection in four, pharyngitis in two, lymphadenitis and liver abscess in one case each. forty-seven out of episodes of s. maltophilia infections ( %) occurred as nosocomial disease, generally in association with advanced immunodeficiency, neutropenia, instrumentation, and prior antimicrobial therapy. bacterial isolates showed an elevated resistance profile against many betalactam compounds, aztreonam, imipenem, and aminoglycosides. conclusion: s. maltophilia represents an emerging opportunistic pathogen in hiv-infected patients extended spectrum beta-lactamases producing germs in intensive care units pm university of medicine and pharmacy 'victor babes ', microbiology, timisoara, romania injury and complications lasted months, demanded for surgical procedures and total cost was comparison of different methods for detection of extended spectrum beta lactamases (esbls) and their genetic relatedness among enterobacteriaceae clinical isolates in a research medical institute pm methods: one hundred out of isolates that were screened positive for esbls were tested with double disk synergy test (ddst), three dimensional test (tdt), e -test-esbl and vitek-esbl test. pulsed field gel electrophoresis (pfge) analysis was applied to esbls; five klebsiella pneumoniae and eight escherichia coli . results: revealed the prevalence of esbls in . % of clinical isolates. the sensitivities of the ddst, tdt, e -test and vitek were , , . and . %, respectively. in the ddst, aztreonam was the most sensitive indicator ( . %). pfge demonstrated that % of k. pneumoniae were derived from a single clone whereas . % of e. coli isolates were derived from two different clones. non-clonal origin was demonstrated in % of k. pneumoniae and . % of e. coli . conclusion: there is an increased prevalence of esbls. the ddst is the most sensitive, practical and cost effective diagnostic method reliable for routine use in our laboratory. both clonal spread and plasmid dissemination contributed to the concurrent nosocomial outbreaks caused by esbl-producing k severe nosocomial infections due to stenotrophomonas maltophilia pm the teaching hospital of infectious diseases total og pts after extensive hysterectomy ( pts), extensive vulvoectomy ( pts) and extensive/combined operations for ovarian cancer ( pts) were analysed. ic developed in pts. one hundred and sixtyseven pts had no ic. twenty-eight of rf analysed were independent rf of ic. most important included: age !/ years (p / . ), grade Á/ obesity (p / . ), diabetes mellitus (p / . ), diagnosis of cervical cancer (p / . ), history of pre-cancer of vulva postpartum endometritis due to group a streptococcus : a case-control study pm unite operationnelle d 'hygiene all isolates were sensitive to vancomycin. among gram-negative bacteria klebsiella spp. was isolated in . % of cases, acinetobacter baumanii in . %, enterobacter spp. in . %, providencia spp. in . %. of the klebsiella spp. isolates % were resistant to amikacin, % to cephalosporins, % to piperacillin/ tazobactam. all were sensitive to imipenem. of the a. baumanii isolates % were resistant to amikacin, aztreonam, cefoperazone, cefotaxime, ceftriaxone, piperacillin; % to ampicillin-sulbactam, % to ceftazidime, and % sensitivity to imipenem antimicrobial activity of selected pharmacopoeial antiseptics analysed according to european standards pm european committee for standardisation approved several european standards (en), describing test methods establishing, whether an antiseptic has or does not have a bactericidal or fungicidal activity under the laboratory conditions defined by en. the aim of the study was to investigate, if some chemical compounds in concentrations recommended by polish pharmacopoeia for skin disinfection, comply european standards requirements. methods: basic bactericidal (en ) and fungicidal (en ) activity were investigated as well as bactericidal activity of products for hygienic and surgical handrub and hand wash used in human medicine (pren ). all methods and used neutralizers were validated. standard strains: staphylococcus aureus , pseudomonas aeruginosa , escherichia coli , e. hirae , candida albicans and a. niger were used, when en standards were evaluated. results: ethanol, izopropanol and n -propanol caused viable microbial count reduction required by ens in pharmacopoeial concentrations the purpose of the study: we collected bacteriological samples from adult and neonate patients who were admitted in intensive care units (icu) . the aim was to observe the colonization status with microbes that may have a nosocomial potential and to establish circulating phenotypes in icus. the results obtained from a total of samples strains of gram negative bacteria (enterobacteriaceae family) were isolated. fourteen strains showed extended spectrum beta-lactamases (esbl) phenotype (eight strains of klebsiella pneumoniae , three of escherichia coli , two of klebsiella ornithynolitica , one of klebsiella oxytoca ). we used both disc diffusion test (extended antibiotic susceptibility test and synergy test to visualize 'champagne stopper' pattern) and mini api † system.the conclusion reached: we put in evidence a massive colonization with germs that may have a nosocomial potential especially microbes that produce esbl ( . % from all enterobacteriaceae isolated) which implies a rational policy in prescribing antibiotics in hospitals from western part of romania.carbapenem activity against nosocomial gram-negative rods pm sawicka-grzelak a a , rokosz a a , meszaros j b , luczak m a . a department of medical microbiology, university medical school, warsaw, poland , b department of general and transplantation surgery, university medical school, warsaw, poland purpose: to determine a susceptibility of nosocomial gramnegative rods to carbapenems.methods: two hundred strains of gram-negative rods were cultured from clinical specimens from hospitalized patients (july Á/november ). identification of strains was performed in the automatic atb system (biomerieux, france). susceptibility of strains to carbapenems: imipenem and meropenem was determined with disc diffusion method according to nccls recommendations. esbl-producing strains were detected with double-disc synergy test (ddst according to jarlier et al., ) or a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd ) (oxoid, england) .results: one hundred and ten strains of enteric rods and strains of non-fermenting rods were cultured. twenty eight ( %) esblpositive strains were detected. carbapenems were active against % of enteric rods. the percentage of non-fermenting rods susceptible to imipenem was and to meropenem */ .conclusions: carbapenems: imipenem and meropenem demonstrated high activity against clinical strains of enteric rods. however, the antibiotics were less active against nosocomial strains of nonfermenting rods.inhaled antibiotics against multiresistant bacteria in bronchial secretions of icu patients: a preliminary report pm horianopoulou m a , kanellopoulou m b , paraskevopoulos i a , valakis k a , kyriakidis a a , lambropoulos s a . a intensive care unit, sismanoglio general hospital, athens, greece , b department of microbiology, sismanoglio general hospital, athens, greece purpose: the aim of this study was to assess the effectiveness of aerosolized ampicillin/sulbactam, ceftazidime and colistin, in icu patients with multiresistant acinetobacter baumannii or pseudomonas aeruginosa colonization of the respiratory tract.methods: fifty-three intubated, mechanically ventilated patients participated in the study. multiresistant a. baumannii , sensitive only to ampicillin/sulbactam, or p. aeruginosa , sensitive to ceftazidime or colistin, were isolated from the bronchial secretions ( Á/ cfu/ ml). all patients were subsequently treated with intravenous ampicillin/sulbactam, ceftazidime or colistin, whereas of them were also given the same antibiotic in aerosolized form.results: a decrease in the number of colonies by Á/ cfu/ml was observed, following Á/ days of combined treatment with both intravenous and inhaled antibiotic. none of the patients developed vap. in the patients who only received the antibiotic intravenously, the decrease ranged from zero to cfu/ml, after days of treatment. two of patients developed vap.conclusions: our results suggest that the administration of aerosolized antibiotics represents an effective means of preventing ventilator-associated pneumonia caused by a. baumanni and p. aeruginosa . introduction: pseudomonas aeruginosa is an important nosocomial pathogen. resistance to certain beta-lactam antimicrobial agents among p. aeruginosa is increasing. despite the development of new antibiotics multiresistant strains of p. aeruginosa represent an important therapeutic problem. the aim of this study was to investigate the activity of imipenem, amikacin, piperacillin, ciprofloxacin, ceftazidime, against clinical isolates of p. aeruginosa . methods: a total of isolates by tracheal aspiration from hospitalized patients, admitted to intensive care units were identified as p. aeruginosa using an algorithm that included: gram stain, pigment, oxidaze ('/ ,) and gram negative identifications microscan walkaway- (dade behring) were used according to the manufactures instructions. minium inhibitory concentrations were determined using walkaway, interpretation based on ncclsm -s , january ' .results: the respiratory tract was the single site of isolation for this study. the best activity was showed by imipenem %, followed by amikacin %, piperacillin, pip/tazobactam, ciprofloxacin had the same sensitivity %.conclusion: a high level resistance to antibiotics was observed to p. aeruginosa isolated from tracheal aspiration. carbapenems seem to be the most active against p. aeruginosa in this study. materials and methods: clinical samples were collected from patients admitted to this hospital. only one isolate per patient was included. antimicrobial susceptibility testing was performed as recommended by the nccls. all bacterial isolates were tested by dd and ad to provide a comparison of both test results. very major error was considered when the strains were resistant (r) by ad and susceptible (s) by dd and major error when s by ad and r by dd. categories of s and r were stablised using the breakpoints suggested by mensura ( ) . colistin r strains was typed by rep-pcr.results: among the strains included in this study ( . %) were s to colistin and five ( . %) were colstin r by ad, of this five colistin r strains four ( . %) were s to colistin by dd and one was r by both methods. all r isolates were similar by rep-pcr.conclusions: most of the ad colistin resistant strains were s when tested by dd indicating that this method is not useful to determine the resistance to colistin. rep-pcr patterns show that the spread of a colistin r clone seems to be involved. methods: gnb were isolated from per-operative biopsies (pob) and/ or from articular punction (ap). patients (pts) received cfp, g bid'/ ofl, mg tid or cip, mg bid intravenously for days, followed by a prolonged oral fq monotherapy. cure was defined as: resolution of all clinical signs of infection, normalization of the biological inflammatory profile at the end of treatment (eot) and absence of infection at the same site during the post-treatment followup period (ptfu).results: all of the studied patients [mean age / years] had hospital acquired bji. seventeen/ had an infected orthopedic device (prosthetic joints / , other orthopedic prosthetic devices / ). culture of pob and ap yielded to pseudomonas sp. ( ), enterobacter cloacae ( ), others ( ). vancomycin was added for six pts co-infected by gnb-mrsa. nineteen/ pts underwent a surgical intervention (debridement / , removal-replacement / , amputation / ). after ptfu period of months (range Á/ ), the overall success rate was / ( . %) without serious adverse events.conclusion: cfp Á/fq combination was safe and efficient in the treatment of hypercase gnb, bji.treatment of posttraumatic mrsa osteomyelitis of the femur with longterm cotrimoxazole */a case report pm the authors report a case of posttraumatic osteomyelitis of the femur caused by methicillin-resistant staphylococcus aureus (mrsa), following the shot injury. relapses of the infection occured in months interval and were treated by revision, debridement, lavage and vancomycin. because of laboratory signs of renal insufficiency vancomycin became contraindicated for treatment of the third relapse of infection and the different approach was employed: classic open treatment of bone infection sec. orr was combined with a long-term administration of high-dose cotrimoxazole. the patient was given cotrimoxazole mg daily divided in four doses ( mg/kg/ h) for months, then for gastrointestinal complaints with lowered dose of g daily for next months. the wound completely healed. during months after the final surgery there was no relaps of infection, but the atrophic pseudoarthrosis of the femur resulted. the patient can walk with a rigid orthesis and two crutches. the whole treatment of the objective: to present a variety of severe nosocomial infections due to stenotrophomonas maltophilia in patients hospitalized in tertiary medical units from cluj.results: during the last year nine strains of s. maltophilia obtained from patients with severe infections and hospitalized in different wards were isolated. all but one were considered nosocomial infections: four cases of pneumonia, one urinary tract infection, three cases of surgical wound infections and one case of endocarditis under surgical treatment. the cases of pneumonia were either primary occurring in a granulocytopenic patient with leukemia or secondary in patients that underwent surgical treatment. in the case of endocarditis the ethiology was established after surgery from the damaged valve in a negative hemoculture patient with a poor outcome under medical treatment. in all cases of surgical wound infection bacteremia occurred diagnosed on clinical basis in the presence of severe sepsis or hematogenous dissemination in the lung. the urinary tract infection occurred in a patient after urinary surgery and having a catheter in place. the immediate evolution was favorable in all cases but treatment was difficult due to the highly resistant strains and to underling diseases.conclusions: s. maltophilia should be considered in nosocomial severe infections and prophylaxis by interrupting environmental transmission has to be promoted. sawicka-grzelak a, rokosz a, luczak m. department of medical microbiology, university medical school, warsaw, poland purpose: to identify and determine the drug-susceptibility of esblpositive strains isolated from urine samples.methods: seven hundred and twelve strains of gram-negative rods were cultured from urine samples from hospitalized patients during months (july Á/november ). identification and susceptibility were performed in the automatic atb system (biomerieux, france) using id e, id gn and atb ur strips. esbl-activity was detected with double-disc synergy test (ddst according to jarlier et al., ) or using a novel method of esbl detection (dd, diagnostic disc) according to appleton ( ) . two discs were applied in this test: with cefpodoxime (cpd) and with cefpodoxime/clavulanic acid (cd ) (oxoid, england).results: five hundred and ninety-five strains ( . %) belonging to enterobacteriaceae family, strains ( . %) of non-fermenting rods and two strains ( . %) of other gram-negative rods were isolated. eighty-two esbl-producing strains ( . % of all strains) were detected. fifty-nine esbl-positive strains were susceptible to nitrofurantoin, -to norfloxacin and ciprofloxacin and -to fosfomycin.conclusions: esbl-positive strains were detected most frequently among enteric rods ( strains). nitrofurantoin and quinolones were the most active in vitro antibacterial agents against examined esblpositive uropathogens. results: the mechanisms of resistance were evaluated phenotypically using different aminoglycosides. a total of aminoglycoside resistant gram-negative strains were studied. one hundred fifty-eight strains were collected in Á/ , in and in . the resistant profiles were determined: enterobacteriaceae */ ; pseudomonas aeruginosa */ ; acinetobacter spp. */ . the most frequently phenotypes were gt (gentamicin, tobramycin) */ % and gtnet (gentamicin, tobramycin, netilmicin) */ %. the gt phenotype due to production ant( ƒ)-i enzyme, the gtnet Á/aac( )-v ( strains), aac( )-iv */one strain and aac( ƒ)-i */one strain. the resistance to amikacin in % strains was due to production aac( ?)-i ( %) and aph( ?)-vi ( %). the most of examined strains were simultaneously resistant to kanamycin and neomycin caused by production of aph( ?)-i ( %). only seven strains were resistant to all aminoglycosides due to impermeability of outer membrane. no substantial differences were observed between years.conclusions: the main mechanism of aminoglycoside resistance is fermentative modification. the high rate to gentamicin and tobramycin was due to production of ant( ƒ)-i and aac( )-v. amikacin and isepamicin were the most active aminoglycosides against gramnegative nosocomial isolates.the incidence of clostridium difficile associated diarrhoea (cdad) increased in our department from january to june .objective: to confirm the out break of cdad, to identify the risk factors and assess the effectiveness of the measures implemented for controlling this outbreak.methods: cdc definitions were used to identify the cases. the scope of the outbreak was defined. cdad incidences during the outbreak period and during the same period in were compared. risk factors (reduced mobility, antibiotic treatments . . .) were studied for patients whom length of hospital stay (lhs) was more than days. contact precautions and environmental cleaning with clona implemented were assessed.results: seventeen episodes of cdad were identified. sex ratio: . , mean age / . , mean lhs / days, mean delay for cdad occurring / days. one hundred and fifty-two patients involved in the study of risk factors. relative risk (r.r.) evaluated were: blactams (r.r. / . , ic %: . Á/ . ), reduced mobility (r.r. / . , ic %: . Á/ . ). incidence of cdad was less than two cases per days of hospitalisation after june .conclusion: we confirmed the outbreak of cdad in our department b-lactams and reduced mobility were identified as risk factors for cdad. measures implemented to control the outbreak were effectiveness.positive heart transport fluid cultures associated with severe infections in heart transplant recipients pm at the mount sinai hospital in new york city heart transplants were performed between and june . cultures were routinely performed on all heart transplant transport fluids. culture data was available for of these patients. in total / ( . %) were positive for bacteria, fungi or both. the organisms isolated included coagulase negative staphylococci ( ), pseudomonas aeruginosa ( ), staphylococcus aureus ( ), acinetobacter baumanii ( ), serratia mercescens ( ), enterobacter cloacae ( ), escherichia coli ( ), proteus mirabilus ( ), enterococcus faecalis ( ), viridans streptococci ( ), and fungi (aspergillus fumigatus ( ), penicillium species ( ), and rhodotorula rubra ( ). two heart transplant recipients had two organisms isolated from the transport fluid. isolation of resistant gram-negative bacilli in the transport fluid was associated with significant infection in / patients ( %) with the same organism. the observed infections were pneumonia secondary to e. cloacae , sternal wound infection secondary to p. aeruginosa , and bacteremia secondary to p. aeruginosa . it appears prudent to provide prophylaxis against resistant gram negative bacilli to prevent infections. zacharof ak, flevaris c, petrogianopoulos c, karachalios g, vroulis j, chartzoulakis g, drakogiorgos g, loizidou a, svoukas g. nd department of internal medicine, hellenic red cross hospital, athens, greece objective: we studied the trends of nosocomial bloodstream infection and calculated the population-attributable risk for death among hospitalized patients. methods: we perform a -year retrospective study for all patients (n / ), admitted to our department between and .results: between and , a total of patients developed episodes of nosocomial bloodstream infection. the crude infection rates increased linearly from . to . per discharges ( . Á/ . episodes per patient-days) during the -year study period. increases in the infection rates were due to gram-positive cocci, yeasts and essentially explained by infections caused by coagulasenegative staphylococci, staphylococcus aureus , enterococci, and candida species, respectively. although the crude mortality in patients with nosocomial bloodstream infections decreased from % in to % in , the in-hospital population-attributable mortality among infected patients increased from . deaths per discharges in to . per discharges in . the etiologic fraction or the proportion of deaths in patients with bloodstream infection to all deaths occurring in the hospital increased from . % in to . % in .conclusions: the incidence and the population-attributable risk for death among patients experiencing nosocomial bloodstream infections increased progressively during the last years in our department.ventilator-associated pneumonia before and after intensive care unit temporary closure pm results: we compared the incidence, causative organisms and mortality of vap in two different time periods. period june to december . period june to december . between those two periods the icu remained closed for months because of reconstruction works.period : sixty-seven consecutive patients (pts) were studied with bronchial secretions cultures at least days after mechanical ventilation (mv) initiation. the vap was diagnosed by clinical, radiological and microbiological criteria in pts ( %). causative organisms included: pseudomonas aeruginosa , acinetobacter , staphylococcus aureus , klebsiella pneum. , enterobacter . in three cases vap was proved polymicrobial. fourteen ( ) episodes of vap ( %) were developed after days mv (late vap) and were attributed to multiresistant microorganisms. mortality of vap was %.period : among consecutive pts, vap was diagnosed in ( %). causative organisms included: acinetobacter , p. aeruginosa , s. aureus , k. pneumoniae , escherichia coli . five ( ) cases were polymicrobial and cases were 'late vap' ( %). causative microorganisms had similar patterns of sensitivity to antibiotics (compared to period one). mortality of vap was %.conclusion: temporary icu closure had no significant influence on the incidence, distribution of causative organisms, their sensitivities to antibiotics and mortality of the vap. efstathiou sp a , pefanis av a , tsioulos di a , tsiakou ag a , zacharos id a , kanavaki s b , mountokalakis td a . a third university department of medicine, sotiria general hospital, athens, greece , b microbiology laboratory, sotiria general hospital, athens, greecepurpose: the aim of this study was to derive a scoring system for the prediction of outcome in adult patients with acute pyelonephritis (ap) severe enough to need hospitalization. therefore, the charts of patients ( men, median age years) were reviewed.results: logistic regression analysis identified in both sexes four independent correlates of in-hospital mortality, the coefficients of which divided by . and rounded to the nearest interval, resulted in the following integer-based scoring system: points (men) / * between concentrations of lps of the two groups were statistically significant on the nd and the th day (p b/ . ). it is concluded that the administration of lactulose may decrease systemic endotoxaemia in the field of obstructive jaundice.nosocomial infections: a prevalence study in the island of crete pm doukakis s a , tzimis l b , perogambrakis g a , kalloniatou m a , christodoulakis n a , evaggelopoulos a a , koutsoumba d a , kastanakis s a . a first medical department, 'saint george ' general hospital, chania, greece , b pharmacy department, 'saint george ' general hospital, chania, greeceprevalence surveillance is a rapid and inexpensive mode to estimate the problem of hospital-acquired infections (hais). to study the problem of nosocomial infections in our hospital, a prevalence study was made from our team in . the study included patients (the total number of hospitalized patients at the time of the study). from these patients were males ( . %) and females ( . %). one hundred and ninety-one patients ( . %) belonged in the groups of age between and years. fifty-seven patients had a urine catheter ( . %). one hundred and fifty-five/ patients ( %) received antibiotics and from these patients received one antibiotic and the remaining patients two or more. a nosocomial infection was found in patients and consequently the prevalence of hais was . %. among these, urinary tract infections were six ( . %), lower respiratory tract infections were three ( . %), surgical site infections were three ( . %), and bloodstream infections was one ( . %). the incidence of multiresistant bacteria was primarily enterococcus spp and secondary, pseudomonas aeruginosa , enterobacter spp, klebsiella pneumoniae , escherirchia coli , staphylococcus aureus , enterobacter cloacae . unfortunately prophylactic chemotherapy of long duration was found despite the suggestions of the infection control committee. regarding age the highest incidence of hais occurred in the third age group. bagirova ns, dmitrieva n. laboratory of microbiology, cancer research center of russia, moscow, russian federation objectives: to determine the pathogens and susceptibility to antimicrobials.methods: blood samples were collected from adult pts ( Á/ ). the bacteraemic episodes were classified according to the definitions of the cdc. laboratory detection of bacteraemia and fungaemia was performed according to cumitech b (blood cultures iii, ). susceptibility testing was performed by disk diffusion method (nccls).results: the total number of blood samples */ , -positive ( . % episodes of significant bacteraemias). bsi was confirmed microbiologically in of febrile pts ( . %). the most frequent pathogens were gram('/) cocci ( . %) (p b/ . ), gram((/) bacilli */ . %, fungi */ . %. coagulase-negative staphylococci (cns) represented . %, staphylococcus aureus . %, streptococcus spp. . %, enterococcus spp. . %, enterobacteriaceae . %, pseudomonas aeruginosa . %, other non-fermenting */ . %, yeast . %, mould . %, anaerobes . %. one hundred percent cns were resistant to penicillin, . % to oxacillin, . % to clindamycin, . % to cefazolin, . % to ceftazidime, . % to ciprofloxacin, . % to gentamicin, and no isolate was resistant to vancomycin. the predominant pathogens in all types of hm were gram('/) cocci (mainly cns). all gram('/) microorganisms were sensitive to vancomycin. katashinsky o a , opriatova t b , tchuev p c . a state clinical hospital, anaesteziology, odessa, ukraine , b state clinical hospital, bacteriology, odessa, ukraine , c medical university, anaesteziology, odessa, ukrainethis investigation was carried out in odessa state clinical hospital during Á/ . of the patients with post-operation complications, % of gram-negative cultures were sensitive to ceftazidime and % to amikacin. sixty-nine percent of gram-positive cultures were resistant to penicillin g, but they were sensitive to vancomycin and nitrofurantoin in and % of cases, respectively. sixty percent of isolates of pseudomonas aeruginosa were sensitive to ceftazidime and % to amikacin. rates of resistance to carbenicillin and gentamicin were and %, respectively. one hundred and fortythree isolates of escherichia coli were studied and % of them were resistant to ampicillin % to cephalothin and % to tetracycline. of the isolates tested against ciprofloxacin, all were sensitive. one hundred and eight isolates of staphylococcus aureus were studied and they were resistant only to penicillin g ( %). staphylococcus aureus was sensitive to erythromycin ( %), tetracycline ( %), oxacillin ( %) and vancomycin ( %). all isolates enterococcus faecalis were sensitive to nitrofurantoin and % to ciprofloxacin. the majority of s. aureus and e. faecalis isolates were susceptible to most other antibiotics, but the majority of e. coli isolates were resistant to the studied antibiotics expect ciprofloxacin.campylobacter foetus bacteraemia in an immunocompromised patient: a case report pm monno r a , ierardi e b , rendina m b , ceci g a , luzzi i c , de vito d a , rizzo g a , francavilla a b . a department of internal medicine and public health, university of bari, bari, italy , b department of emergency and organ transplantation, university of bari, bari, italy , c istituto superiore di sanità, rome, italy a -year-old woman was admitted for recurrent fever. the patient underwent a liver transplantation and splenectomy in . she had followed immunosuppressive therapy until when tacrolimus was added for chronic rejection. in non-hodgkin lymphoma was diagnosed and chemotherapy was started. six months later because of the presence of two metastatic encephalic foci affecting the optic chiasm, a new chemotherapy course was started with the regression of lesions. in january she was treated with steroid recycle and cyclosporine Á/azathioprine Á/prednisone reintroduction. fever occurred after months and cytomegalovirus (cmv) infection was diagnosed. treatment with ganciclovir was started with clinical remission. in november cmv infection recurred and blood cultures were positive for a bacterium that was identified as campylobacter fetus . the patient was successfully treated with intravenous ciprofloxacin. bacteremia frequently occurs in cancer patients. bacteremia due to c. fetus are rare, occurring mainly in immunocompromised patients. c. fetus expresses a proteinaceous surface layer that confers serum resistance. in our patients steroid and immunosuppression may have contributed to the development of lymphoma. all of these factors and chemotherapy have contributed to cmv infection and all have made the patient susceptible to bacteremia with this infrequently found bacterium. the clinical microbiologist should be aware of this infection in immunocompromised host. minenko sv, dmitrieva nv, sokolova en, ptushkin vv. bone marrow transplantation department, n.n. blokhin cancer research center, moscow, russian federation c'/a is the standard regimen as empirical therapy for febrile neutropenia (fn). activity of c against g'/ bacteria and g(/ bacteria, producing chromosomally-mediated b-lactamases (e.g. ampc) is suboptimal. cep is active against a broad range of g'/ and g(/, including ampc producing bacteria. the purpose of the study was to compare the efficacy of two regimens in the treatment of fn.methods: patients with fn received either cep ( g/ h) or c ( g/ h) plus amikacin ( mg/kg/day) or netilmicin ( mg/kg/day). data were collected prospectively.results: a total of pts with episodes ( / ) of fn were included. fifteen/ in cep group and / in c'/a group. the median duration of neutropenia grade , distribution of age, sex and underlying disease were comparable in both arms. mdi was in and %, cdi in and % fuo in and % in fep and c'/a groups correspondingly. response to the initial empirical regimen according to ihs criteria was in % of cep and . % of c'/a groups (p / . ). modification of therapy with a change of cep or c to carbapenem took place in and % of cep and c'/a groups (p / . ). no patient in either treatment group died due to the presenting infection. tolerability of cep was good and no laboratory abnormalities took place. transient elevation of serum creatinin level was observed in two patients c'/a group.conclusions: cep monotherapy is as effective as c'/a combination in the treatment of patients with fever and granulocytopenia purpose of the study: retrospectively, we analysed patients with intraabdominal infection for aetiology, risk factors, and outcome from hospitals in slovak republic within year (march Á/march ). results: in this group significantly more frequent were older patients ( !/ years) with cancer ( vs. %, p b/ . ). acinetobacter spp. ( vs. %, p b/ . ) and enterobacteriaceae ( vs. %, pb/ . ) were predictive for monoinfection. pre-treated patients with other antibiotics had inferior prognosis and more risk factors: permanent urinary catheter ( vs. %, pb/ . ), ventilation or intubation ( vs. %, pb/ . ) and polymicrobial infection ( vs. %, pb/ . ). the risk factors with poor prognosis were enterobacteriaceae ( vs. %, pb/ . ), diabetes mellitus as underlying disease ( vs. %, p b/ . ) and uraemia ( vs. %, p b/ . ). surprisingly, negative prognostic factor was also non-effective previous antibiotic therapy. failed patients died significantly more frequently patients due to underlying disease. cefoperazone/sulbactam was shown to be useful, effective and well tolerated also in one group of patients with % efficacy of treatment, and it belongs to a group of antibiotics suitable for treatment of nosocomial infections.