key: cord-302277-c66xm2n4
authors: Bakaletz, Lauren O.
title: Developing animal models for polymicrobial diseases
date: 2004
journal: Nat Rev Microbiol
DOI: 10.1038/nrmicro928
sha: 
doc_id: 302277
cord_uid: c66xm2n4

Polymicrobial diseases involve two or more microorganisms that act synergistically, or in succession, to mediate complex disease processes. Although polymicrobial diseases in animals and humans can be caused by similar organisms, these diseases are often also caused by organisms from different kingdoms, genera, species, strains, substrains and even by phenotypic variants of a single species. Animal models are often required to understand the mechanisms of pathogenesis, and to develop therapies and prevention regimes. However, reproducing polymicrobial diseases of humans in animal hosts presents significant challenges.

There is now compelling evidence that many infectious diseases of humans (FIG. 1) and animals (TABLE 1) are caused by more than one microorganism. The mixed microbial nature of these diseases has been recognized since the early 1920s but there has been renewed interest in this topic since the 1980s 1 , signalled by the publication of four important reviews from 1982 to the present date. Polymicrobial diseases (see BOX 1 for nomenclature) can be caused by the synergistic or sequential action of infectious agents from either the same or different kingdoms, genera, species, strains or substrains, or by different phenotypic variants of a single species 6 . Polymicrobial diseases share underlying mechanisms of pathogenesis, such as common predisposing factors (BOX 2) , but each disease has unique aspects. Although the molecular mechanisms of some polymicrobial infections are known, other polymicrobial diseases are not well understood. Owing to their complexity, the study of polymicrobial infections requires a multidisciplinary approach and specific in vitro methodologies and animal models. The development of assay systems and treatment and prevention regimes is needed.

Multiple diverse in vitro systems have been used to study polymicrobial diseases (BOX 3) . Although in vitro methods are crucial for understanding polymicrobial diseases, rigorous, reproducible and relevant animal models of human diseases are essential for the prevention and treatment of these co-infections [7] [8] [9] [10] .All animal models of human diseases have inherent limitations but they also have important advantages over in vitro methods, including the presence of organized organ systems, an intact immune system and, in inbred mice, specific genetic backgrounds, and the availability of many reagents for characterizing the immune response to sequential or co-infecting microorganisms. The availability of mice with specific genetic backgrounds can have a pivotal role in understanding the mechanisms of pathogenesis of polymicrobial diseases, as exemplified by studies on septic peritonitis 11-17 , periodontal disease 18 and Lyme arthritis 19, 20 . Understanding the molecular mechanisms underlying polymicrobial diseases of veterinary importance has also been facilitated by the use of animal models. These veterinary systems are useful examples for those researchers attempting to develop animal models of complex human diseases.

So far, most animal models for human polymicrobial diseases are rodents, usually mice, but also rats, gerbils, cotton rats and chinchillas. Other animal models include non-human primates, which are useful for modelling diseases that are caused by microorganisms with a restricted host range. For most human viral co-infections of clinical importance, good animal models and culture systems are lacking and are urgently required.

This review provides an overview of the pathogenesis of selected polymicrobial diseases, the molecular basis for some of these co-infections and describes animal models that have been developed to mimic these diseases.

Human co-infections with multiple hepatotropic viruses from the hepatitis virus group are well documented. Co-infection with multiple hepatitis viruses is possible owing to their similar routes of transmission and ability to chronically infect the host. Hepatitis A virus (HAV) co-infection of individuals that are chronically infected with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) results in a disease of increased severity and risk of death. Moreover, HBV-HCV co-infection occurs in 10-15% of HBV patients, and hepatitis G virus (HGV)-HCV co-infection occurs in 10-20% of individuals with chronic HCV infection; however, HBV-hepatitis D virus (HDV) coinfection occurs only in the setting of co-infecting HBV. VIRAL INTERFERENCE, in which replication of one virus is suppressed by another virus, is an intriguing aspect of triple HBV-HCV-HDV infection -HDV can suppress both HBV and HCV replication 29 . In a retrospective study of patients with hepatitis virus co-infections, HDV was dominant by RT-PCR detection of HDV RNA in triple co-infections, but in dual co-infections there were alternating dominant roles for either HBV or HCV. Multiple hepatotropic viral infections are associated with reduced HCV replication but increased pathology. Patients with dual or triple co-infections have more severe liver disease pathologies than patients that are

In cattle, infections with bovine viral diarrhoea virus (BVDV) can be clinically asymptomatic or can cause severe symptoms. The outcome depends on whether the primary infection occurred in utero or after birth 21 and whether the primary infection was with a cytopathic or non-cytopathic biotype of BVDV. Milder, congenital persistent infection follows foetal infection with noncytopathic BVDV. Death, or culling from the herd within 1 year of birth after failure to thrive, is common; however some persistently infected calves seem healthy at birth and survive for several years. Mucosal disease follows congenital persistent infection and is due to coinfection with cytopathic and non-cytopathic BVDV in utero. Conversely, acute bovine diarrhoeal disease is induced by primary post-natal infection with either of 

In some instances production of a virulence factor by one microorganism can increase the risk of infection or colonization by a second microorganism. This might include sharing virulence factors, such as adhesins; for example, H. influenzae shows enhanced adherence when pretreated with Bordetella pertussis adhesins.

Infection with a microorganism that results in an impaired immune system predisposes the affected individual to infection with other microorganisms, or can allow infection of a niche that is usually protected in the body. [56] [57] [58] [59] [60] [61] . BRDC pathology results from the effects of pathogen and host virulence factors. M. haemolytica produces multiple virulence factors, including a leukotoxin of the repeat in toxin (RTX) family that activates PMNs, induces production of inflammatory cytokines, results in cytoskeletal changes and causes apoptosis. Leukotoxin-activated PMNs are crucial to pathogenesis and inflammatory mediators released by neutrophils are thought to be essential because inflammation and most of the pathology in BRDC is absent human viruses, in contrast with rodent hosts, the use of greater primates for modelling human viral disease is limited by differences in the clinical presentation of disease -some diseases are asymptomatic in primates -and the expense of using primate models in research 40 . Given the difficulties of modelling diseases caused by individual viruses, it is not surprising that models of virus co-infections, such as HIV and HCV, have not been established.

A variety of small animal and lower-order nonhuman primate model systems have been developed to model human viral co-infections. Mice and ferrets have been used to study interference between influenza A virus (IAV) strains, as well as interference between cold-adapted influenza A and B vaccine reassortants and wild-type viruses [41] [42] [43] . Murine hosts have been used to study how one retrovirus can block infection by a second retrovirus 44 , and to define the role of the tissue tropisms of HELPER VIRUSES on the disease specificity of a co-infecting oncogene-containing retrovirus such as the type of tumour that is induced 45 . BALB/c and NIH Swiss mice have been used as models to analyse a putative pathogenic interaction between a murine leukaemia virus and a polyomavirus 46 . Rabbits have been used to produce models of mixed HTLV-1 and HIV-1 co-infection 47 and co-infection with HTLV types I and II 48 .

Rhesus and pig-tailed macaque monkeys have been used to model co-infection with simian immuno deficiency virus (SIV) and simian acquired immunodeficiency syndrome retrovirus type 1 (SRV-1) 49 . More recently, macaques have been used to define the susceptibility to co-infection with two human HIV-2 isolates 50 . In this model, co-infections were established in macaques that were simultaneously exposed to both viruses, whereas in macaques that were sequentially CHALLENGED, co-infections were only observed if challenge with the second HIV-2 isolate occurred early after challenge with the first HIV-2 isolate and before full SEROCONVERSION. Chimpanzees have also been used to study HIV-1 subtype B strain co-infections 51 

There are several new in vitro methods, including: • Genomic sequencing of individual microorganisms and mixed microbial ecosystems and the use of meta-genomics to study the genomes of uncultured microbial communities.

• Molecular phylogenetic studies, such as genotyping or 16S rRNA analyses, to determine the genetic relatedness or diversity of microbial community members.

• Genome-wide transcription profiling using microarrays to assess the rates of transcription during polymicrobial infection.

• Fluorescence-based imaging and detection methods such as laser confocal microscopy using fluorescent probes, fluorescent in situ hybridization (FISH) using species-specific 16S rRNA-directed oligonucleotide probes and the use of transcription and translation reporter gene constructs.

• Analyses of inter-genera bacterial signalling such as quorum sensing.

• Use of biofilm chambers and continuous culture flow cell reactors to study polymicrobial diseases.

• Co-infections of cell lines, tissue and organ cultures and extracted teeth.

• Laser capture microdissection of colonized infected tissues.

The mechanisms of synergy between pathogens in OM have been analysed using in vitro methods and animal models (reviewed in REF. 7 ). Briefly, viral infection compromises the protective functions of the Eustachian tube, alters respiratory-tract secretions, damages the mucosal epithelial lining, interferes with antibiotic efficacy, modulates the immune response and enhances bacterial adherence 77 and colonization 78 to predispose the host to bacterial OM. Influenza and parainfluenza viruses have neuraminidases that remove sialic acids from host-cell glycoproteins, which results in the exposure of receptors for pneumococci. The activity of neuraminidases allows the adherence of and/or colonization by S. pneumoniae 79 , which is one of the primary aetiological agents of acute OM.

Although all upper respiratory tract viruses can disrupt the host respiratory tract defences, each virus has a specific pathology. Not surprisingly, there are specific partnerships between viruses and bacteria in OM. In the chinchilla model (FIG. 2) IAV predisposes the host middle ear to S. pneumoniae-induced or pneumococcal OM and adenovirus infection predisposes the host middle ear to NTHI OM. IAV does not predispose the chinchilla host to NTHI-induced OM, nor does adenovirus predispose the host to either M. catarrhalis-induced OM 80 or to pneumococcal OM 78, 81 .Virus and bacteria synergy seems to be maintained in adults and children. The oropharynges of 15% of adults with experimental IAV infection were heavily colonized with S. pneumoniae six days after viral challenge 82 , whereas isolation rates for other middleear pathogens were unaffected. In children, S. pneumoniae is cultured more often from middle-ear fluids that contain IAV than from those that are culture-positive for either RSV or parainfluenza virus 83 .

Cystic fibrosis polymicrobial diseases. Upper respiratory viruses predispose the host to bacterial invasion of the lower respiratory tract and are often detected in patients with COPD 84 or cystic fibrosis (CF). In addition to bacterial factors, host determinants also have a role in co-infections of the CF lung. CF patients do not have a higher incidence of viral disease compared with non-CF individuals, but viral disease produces more significant pathology. It has been proposed that CF patients have impaired innate immunity, which allows increased virus replication and upregulated cytokine production. In turn, this results in increased bacterial colonization of the lung. Zheng and co-workers 85 showed that increased virus replication in CF patients is due to the absence of the antiviral nitric oxide synthesis pathway. This was attributed to impaired activation of signal transducer and activator of transcription (STAT1), which is an important component of the antiviral defences of the host. Compromising innate immunity provides a mechanism for the severity of viral disease in CF and the establishment of bacterial co-infections.

Expression of virulence determinants by Pseudomonas aeruginosa, a pathogen of CF patients, can depend on signals produced by other bacteria. Transcriptional profiling in vitro coupled with research in an animal model showed that adding exogenous signalling molecules, in neutrophil-depleted animals. In pigs, porcine respiratory disease complex (PRDC) is a similar disease complex that is caused by co-infection with one of several porcine respiratory tract viruses and members of the Pasteurellaceae family [62] [63] [64] [65] [66] .

Porcine reproductive and respiratory syndrome. PRRS is caused by PRRSV co-infection with multiple bacterial pathogens including Streptococcus suis type II 67 , Bordetella bronchiseptica 68 , Mycoplasma hyopneumoniae 69 and Actinobacillus pleuropneumoniae 70 .

In turkeys, PEMS is caused by turkey coronavirus, avian pneumovirus or Newcastle disease virus co-infection with enteropathogenic Escherichia coli 71, 72 . 1) . Despite the diverse spectrum of diseases and anatomical niches, there are common underlying mechanisms involved in these co-infections. Often, viral disruption of host defences has a role in the development of bacterial co-infections.

In otitis media, which is a middle ear infection, a synergistic interaction that results in disease owing to co-infection with an upper respiratory tract virus and three bacterial species -Streptococcus pneumoniae, nontypeable Haemophilus influenzae (NTHI) and Moraxella catarrhalis -is well documented. However, certain viruses such as respiratory syncytial virus (RSV) and rhinovirus seem to predispose affected individuals more often to bacterial OM. The saying that children ''get a cold and a week later develop OM'' is substantiated by epidemiological data that indicate a seasonal influence on the coincidence of 'colds' and OM, as well as evidence for a peak incidence of virus isolation that is coincident with, or immediately preceding, peak incidence of OM (FIG. 2) . In the recent Finnish OM Cohort Study and Finnish OM Vaccine Trial, the relationship between viruses and OM was supported by data that showed the presence of a virus in either nasopharyngeal aspirates or middle-ear fluid specimens in 54% or 67% of OM cases in these studies, respectively 76 . Rhinovirus was the most commonly isolated virus, followed by enterovirus and RSV. A specific virus was detected in two-thirds of all cases of acute OM in young children, but only those viruses that are tested for can be detected, so this figure is likely to underestimate the proportion of acute OM events with viral co-infection.

A sequential inoculation model has been developed in mice to probe the mechanisms of the interaction between S. pneumoniae and IAV. Mice infected simultaneously with S. pneumoniae and IAV displayed gradual weight loss and increased mortality, commensurate with an additive effect. Conversely, mice infected with S. pneumoniae seven days after IAV infection uniformly died within 24 hours and had significant bacteraemia -lethality was due to overwhelming pneumococcal septicaemia 89 . This model is being used to define the molecular mechanisms of the lethal synergy of IAV with S. pneumoniae 90 . The activity of viral neuraminidase was found to be crucial to this synergistic relationship 91 and, in common with OM, has an important role in predisposing both the upper and lower respiratory tracts to invasion by S. pneumoniae.

such as autoinducer-2, or the production of this molecule by the oropharyngeal bacterial flora upregulated the expression of genes that encode virulence factors 87 . Modulation of gene expression by interspecies communication between normal flora and pathogenic bacteria could therefore have a role in polymicrobial diseases.

Periodontitis. Some herpesviruses, including human cytomegalovirus (HCMV), Epstein-Barr virus type 1 (EBV-1) and HSV, have been implicated in the pathogenesis of a severe and highly aggressive form of periodontitis through co-infection with Porphyromonas gingivalis 87, 88 . HCMV and HSV were detected at significant levels using PCR in periodontal disease and were shown to be good predictors of the presence of P. gingivalis. EBV-1 was not linked to isolation of P. gingivalis but was also predictive of active disease 87 . A common theme has emerged from these models that upper respiratory tract viruses of both animal and human hosts can predispose the respiratory tract to infection by pasteurellaceae in BRDC and PRDC in animals and periodontitis, sinusitis, COPD and OM in humans.

A mouse model has been developed to evaluate the role of respiratory dendritic cells (RDCs) in viral-bacterial co-infections 105 . RDC migration from the lungs to the secondary lymph nodes after infection with pulmonary virus is monitored by the use of a fluorescent dye. After inoculation with influenza virus, the rate of RDC migration to the draining peribronchial lymph nodes increased, but this only occurred during the first 24 hours after virus infection. After 24 hours, RDCs did not migrate, despite virus replication and pulmonary inflammation. Moreover, viral infection suppressed additional RDC migration in response to either a second pulmonary virus infection or administration of BACTERIAL CPG DNA. In addition to suppressed RDC migration, there was also suppression of an antiviral pulmonary CD8 + T-cell response. It seems likely that the transient suppression of RDC migration and the delayed development of an effective adaptive immune response to a second infection might be another mechanism by which influenza virus predisposes the host to bacterial co-infection.

Atrophic rhinitis. Infection with more than one bacterial species is common in animals and man. In pigs, atrophic rhinitis (AR), which is characterized by severe atrophy of the nasal TURBINATES, is caused by co-infection with strains of B. bronchiseptica and heat-labile toxin-producing strains of P. multocida [106] [107] [108] . P. multocida can adhere to respiratory tissues, but co-infection with B. bronchiseptica allows more efficient colonization by P. multocida. P. multocida produces a dermonecrotic toxin called PMT (for P. multocida toxin), which interferes with normal bone modelling in both the nasal turbinates and long bones in swine, and is distinct from the B. bronchiseptica dermonecrotic toxin (DNT). In porcine models, PMT causes a more serious form of AR known as progressive AR, whereas B. bronchiseptica infection alone induces a milder, or non-progressive, form of the disease.

Bacterial co-infections of humans include orofacial infections 109 , adenotonsillitis 110 , persistent osteomyelitis 111 , peritonitis 112 , chronic sinusitis 113 , abscesses 114, 115 , necrotizing fasciitis and approximately one-third of urinary tract infections (UTIs) in the elderly 116 and in renal transplant patients 117 . Two important bacterial co-infections are periodontitis and vaginosis.

Periodontitis. Periodontal disease causes tooth loss and is associated with systemic vascular diseases such as atherosclerosis and carotid coronary stenotic artery disease 118 . Periodontitis in an expectant mother can contribute to both low birth weight and pre-term labour 119 . Periodontal disease is initiated by the formation of a

A murine model has been developed to reproduce the pathogenesis of human meningococcaemia, which often results in serious symptoms or death 92 . In this model, adult BALB/c mice are infected intranasally with a mouse-adapted IAV, then seven or ten days later, are co-infected with Neisseria meningitidis. Fatal meningococcal pneumonia and bacteraemia occurred in mice challenged at seven, but not ten, days after IAV infection. Meningococcal pneumonia and bacteraemia did not develop in mice that were not co-infected with IAV. Susceptibility to lethal infections correlated with peak interferon-γ production in the lungs and decreased IAV load and production of IL-10, which indicates that transient IAV-induced modulation of host immunity has a role in susceptibility to N. meningitidis co-infection.

The only viral-bacterial co-infection model for OM is the chinchilla. Before 1980, most OM studies in the chinchilla used inoculation of pathogens directly into the middle ear. Although this induces disease in almost all of the animals inoculated and is therefore extremely useful for studies of therapeutics and surgical intervention strategies, it bypasses all of the early steps in the development of the pathogenesis of the disease, including colonization of the nasopharynx, ascension of the Eustachian tube and initiation of infection in the middle ear. Giebink and co-workers 93 , developed a clinically relevant model in which chinchillas were challenged intranasally with both S. pneumoniae and IAV. This study showed that 4% of the chinchillas that were infected with IAV alone, and 21% of those inoculated with S. pneumoniae alone, developed OM, but of the animals that were coinfected with both microorganisms,~67% developed OM (FIG. 2) . This model has been useful in defining the molecular mechanisms of IAV predisposition to pneumococcal OM [94] [95] [96] and to investigate the role of pneumococcal virulence determinants in OM 81 .

To study the pathogenesis of OM mediated by NTHI, a chinchilla model that uses a co-challenge method was developed. In this model, adenovirus infection can predispose the chinchilla to NTHI invasion of the middle ear 97 (FIG. 2) ; however, IAV infection has no effect. This adenovirus-NTHI co-infection model has been used to study the mechanisms of adenovirus predisposition to NTHI-induced OM 98, 99 , to identify new NTHI virulence determinants 100 and to assay the relative efficacies of different NTHI-derived vaccine candidates for OM [101] [102] [103] . A cotton rat model of RSV and NTHI co-infection has also been developed to study co-infections of the respiratory tract 104 . In the cotton rat, colonization of the respiratory tract with NTHI increased to a maximum level four days after infection with RSV and colonization was increased compared with rats that had not been infected with RSV. NTHI colonization of the respiratory tract was increased by RSV co-infection and, although the mechanisms underlying this relationship are not understood, this model might be useful to determine the mechanisms of RSV predisposition to bacterial OM. BACTERIAL formed, in terms of microbial constituents, is the main predictor for periodontal disease.

Actinobacillus actinomycetemcomitans, P. gingivalis and Bacteroides forsythus are the main periodontal pathogens. Periodontal disease covers a range of clinical symptoms and there are multiple forms of periodontitis in children and adults. In individuals under 20 years of age, A. actinomycetemcomitans is the main bacterial pathogen, whereas in adults aged 35 years or older, periodontitis has been linked to P. gingivalis and B. forsythus. Spirochaetes, especially Treponema denticola, have been implicated in periodontitis despite the fact that most oral spirochaetes have not been successfully cultured. Recent studies using molecular phylogenetic techniques have implicated new bacterial biofilm on the tooth surface followed by bacterial invasion of gingival tissues 119 . Teeth have a non-shedding surface and are located in a warm, moist environment, so are a particularly suitable niche for biofilm formation by the oral microbial flora. In periodontitis, coaggregation -a process in which genetically distinct bacteria become interconnected by specific adhesins -is central to the formation of complex multispecies biofilms 120 (FIG. 3) . In dental plaque, primary colonizers such as Streptococcus gordonii, and other oral streptococci that express adhesins, provide a film on which other bacterial colonizers assemble the biofilm. It was thought that the abundance of plaque that formed was responsible for the induction of periodontitis but, at present, the favoured hypothesis is that the quality of the plaque 

Periodontal disease. In most individuals, periodontal pathogens trigger an inflammatory response that effectively prevents microbial colonization and invasion of adjacent gingival tissues. However, individuals that have specific IL-1 polymorphisms that result in increased levels of IL-1 expression are predisposed to periodontal disease. Using this criterion, a mouse model of polymicrobial-induced osteoclastogenesis, bacterial penetration, leukocyte recruitment and softtissue necrosis has been developed to clarify the role of cytokines in periodontal disease. In this model, the dental pulp of the first mandibular molars is exposed by surgically clipping the mesial cusps and then a mixture of putative oral pathogens is inoculated into the dental pulp (FIG. 4a) . By monitoring the size of osseous lesions, tissue necrosis, osteoclastogenesis, osteoclastic activity, inflammatory cell recruitment and bacterial penetration into tissue periodontal disease, pathogenic mechanisms can be investigated 135 . IL-1 or TNF receptor signalling does not seem to be required for bacteria-induced osteoclastogenesis and bone loss in this model, but does have a crucial role in protecting the host against anaerobic co-infections.

A rat model of periodontitis was developed to test adherent (rough) and non-adherent (smooth) variants of A. actinomycetemcomitans for virulence, as well as to assess phenotypic reversion in vivo 136 . In this model, the normal flora of the oral cavity of Sprague-Dawley rats is reduced by antibiotic treatment, after which rats are inoculated with A. actinomycetemcomitans by either normal ingestion of food layered with bacterial cultures, oral swabbing or gastric lavage (FIG. 4b) . When clinical isolates of A. actinomycetemcomitans were compared with laboratory-adapted variants, Fine et al. 136 found that the clinical strains were more efficient at colonization and persisted longer in the rat oral cavity than laboratory strains. Rough variants were more efficient colonizers of the rat oral cavity than smooth variants, regardless of the method of inoculation, although feeding was the preferred method owing to the similarity with human disease. Importantly, rats that were orally infected with A. actinomycetemcomitans by feeding developed immunoglobulin G (IgG) antibodies to the bacteria and had bone loss that was typical of periodontitis. This model has not been used to study the process of bacterial co-infection in periodontitis, but has been used to identify a gene locus that is important in virulence and which mediates tight adherence by A. actinomycetemcomitans 137 .

A primate model (Macaca fascicularis) of periodontal disease uses silk ligatures tied around the posterior teeth to induce plaque accumulation and the initiation of periodontitis 138 . So far, this model has only been used for single pathogen studies, but is considered to be a relevant animal model of periodontal disease owing to the similarity of clinical and histological features with those of periodontal disease of humans, and because, in this model, periodontal destruction is clearly triggered by bacterial infection 139 . species or phylotypes -including members of the uncultivated bacterial division TM7 -in periodontitis, dental caries and halitosis [121] [122] [123] [124] [125] [126] [127] . In many of these studies, not only were new species and phylotypes identified, but bacteria that are known to be oral pathogens were found to be numerically minor, which was expected because ~50% of oral flora have not been cultivated 123 .

In localized juvenile periodontitis (LJP), the leukotoxin of A. actinomycetemcomitans, like that of M. haemolytica, is the best-studied virulence factor. This leukotoxin selectively kills PMNs and macrophages in vitro, and PMNs and macrophages are important components of the host defence in vivo. Expression of this leukotoxin is variable among A. actinomycetemcomitans isolates and the leukotoxin-expression phenotype correlates with differences in the promoter region of the leukotoxin gene operon 128 . A subset of leukotoxin-overproducing strains are more virulent and are associated with LJP in humans.

Neutrophil abnormalities seem to be an important predisposing condition for periodontal disease. In addition, loss of tooth attachment and bone resorption, which are important events in periodontal disease, occur together with increased IL-1 and tumour-necrosis factor (TNF) activities. The production of IL-1 and TNF (both of which are pro-inflammatory cytokines) has been correlated with the spread of inflammatory cells to connective tissues, the loss of connective tissue attachment, osteoclast formation and the loss of alveolar bone. An overzealous host response to periodontal pathogens, resulting in excessive production of IL-1 and TNF, is hypothesized to be responsible for much of the damage that occurs in periodontal disease 129 .

ial co-infection. The mucosal environment of the vagina is influenced by developmental and hormonal changes 131 . The most common bacterial constituents of the vaginal microflora are lactobacilli, including Lactobacillus crispatus and Lactobacillus jensenii 131 . When these hydrogen peroxide (H 2 O 2 )-producing lactobacilli are outcompeted by anaerobic and facultatively anaerobic members of the vaginal flora, BV develops with a concomitant rise in vaginal pH, which further promotes the growth of the resident lactobacilli. BV is common, occurring in 5-51% of the global female population 130 and the role of lactobacilli in the maintenance of vaginal homeostasis has been well studied. Women with stable bacterial colonization have a reduced risk of developing BV 132 . Normal vaginal flora has a role in defence against the acquisition of other pathogenic microorganisms, including those that are responsible for sexually transmissible diseases (STDs), and BV is a strong predictor of STD acquisition 133 . Compared with subjects with normal vaginal flora, subjects that have BV are more likely to test positive for Neisseria gonorrhoea and Chlamydia trachomatis. Recently, BV has also been found to be associated with an increased risk of HSV-2 infection 134 .

Candida species. Co-infection with multiple Candida strains and SUBSTRAINS are also found. Regardless of the co-pathogens, mycotic co-infections of the oral and vaginal cavities, on indwelling prosthetic devices, or systemic infection of the blood can present significant therapeutic challenges. Difficulty in treating some of these infections is partly attributed to the formation of biofilms by Candida spp. 152 Biofilm formation on devices such as prosthetic heart valves and catheters has been studied in vitro 95 . When cultured on a variety of catheter materials, Candida spp. form biofilms comprising a matrix of microcolonies of both the yeast and the filamentous hyphal forms. In studies of mixed microbial populations, Candida spp. form biofilms with several bacterial species, including Staphylococcus epidermidis and oral streptococcal species. The receptor for Candida albicans co-aggregation with S. gordonii is a complex cell surface polysaccharide that is expressed on the surface of the bacterium. The interaction between yeast cells and oral streptococci or other bacteria has important implications for the mechanisms of yeast infections of the oral cavity, in addition to promoting biofilm formation on a variety of surfaces. In the oral cavity, Candida-bacterial interactions are responsible for denture stomatitis, angular cheilitis and gingivitis, and also have a role in periodontitis 153 .

Although the role of pathogenic enterococci and their role in peritonitis is not understood, many putative virulence factors have been identified using animal models. Available animal models include systemic infection in mice and compartmentalized infection in rats, and the bacterial virulence factors that have been identified using each model differ 140 . This indicates that both host and pathogen factors contribute to peritonitis and, perhaps, that the animal models are quite different. Nevertheless, these models have identified a role for cytokines in septic shock, a protective role for IL-10 against lethal shock 141 , a role for STAT4 in the mortality seen in bacterial co-infection sepsis 142 and helped to define the role of the classical pathway of complement activation in defence against polymicrobial peritonitis 143 . Animal models of bacterial co-infection peritonitis and/or sepsis can involve any of the following methods for induction of infection: peritoneal implantation of microbe-filled gelatin capsules 140, 141 ; intraperitoneal injection of faecal suspensions 17 or caecal ligation and puncture 112, [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] .

Candida and mixed infections. As defined by Soll and his colleagues 6 Bacterial suspensions that are being tested for the ability to cause periodontal disease are injected into the dental pulp and the mouse is monitored for signs of periodontal disease by methods that include examination of osseous lesions, tissue necrosis, inflammatory cell recruitment, bacterial tissue penetration and osteoclastogenesis. b | In the rat model bacteria are grown using standard laboratory procedures and washed 3 times with phosphate-buffered saline (PBS) supplemented with 3% sucrose. The rats are pretreated with antibiotics and the mouth is swabbed with chlorhexidine to deplete the oral flora. The bacterial suspension is mixed into the rat's food so that this animal model replicates, as far as possible, the natural route of infection for periodontal disease. After daily inoculation the rats are assessed for bacterial colonization and bone loss. Using this model, different strains of bacteria and the contribution of different virulence loci can be tested. cfu, colony-forming units. of these genetic manipulations 159 . A murine host has been used as a model of systemic candidiasis 160 and there is also a murine model for C. glabrata-induced vaginitis 161 . In the C. glabrata model, the increased susceptibility of non-obese diabetic mice to C. glabratainduced vaginitis compared with their non-diabetic counterparts indicates a link between susceptibility to diabetes and infection with C. glabrata. In addition to studies of Candida genetics and pathogenicity 162 , this model is useful for the evaluation of the relative efficacy of antimycotic agents and probiotics for the prevention of vaginitis.

An animal model of haematogenously disseminated candidiasis has recently been developed 163 that can investigate the role of phenotype switching in candidiasis. In this model (FIG. 5) mice were injected with engineered C. albicans strains in which the transition between yeast and filamentous forms is under the control of a doxycycline-regulated promoter. Mice that were infected with strains that switched to the filamentous form died, whereas those infected with strains that could not switch from the yeast to the filamentous form survived, despite the fact that the fungal burdens in both groups were nearly identical. These data indicate that the filamentous form is important for mortality but that the yeast form of C. albicans is important for dissemination to deeper tissues.

Parasite-parasite co-infections. Several human diseases have mixed parasitic aetiologies, including co-infections with Plasmodium spp. and nematodes. In some studies, co-infection with a helminth seemed to confer protection against severe complications of malaria 164 , but this is not always the case. When infected controls with a low helminth burden were compared with those with circulating helminth schizonts, co-infection with Ascaris lumbricoides was found to be associated with protection from cerebral malaria. In addition, a later study showed a significant association between Ascaris infection and the risk of co-infection with Plasmodium falciparum and Plasmodium vivax, indicating that pre-existing Ascaris infection might increase host tolerance to coexisting Plasmodium spp. 165 Subsequently, helminthinfected patients were found to be more likely to develop falciparum malaria compared with those that were not co-infected 166 . Moreover, the risk of developing falciparum malaria increased with the number of coinfecting helminth species. Collectively, these findings indicate that a helminth-mediated helper T cell 2 (T H 2) shift (an immune response that is biased towards that which is characteristic of a T H 2-mediated response) might have a complex impact on malaria co-infection -decreasing antisporozoite immunity but inducing a protective outcome against severe complications of malaria. Although the underlying mechanism is less clear, Mwatha et al. 169 showed that exposure of Schistosoma mansoni-infected children to P. falciparum had a significant influence on the severity of hepatosplenomegaly (enlargement of the liver and spleen) that was observed in co-infected children.

A new category of polymicrobial diseases has been proposed for Candida spp. in which the infection is due to phenotypic heterogeneity 154 . In addition to the hypha-bud transition, C. albicans has a reversible, high-frequency phenotype switch that can be identified by differences in colony morphology. C. albicans cells of two phenotypic phases have different virulence characteristics. The ability of this human pathogen to rapidly switch between phenotypes could be a higher-order pathogenic trait. Support for this hypothesis comes from studies in which strains that cause deep tissue mycoses were shown to switch at higher frequencies than those that cause superficial infections. Furthermore, pathogenic C. albicans strains that were isolated from the oral cavity switch at higher rates than commensal strains that were isolated from the same site. A clinically relevant example of the role of both phenotype and mating-type switching in disease was characterized by Brockert et al. 155 , who investigated oral cavity and vaginal isolates of C. glabrata in three patients with vaginitis. The results of this study showed that switching occurs at sites of infection, that different switch phenotypes of the same strain can dominate in different anatomical locations in the same host and that mating-type switching occurs in vivo.

These co-infections can cause disease in the lower respiratory tract. In CF patients, clinical specimens that also harbour C. albicans contain nine times the amount of P. aeruginosa compared with patients that do not harbour C. albicans 156 . Moreover, sputum samples of 6-70% of CF patients contain C. albicans in addition to P. aeruginosa. Hogan and Kolter 157 showed that P. aeruginosa forms a dense biofilm on C. albicans filaments in vitro and, in doing so, kills the fungus. P. aeruginosa fails to bind to, or kill, the yeast form of C. albicans. It is unclear if a similar relationship between these two pathogens functions in vivo but, as several P. aeruginosa virulence factors that are important in human disease are also involved in killing the fungal filaments, this co-culture system could prove useful for the study of the pathogenesis of P. aeruginosa-induced disease.

Owing to the ability of Candida spp. to switch between bud and hypha (or hypha-like) forms as well as to switch phenotype, all animal models of Candida infection are likely to represent one or another of the multiple polymicrobial states that have been proposed for this microorganism. A rat model of oral colonization has been used to compare the relative pathogenicity of different Candida strains as well as to determine the effect of chemotherapeutic immunosuppression on the ability of Candida spp. to switch from a commensal to an invasive phenotype 158 . A rat model of oral candidiasis has also been developed and used to assay isogenic derivatives of a virulent C. albicans strain for the biological consequences

Co-infection with Schistosoma species and Plasmodium species has been modelled in field voles and mice since 1956 (REF. 172) , with conflicting observations concerning the ability of one parasite to suppress the capacity of the other to infect the host 173, 174 . Results obtained seem to depend on the Plasmodium species used as well as the immune status of the host. S. mansoni is, however, a potent inducer of a T H 2 dominant response, not only to itself but also to other BYSTANDER ANTIGENS that are present in a host, so it does have an influence on the clinical outcome in these co-infections 175, 176 . Synergistic interactions between specific protozoans and helminths are often ascribed to the immunosuppression that is characteristic of protozoan infections 177 and which is observed in the mouse, which is the main model for these infections A mouse model has been used to model the arthritis and carditis that can occur in co-infections with B. microti and B. burgdorferi 19 . Co-infection resulted in a significant increase in symptoms of arthritis. This increase was correlated with a reduction in concentrations of the cytokines IL-10 and IL-13. A mouse model for tick-borne Lyme arthritis mediated by co-infection with B. burgdorferi and a causative agent of human granulocytic ehrlichiosis (HGE) has been developed 178, 179 (FIG. 6) . Co-infection results in increased titres of both pathogens and more severe arthritis than does infection with B. burgdorferi alone. Co-infection resulted in reduced concentrations of IL-12, IFN-γ and TNF-α and increased concentrations of IL-6. IFN-γ expression in macrophages was suppressed, which might indicate a reduction in phagocytic activity in co-infection. These models will allow us to define the modulation of host immune responsiveness that occurs in those individuals that are simultaneously or sequentially infected with multiple tick-borne pathogens 179 .

Co-infections can arise as a result of the virus-induced immunosuppression that is characteristic of a subset of human viral pathogens, the best characterized of which is HIV.

Schistosomiasis is a chronic helminth infection that is caused by S.mansoni. In HCV and S. mansoni co-infection, there is a higher incidence of viral persistence and accelerated damage to the liver than when the patient is infected with either infectious agent alone. In a recent study, stimulation of CD4 + T cells with HCV antigens produced a type 1 cytokine profile in patients infected with HCV, whereas in patients that were co-infected with HCV and S. mansoni, a type 2 cytokine predominance was evident despite the fact that T cells that were recovered from both patient populations responded in the same manner to stimulation with schistosomal antigens 168 . The helminth-induced inability to generate an HCV-specific CD4 + /T H 1 T-cell response has been shown to have a role in the persistence and severity of HCV infection, which indicates that the induction of a strong cellular immune response through new therapeutic approaches might limit subsequent liver damage in those individuals with chronic HCV infection 169 .

Parasite-bacteria co-infections. One example of coinfection with a parasite and bacterium in humans is that of Borrelia burgdorferi (the causative agent of Lyme disease) and the intra-erythrocytic parasite Babesia microti. Both of these pathogens are transmitted by the tick vector Ixodes scapularis. Co-infection can occur by a bite from a single tick carrying multiple pathogens, or from multiple tick bites. The first cases of Lyme borreliosis and babesiosis co-infection were reported in the mid 1980s, with parasite-bacteria co-infection rates of up to 33% among those with confirmed tick-borne infection in certain populations. Although ticks can also harbour the human pathogen Anaplasma phagocytophilum, Lyme borreliosis and babesiosis coinfection accounts for ~80% of polymicrobial disease in the eastern United States. Consistent with the theme for other co-infections involving a parasite, patients that harbour both of these pathogens had more severe and longer-lasting symptoms than those with Lyme borreliosis alone 170 (NUG) and is characterized by a surface biofilm of mixed microbial flora overlying a subsurface flora comprising dense aggregates of spirochaetes. In contrast to NUG, high levels of yeast and herpes-like viruses were observed using transmission electron microscopy examination of tissues recovered from the former patient group. Herpes-like particles were observed in 56.5% of biopsies obtained from HIV-infected patients with NUP. These findings correlate well with those of Contreras and co-workers 183, 184 in which co-infection with herpesvirus was associated with high levels of periodontopathic bacteria. The role of viruses in the pathogenesis of NUP or periodontitis is not known but, in addition to inducing immunosuppression, it has been suggested that viruses might promote the overgrowth of bacterial pathogens and/or induce the release of tissuedestroying cytokines by host cells 185 . 32, 180 .

In addition to systemic diseases, localized infections with Candida spp., such as thrush in the oral cavity, are common co-infections in HIV-infected individuals 181 . The commensal oral flora acquires an invasive phenotype in the HIV-infected host, and C. albicans is indicative of a defect in host T-cell immunity in HIV infection 182 . Oropharyngeal candidiasis develops iñ 20-50% of HIV-infected patients and often precedes the development of a more invasive Candida infection, oesophageal candidiasis. The progressive immunosuppression that is characteristic of HIV infection provides a mechanism for the development of oesophageal candidiasis, which is a reportable AIDSdefining opportunistic illness.

Another disease of the oral cavity in HIV-seropositive patients is necrotizing ulcerative periodontitis (NUP), which is a disease that is characterized by ulcerated gingival papillae 122 Figure 6 | Animal model for Lyme disease and human granulocytic ehrlichiosis (HGE) co-infection. These diseases share a tick vector, Ixodes scapularis, and to analyse whether Ehrlichia sp. and Borrelia burgorferi (the causative agents of HGE and Lyme disease, respectively) co-infection leads to increased severity of spirochaete-induced Lyme arthritis a mouse model has been developed. Mice are infected intradermally with either spirochaetes (B. burgdorferi cultured in vitro) or HGE (blood culture from a SCID mouse, see inset panel) 178 . Arthritis and presence of the two pathogens can then be determined through histopathology, PCR to detect bacterial DNA and by assessing immune responses. Ticks were allowed to feed on all groups of mice to assess transmission of the pathogens. After feeding, PCR (HGE) and immunofluorescence (B. burgdorferi) were used for pathogen detection.

and not due to either increased apoptosis or altered distribution of these cells between the spleen, blood or lymphatics. The ability of measles virus to suppress both innate and adaptive immune responses is thought to be responsible for the increased susceptibility to bacterial co-infection.

The future of polymicrobial disease research Molecular methods are now being used together with conventional culture techniques to determine the identity of the full complement of microorganisms that are involved in co-infections and to determine the interactions between these microorganisms. As a result, additional diseases of polymicrobial origin will be identified. This will necessitate the development of new animal models and new in vitro methods for the study of polymicrobial diseases. Uncovering the molecular mechanisms that are involved in the pathogenesis of complex diseases might show that changes in lifestyle, such as smoking cessation or dietary changes, could prevent co-infections. Developing methods to disrupt biofilms are one target for researchers. New antimicrobials and vaccine candidates for both the predisposing and the co-infecting microorganisms will be sought. Therapeutic approaches for polymicrobial diseases might include the use of probiotics for the treatment or prevention of vaginal infections, gastroenteritis, inflammatory bowel disease, UTIs and periodontitis. Moreover, advances in nanotechnology and biomedical engineering will allow the development of new ways to deliver these therapeutic or preventative agents in a disease-or site-specific manner such as the design and use of 'intelligent implants' 193 . These indwelling devices might be embedded with sensors to detect the biofilm-forming microorganisms and signal the release of antimicrobial agents stored in an internal reservoir. As the organizers of the first satellite conference on diseases of mixed microbial aetiology (see the online links box) stated -polymicrobial diseases are ''a concept whose time has come'' 1 .

virus cannot present antigen to T cells and have a diminished capacity to secrete Ig or proliferate. Susceptibility to bacterial co-infection is likely owing to these underlying immune defects that result in the hallmark of measles virus infection -inhibition of the proliferation of CD4 + and CD8 + T cells [186] [187] [188] [189] .

A primate model of HIV-induced immunosuppression that developes cutaneous leishmaniasis has been developed in rhesus macaques. In this model, macaques are chronically infected with SIV, and then co-infected with Leishmania major metacyclic promastigotes by intradermal injection. Lesion size, parasite load and SIV viraemia are measured weekly. This model has been used to assay both the synergistic relationship between these two pathogens and the responsiveness to, and relative protective efficacy of, CpG oligodeoxynucleotides delivered to co-and mono-infected macaques 190 . Recently, a rhesus monkey model for SIV predisposition to Mycobacterium leprae co-infection has been developed, which showed that co-infection increases the susceptibility to leprosy regardless of the timing between the two infections 191 .

As mentioned earlier, measles virus-induced immunosuppression often leads to bacterial co-infection. To understand the mechanisms of co-infection, a murine model of combined measles virus and Listeria monocytogenes infection was developed 192 . In this model system, transgenic mice expressing the human measles virus receptor CD46 are co-infected with measles virus and L. monocytogenes, or are challenged with the bacterial pathogen alone. Mice co-infected with measles virus were more susceptible to infection with L. monocytogenes and this susceptibility corresponded with a reduction in the macrophage and PMN populations in the spleen, as well as a reduction in IFN-γ production by CD4 + T cells. A reduction in CD11b + macrophages and IFN-γ producing T cells was found to be due to reduced proliferative expansion

Introduces and provides a concise overview of polymicrobial diseases

The role of microbial interactions in infectious disease

Mechanisms of bacterial superinfections in viral pneumonias

Respiratory viral infection predisposing for bacterial disease: a concise review

Molecular pathogenesis of pneumococcal pneumonia

Polymicrobial Diseases

Polymicrobial Diseases

Regulation of gene expression by cell-to-cell communication: acylhomoserine lactone quorum sensing

Bacterial biofilms: an emerging link to disease pathogenesis

endotoxemia and polymicrobial septic peritonitis

Resistance to acute septic peritonitis in poly(ADP-ribose) polymerase-1-deficient mice

Interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury

CD40 contributes to lethality in acute sepsis: in vivo role for CD40 in innate immunity

Role of the classical pathway of complement activation in experimentally induced polymicrobial peritonitis

Mice lacking monocyte chemoattractant protein 1 have enhanced susceptibility to an interstitial polymicrobial infection due to impaired monocyte recruitment

Provided the background for the development of an animal model of parasitic co-infection, as well as demonstrating the potential importance of mouse strain -genetic background -in disease outcome

Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis suppresses IL-2 and IFN-γ production and promotes an IL-4 response in C3H/HeJ mice

Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study

Pathogenesis and clinical aspects of a respiratory porcine reproductive and respiratory syndrome virus infection

Dual infections of PRRSV/influenza or PRRSV/Actinobacillus pleuropneumoniae in the respiratory tract

Experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus

Induction of dual infections in newborn and three-week-old pigs by use of two plaque size variants of porcine reproductive and respiratory syndrome virus

Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus

Pathogenesis of postweaning multisystemic wasting syndrome reproduced by co-infection with Korean isolates of porcine circovirus 2 and porcine parvovirus

Replication status and histological features of patients with triple (B, C, D) and dual (B, C) hepatic infections

Polymicrobial Diseases

Hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infections in alcoholics

HIV/HCV co-infection: putting the pieces of the puzzle together

Polymicrobial Diseases

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8): key aspects of epidemiology and pathogenesis

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) as a tumour virus

HIV and herpes co-infection, an unfortunate partnership

Epidemiology of herpes and HIV co-infection

The interaction between herpes simplex virus and human immunodeficiency virus

The role of sexually transmitted diseases in HIV transmission

G protein-coupled receptors in HIV and SIV entry: new perspectives on lentivirus-host interactions and on the utility of animal models

Interference between cold-adapted (ca) influenza A and B vaccine reassortants or between ca reassortants and wild-type strains in eggs and mice

Interference by a non-defective variant of influenza A virus is due to enhanced RNA synthesis and assembly

Interference following dual inoculation with influenza A (H3N2) and (H1N1) viruses in ferrets and volunteers

Post-entry restriction of retroviral infections

The helper virus envelope glycoprotein affects the disease specificity of a recombinant murine leukemia virus carrying a v-myc oncogene

A model for mixed virus disease: coinfection with Moloney murine leukemia virus potentiates runting induced by polyomavirus (A2 strain) in BALB/c and NIH Swiss mice

Evidence for dual infection of rabbits with the human retroviruses HTLV-I and HIV-1

Dual infection of rabbits with human T cell lymphotropic virus types I and II

Infection of macaque monkeys with simian immunodeficiency virus from African green monkeys: virulence and activation of latent infection

Identification of a window period for susceptibility to dual infection with two distinct human immunodeficiency virus type 2 isolates in a Macaca nemestrina (pig-tailed macaque) model

One of the few examples of an attempt to model a human viral co-infection in which viral host restriction presents significant challenges

Extensive diversification of human immunodeficiency virus type 1 subtype B strains during dual infection of a chimpanzee that progressed to AIDS

Interference between non-A, non-B and hepatitis B virus infection in chimpanzees

Hepatitis δ-virus cDNA sequence from an acutely HBV-infected chimpanzee: sequence conservation in experimental animals

Molecular genetic analysis of virulence in Mannheimia (Pasteurella) haemolytica

Coinfection with BHV-1 modulates cell adhesion and invasion by P. multocida and Mannheimia (Pasteurella) haemolytica

Smoke and viral infection cause cilia loss detectable by bronchoalveolar lavage cytology and dynein ELISA

Ultrastructural features of lesions in bronchiolar epithelium in induced respiratory syncytial virus pneumonia of calves

Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: immunofluorescent and electron microscopic studies

Pathological study of experimentally induced bovine respiratory syncytial viral infection in lambs

Role of α/β interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins

Nonstructural proteins NS1 and NS2 of bovine respiratory syncytial virus block activation of interferon regulatory factor 3

Bovine viral diarrhea virus isolated from fetal calf serum enhances pathogenicity of attenuated transmissible gastroenteritis virus in neonatal pigs

Effects of intranasal inoculation with Bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with Pasteurella multocida in pigs

Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus

Association of porcine circovirus 2 with porcine respiratory disease complex

In utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by Streptococcus suis type II

Effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, Bordetella bronchiseptica, or a combination of both organisms in pigs

Differential production of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection model

Dual infections of PRRSV/influenza or PRRSV/Actinobacillus pleuropneumoniae in the respiratory tract

High mortality and growth depression experimentally produced in young turkeys by dual infection with enteropathogenic Escherichia coli and turkey coronavirus

Experimental infection of turkeys with avian pneumovirus and either Newcastle disease virus or Escherichia coli

Invasive group A streptococcal disease in children and association with varicella-zoster virus infection. Ontario group A streptococcal study group

Risk factors for invasive group A streptococcal infections in children with varicella: a casecontrol study

Invasive group A streptococcal infections in children with varicella in Southern California

Presence of specific viruses in the middle ear fluids and respiratory secretions of young children with acute otitis media

Adenovirus infection enhances in vitro adherence of Streptococcus pneumoniae

Effect of adenovirus type 1 and influenza A virus on Streptococcus pneumoniae nasopharyngeal colonization and otitis media in the chinchilla

Reviews the evidence in support of the crucial role of a viral virulence factor in predisposing both the upper and lower respiratory tract to bacterial secondary infections

Adenovirus serotype 1 does not act synergistically with Moraxella (Branhamella) catarrhalis to induce otitis media in the chinchilla

Comparison of alteration of cell surface carbohydrates of the chinchilla tubotympanum and colonial opacity phenotype of Streptococcus pneumoniae during experimental pneumococcal otitis media with or without an antecedent influenza A virus infection

Effect of experimental influenza A virus infection on isolation of Streptococcus pneumoniae and other aerobic bacteria from the oropharynges of allergic and nonallergic adult subjects

Prevalence of various respiratory viruses in the middle ear during acute otitis media

Infectious exacerbations of chronic obstructive pulmonary disease associated with respiratory viruses and non-typeable Haemophilus influenzae

Impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis

Modulation of Pseudomonas aeruginosa gene expression by host microflora through interspecies communication

The herpesvirus-Porphyromonas gingivalis-periodontitis axis

Herpesviral-bacterial interactions in aggressive periodontitis

A mouse model of dual infection with influenza virus and Streptococcus pneumoniae

Lethal synergism between influenza virus and Streptococcus pneumoniae: characterization of a mouse model and the role of plateletactivating factor receptor

Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae

A model of meningococcal bacteremia after respiratory superinfection in influenza A virus-infected mice

The chinchilla superinfection model developed in this study was the first animal model to demonstrate conclusively the important role of the upper respiratory tract viruses

Eustachian tube histopathology during experimental influenza A virus infection in the chinchilla

Different virulence of influenza A virus strains and susceptibility to pneumococcal otitis media in chinchillas

Polymorphonuclear leukocyte dysfunction during influenza virus infection in chinchillas

Synergistic effect of adenovirus type 1 and nontypeable Haemophilus influenzae in a chinchilla model of experimental otitis media

Evidence for transudation of specific antibody into the middle ears of parenterally immunized chinchillas after an upper respiratory tract infection with adenovirus

Kinetics of the ascension of NTHi from the nasopharynx to the middle ear coincident with adenovirus-induced compromise in the chinchilla

Nontypeable Haemophilus influenzae gene expression induced in vivo in a chinchilla model of otitis media

Protection against development of otitis media induced by nontypeable Haemophilus influenzae by both active and passive immunization in a chinchilla model of virus-bacterium superinfection

Passive transfer of antiserum specific for immunogens derived from a nontypeable Haemophilus influenzae adhesin and lipoprotein D prevents otitis media after heterologous challenge

Relative immunogenicity and efficacy of two synthetic chimeric peptides of fimbrin as vaccinogens against nasopharyngeal colonization by nontypeable Haemophilus influenzae in the chinchilla

Effect of respiratory syncytial virus on adherence, colonization and immunity of non-typable Haemophilus influenzae: implications for otitis media

Accelerated migration of respiratory dendritic cells to the regional lymph nodes is limited to the early phase of pulmonary infection

Evaluation of vaccines for atrophic rhinitis -a comparison of three challenge models

The pathological effect of the Bordetella dermonecrotic toxin in mice

Immunopathological changes in mice caused by Bordetella bronchiseptica and Pasteurella multocida

The virulence of mixed infection with Streptococcus constellatus and Fusobacterium nucleatum in a murine orofacial infection model

Bacteriology of adenoids and tonsils in children with recurrent adenotonsillitis

Bacterial biofilms: a common cause of persistent infections

An excellent review of the role of polymicrobial biofilms in diverse anatomical niches that are involved in persistent and chronic human diseases

Handbook of Animal Models of Infection

Bacteriologic findings in patients with chronic sinusitis

Microbiology of polymicrobial abscesses and implications for therapy

Synergistic effect of bacteroides, Clostridium, Fusobacterium, anaerobic cocci, and aerobic bacteria on mortality and induction of subcutaneous abscesses in mice

The etiology of urinary tract infection: traditional and emerging pathogens

Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients

Involvement of periodontopathic biofilm in vascular diseases

Periodontal disease: bacterial virulence factors, host response and impact on systemic health

Bacterial coaggregation: an integral process in the development of multi-species biofilms

An overview of the molecular mechanisms by which bacteria interact with one another to construct complex multispecies biofilms

Phylogeny of Porphyromonas gingivalis by ribosomal intergenic spacer region analysis

Association of Bacteroides forsythus and a novel Bacteroides phylotype with periodontitis

Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients

Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease

Single-cell enumeration of an uncultivated TM7 subgroup in the human subgingival crevice

Genetic relatedness and phenotypic characteristics of Treponema associated with human periodontal tissues and ruminant foot disease

New bacterial species associated with chronic periodontitis

Beyond the specific plaque hypothesis: are highly leukotoxic strains of Actinobacillus actinomycetemcomitans a paradigm for periodontal pathogenesis?

The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction

The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species

Hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections

Bacterial vaginosis is a strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infection

Association between acquisition of herpes simplex virus type 2 in women and bacterial vaginosis

Interleukin-1 and tumor necrosis factor receptor signaling is not required for bacteria-induced osteoclastogenesis and bone loss but is essential for protecting the host from a mixed anaerobic infection

Provided the background for the development of an animal model of the relative bacterial colonization and persistence by single phenotypic variants of a bacterium

Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model

Inflammation and tissue loss caused by periodontal pathogens is reduced by interleukin-1 antagonists

Non-human primates used in studies of periodontal disease pathogenesis: a review of the literature

Exemplifies the potential for different animal models to provide disparate data and shows that there can be species and model dependency to our ability to define and characterize microbial virulence determinants in polymicrobial diseases origin

Microbiological and inflammatory effects of murine recombinant interleukin-10 in two models of polymicrobial peritonitis in rats

STAT4 is required for antibacterial defense but enhances mortality during polymicrobial sepsis

Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture

Early activation of pulmonary nuclear factor κB and nuclear factor interleukin-6 in polymicrobial sepsis

Granulocyte colony-stimulating factor and antibiotics in the prophylaxis of a murine model of polymicrobial peritonitis and sepsis

Antibiotics delay but do not prevent bacteremia and lung injury in murine sepsis

Mortality in murine peritonitis correlates with increased Escherichia coli adherence to the intestinal mucosa

Polymicrobial sepsis induces organ changes due to granulocyte adhesion in a murine two hit model of trauma

Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis

The importance of systemic cytokines in the pathogenesis of polymicrobial sepsis and dehydroepiandrosterone treatment in a rodent model

The activity of tissue factor pathway inhibitor in experimental models of superantigen-induced shock and polymicrobial intra-abdominal sepsis

Candida biofilms and their role in infection

Adherence of Candida albicans to a cell surface polysaccharide receptor on Streptococcus gordonii

Reviews the pathogenic potential and phenotypic variability of C. albicans and illustrates that the coinfecting microorganisms can be phenotypic variants of a single microbial strain

Phenotypic switching and mating type switching of Candida glabrata at sites of colonization

ASM Conference on Polymicrobial Diseases Abstract 14

Pseudomonas-Candida interactions: an ecological role for virulence factors

The relative pathogenicity of Candida krusei and C. albicans in the rat oral mucosa

Avirulence of Candida albicans auxotrophic mutants in a rat model of oropharyngeal candidiasis

In vivo pathogenicity of eight medically relevant Candida species in an animal model

A murine model of Candida glabrata vaginitis

The protective immune response against vaginal candidiasis: lessons learned from clinical studies and animal models

Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection

Provided the background for the development of a murine model of fungal co-infection that allowed the demonstration of the importance of the ability of Candida albicans to switch morphology from a yeast to a filamentous form in pathogenesis

Ascaris lumbricoides infection is associated with protection from cerebral malaria

Contemporaneous and successive mixed Plasmodium falciparum and Plasmodium vivax infections are associated with Ascaris lumbricoides: an immunomodulating effect?

Intestinal helminth infections are associated with increased incidence of Plasmodium falciparum malaria in Thailand

Specific cellular immune response and cytokine patterns in patients coinfected with hepatitis C virus and Schistosoma mansoni

Kinetics of intrahepatic hepatitis C virus (HCV)-specific CD4 + T cell responses in HCV and Schistosoma mansoni coinfection: relation to progression of liver fibrosis

Associations between anti-Schistosoma mansoni and anti-Plasmodium falciparum antibody responses and hepatosplenomegaly, in Kenyan schoolchildren

Epidemiology and impact of coinfections acquired from Ixodes ticks. Vector Borne Zoonotic Dis

Coinfection in patients with lyme disease: how big a risk?

Some aspects of concomitant infections of plasmodia and schistosomes. I. The effect of Schistosoma mansoni on the course of infection of Plasmodium berghei in the field vole (Microtus guentheri)

Infection of mice concurrently with Schistosoma mansoni and rodent malarias: contrasting effects of patent S. mansoni infections on Plasmodium chabaudi, P. yoelii and P. berghei

Suppression of schistosome granuloma formation by malaria in mice

Altered immune responses in mice with concomitant Schistosoma mansoni and Plasmodium chabaudi infections

Schistosoma mansoni infection cancels the susceptibility to Plasmodium chabaudi through induction of type 1 immune responses in A/J mice

Heterologous synergistic interactions in concurrent experimental infection in the mouse with Schistosoma mansoni, Echinostoma revolutum, Plasmodium yoelii, Babesia microti, and Trypanosoma brucei

Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis alters murine immune responses, pathogen burden, and severity of Lyme arthritis

Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis suppresses IL-2 and IFN-γ production and promotes an IL-4 response in C3H/HeJ mice

Tuberculosis in HIV-infected patients: a comprehensive review

A TEM/SEM study of the microbial plaque overlying the necrotic gingival papillae of HIV-seropositive, necrotizing ulcerative periodontitis

Molecular epidemiology of Candida albicans strains isolated from the oropharynx of HIV-positive patients at successive clinic visits

Relationship between herpesviruses and adult periodontitis and periodontopathic bacteria

Herpesviruses in human periodontal disease

Herpesviruses: a unifying causative factor in periodontitis?

Modulation of immune system function by measles virus infection: role of soluble factor and direct infection

Modulation of immune system function by measles virus infection. II. Infection of B cells leads to the production of a soluble factor that arrests uninfected B cells in G 0 /G 1

Manganese superoxide dismutase induction during measles virus infection

Suppression of antigen-specific T cell proliferation by measles virus infection: role of a soluble factor in suppression

CpG oligodeoxynucleotides protect normal and SIV-infected macaques from Leishmania infection

Interactions between Mycobacterium leprae and simian immunodeficiency virus (SIV) in rhesus monkeys

This paper reviews how virus-induced immunosuppression of both innate and adaptive immune responses can provide an underlying mechanism for bacterial co-infection

This paper introduces the concept of one approach to treat or prevent polymicrobial diseases by using bioengineering and nanotechnology to specific anatomic sites and crucial time points in the disease course for intervention and/or prevention

Communication among oral bacteria. Microbiol

The author would like to thank J. Neelans for help with manuscript preparation.

Discusses our increasing understanding of the link between biofilms and the pathogenesis of human disease, as well as identifying the need for relevant animal models with which to study these infectious states. 10. Brogden