ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2005, p. 833–835 Vol. 49, No. 2 0066-4804/05/$08.00�0 doi:10.1128/AAC.49.2.833–835.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved. Extended-Spectrum �-Lactamases in Escherichia coli Isolated from Dogs and Cats in Rome, Italy, from 2001 to 2003 Alessandra Carattoli,1 Sarah Lovari,2 Alessia Franco,2 Gessica Cordaro,2 Paola Di Matteo,2 and Antonio Battisti2* Istituto Superiore di Sanità1 and Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana,2 Rome, Italy Received 23 July 2004/Returned for modification 6 September 2004/Accepted 12 October 2004 We report expanded-spectrum cephalosporin resistance in Escherichia coli from dogs and cats in Rome, Italy. Three major �-lactamases (CMY-2, SHV-12, and CTX-M-1) are reported for the first time in E. coli from sick and healthy dogs and cats. Molecular characterization suggests the presence of several combinations of �-lactamase genes in E. coli from companion animals. Escherichia coli is a common microorganism found in the intestinal flora of humans and animals, although pathogenic strains cause serious diseases, including urinary and wound infections and septicemia. While antimicrobial use in produc- tion animals has been shown to lead to the emergence of resistant bacteria throughout the food chain (5), little is known about the development of resistance in companion animals (9). The objective of this study was to assess the presence of ex- panded-spectrum cephalosporin resistance in E. coli recovered from dead, sick, and healthy dogs and cats living in kennels or with private owners. Over a 3-year period (2001 to 2003), 298 E. coli isolates obtained from specimens from 204 dogs and 61 cats submitted for routine diagnostic investigation were collected at the Isti- tuto Zooprofilattico Sperimentale delle Regioni Lazio e To- scana, Rome, Italy. Of a total 226 canine isolates, 144 were obtained from necropsies (86 from gut contents and 58 from infected organs), 33 were from diagnostic samples, and 49 were from fecal samples from healthy animals submitted for parasite screening. A total of 72 E. coli isolates of feline origin were obtained, 51 of which were from necropsy specimens (29 from gut contents and 22 from infected organs), while 6 and 15 isolates were from diagnostic samples and fecal samples from healthy animals, respectively. Two-thirds (67%) of the dogs investigated were from private owners, and the rest were from five different municipal facilities for unclaimed stray or lost dogs and from authorized private animal shelters. The cats tested belonged mainly to private owners (57%) and colonies of abandoned cats (38%) that are cared for by volunteers. An additional E. coli isolate was obtained from the gut of a brown rat (Rattus norvegicus) found dead in a kennel in which dogs had also been tested. All strains were screened by antimicrobial susceptibility test- ing performed by the agar diffusion method with 16 different antimicrobial drugs. Sensitivity testing for ampicillin, amikacin, amoxicillin-clavulanic acid, cefotaxime, cephazolin, chloram- phenicol, enrofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfonamides, tetracycline, and trimethoprim- sulfamethoxazole were interpreted in accordance with the rec- ommendations of the National Committee for Clinical Labo- ratory Standards (NCCLS) (7, 8). For colistin, breakpoint diameters of 8 mm for resistance and 11 mm for sensitivity were used. Twenty-one strains (7%) from healthy, dead, and diseased dogs and cats, and from the rat, showed resistance to cefo- taxime (20 [6.7%] of 298) and/or cefoxitin (13 [4.4%] of 298). Twelve strains (4%) also showed resistance to �-lacta- mase inhibitors (amoxicillin-clavulanic acid), and all strains showed resistance to several different antimicrobials, includ- ing nalidixic acid (20.1%), enrofloxacin (15.1%), aminoglyco- sides (gentamicin [8.1%], kanamycin [15.4%], streptomycin [37.2%], and amikacin [0.7%]), trimethoprim-sulfamethox- azole (33.9%), chloramphenicol (18.1%), and tetracyclines (45.0%). Resistance to extended-spectrum cephalosporins was defined on the basis of conventional NCCLS breakpoints; thus, the number of expanded-spectrum �-lactamase producers might have been underestimated in this collection. The characteristics of the 21 E. coli strains showing resis- tance to expanded-spectrum cephalosporins are shown in Ta- ble 1. This wide spectrum of antimicrobial resistance, especially toward extended-spectrum cephalosporins, prompted further characterization of the isolates. To investigate the genetic re- lationship among the isolates, we analyzed the chromosomal patterns obtained by pulsed-field gel electrophoresis (PFGE) after digestion with the XbaI restriction enzyme. Twelve dif- ferent PFGE profiles were obtained (PFGE patterns differing for more than three DNA fragments were classified as different profiles and are designated A to N in Table 1), demonstrating that there was not a unique resistant E. coli clone spreading among the animals (12). However, five strains, four of them isolated from dogs from the same kennel, show similar chro- mosomal patterns (pattern G in Table 1), differing by one or two bands, indicating the diffusion of this strain among animals living in kennel C. E. coli strains were analyzed by PCR for the presence of the blaSHV-, blaTEM-, blaCTX-M-, blaAmpC-type genes with previ- ously described primer pairs (blaSHV and blaAmpC gene prim- ers in reference 6, CTX-MA and CTX-MB primers in refer- * Corresponding author. Mailing address: Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Rome, Italy. Phone: 390679099469. Fax: 390679340724. E-mail: abattisti@rm.izs.it. 833 o n A p ril 5 , 2 0 2 1 a t C A R N E G IE M E L L O N U N IV L IB R h ttp ://a a c.a sm .o rg / D o w n lo a d e d fro m http://aac.asm.org/ ence 2, and blaTEM gene primers in reference 4). The amplicons obtained for the blaSHV-, blaCTX-M-, and blaAmpC- type genes were sequenced, and a comparative analysis of the nucleotide sequences was performed with advanced BLAST search program 2.0 within the QBLAST system at the National Center for Biotechnology Information website (www.ncbi.nlm .nih.gov/BLAST/). We found different combinations of �-lactamase genes in the E. coli strains in our collection (Table 1). Sixteen isolates were positive by PCR, and confirmed by DNA sequencing, for the blaCTX-M-1 gene. E. coli isolates with the blaCTX-M-1 gene also frequently possessed the TEM �-lactamase. E. coli pro- ducing plasmid-mediated CTX-M �-lactamase have been re- ported in cattle from Japan (11), but to our knowledge, E. coli isolates carrying the blaCTX-M-1 gene have never been de- scribed from healthy or diseased companion animals. Three epidemiologically and genetically unrelated strains (identification no. 18196, 16117, and 31038) were positive to a blaCMY-like gene, and the DNA sequence of the amplicons revealed the presence of the blaCMY-2 gene (1). Two of the blaCMY-2-positive strains were isolated from dogs coming from different kennels (A and B): one was from infected organs of a necropsied animal, while the other was from the feces of a healthy animal. Interestingly, the third blaCMY-2-positive iso- late, also showing the presence of the blaCTX-M-1 gene, was from the rat found dead in kennel B, showing a PFGE profile different from that of the E. coli isolates from the dogs that were tested in the same facility, thus suggesting that diffusion of the blaCMY-2 gene may have occurred in this kennel. This is the first evidence of community-acquired E. coli isolates car- rying genes encoding CMY-2 from pets, although blaCMY-2- positive E. coli strains were previously reported to be associ- ated with nosocomial infections in dogs (10). A significant extended-spectrum �-lactamase was also found in animals coming from kennel C. In this case, E. coli strains showed only two PFGE profiles (patterns G and H) and three of the strains isolated from necropsy specimens (identification no. 1599B, 1599C, and 1599D), from gut contents and from diseased organs, were positive for the same blaSHV amplicon that, after sequencing, was identified as the blaSHV-12 gene (6) (Table 1). A fourth isolate (identification no. 11361) from a private owner’s necropsied dog, was also positive for the blaSHV-12 gene (Table 1). The SHV-12 �-lactamase has previ- ously been described in clinical E. coli isolates from humans, healthy production animals, and a dog with recurrent urinary tract infections (3, 13). In our study, the isolation of SHV-12- positive E. coli strains from lesions of dead animals from the same municipal facility suggests a community-acquired infec- tion, probably favored by the high animal density in the kennel. However, it is of concern that the same blaSHV-12 gene was also TABLE 1. Characteristics of E. coli isolates recovered from sick and healthy dogs and cats in Rome, Italy from 2001 to 2003 Isolate Species Sourcea Origin Resistance patternb PFGE profilec blaSHV-12 blaCMY-2 blaCTX-M-1 blaTEM d 18196 Dog Organs Kennel A AMP AMC CTX FOX KAN SUL SXT TET A � � � � 16117 Dog Feces Kennel B AMP AMC CHL CTX FOX GEN B � � � � 20432 Dog Organs Kennel B AMP AMC CHL CTX ENO FOX KAN NAL STR SUL SXT TET C � � � � 14083 Dog Organs Kennel B AMP AMC CHL CTX ENO FOX KAN NAL STR SUL SXT TET ND � � � � 331 Dog Feces Kennel B AMP AMC CHL CTX ENO FOX GEN KAN NAL STR SUL SXT TET D � � � � 1092 Dog Infection Kennel B AMP AMC CHL CTX FOX GEN KAN NAL SPT STR SUL SXT TET E � � � � 31038 Rat Gut contents Kennel B AMP AMC CTX FOX GEN STR SUL SXT TET F � � � � 24623 Dog Feces Kennel B AMP AMC CHL CTX ENO FOX NAL SUL SXT TET G � � � � 1599B Dog Gut contents Kennel C AMP CTX ENO GEN KAN NAL STR SUL SXT TET G � � � � 1599C Dog Organs Kennel C AMP CTX ENO GEN NAL SUL SXT TET G � � � � 1599D Dog Organs Kennel C AMP CTX ENO GEN NAL STR SUL SXT TET G � � � � 17795 Dog Gut contents Kennel C AMP CHL CTX ENO NAL SUL SXT TET G � � � � 1599A Dog Gut contents Kennel C AMP CHL CTX ENO NAL STR SUL SXT TET H � � � � 1599E Dog Organs Kennel C AMP CHL CTX ENO NAL STR SUL SXT TET H � � � � 322 Dog Organs Kennel D AMP CTX STR SUL SXT TET I � � � � 11361 Dog Organs Private owner AMP CHL CTX KAN SPT STR SUL SXT TET L � � � � 362 Dog Feces Private owner AMP AMC CHL CTX ENO FOX KAN NAL SUL SXT TET ND � � � � 17419 Cat Organs Private owner AMP AMC CHL CTX ENO FOX KAN NAL STR SUL SXT TET C � � � � 3050 Dog Organs Private owner AMP AMC CTX ENO FOX NAL STR SUL SXT TET M � � � � 34430 Cat Organs Private owner AMP CTX ENO FOX NAL SUL SXT TET M � � � � 8113 Cat Organs Private owner AMP AMC CHL ENO FOX KAN NAL SPT STR SUL SXT TET N � � � � a Gut contents and organs are from necropsy specimens. b AMP, ampicillin; AMC, amoxicillin-clavulanic acid; CHL, chloramphenicol; CTX, cefotaxime; ENO, enrofloxacin; FOX, cefoxitin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; SPT, spectinomycin; STR, streptomycin; SXT, sulfometoxazole-trimethoprim; SUL, sulfonamides; TET, tetracycline; c ND, not determined. PFGE patterns differing for more than three DNA fragments were classified as different profiles. d blaTEM genes were identified by PCR, although several amplicons were sequenced identifying blaTEM-1a and blaTEM-1b gene variants. 834 NOTES ANTIMICROB. AGENTS CHEMOTHER. o n A p ril 5 , 2 0 2 1 a t C A R N E G IE M E L L O N U N IV L IB R h ttp ://a a c.a sm .o rg / D o w n lo a d e d fro m http://aac.asm.org/ found in the dog of a private owner, indicating the possible future appearance of this resistance gene in other companion animals. In several strains, the observed phenotype of resistance to cefoxitin or amoxicillin-clavulanic acid cannot be completely explained by the identified �-lactamase genes, suggesting the presence of additional mechanisms of resistance in these strains, such as inhibitor-resistant blaTEM or blaOXA-1 genes or overproduction of non-inhibitor-resistant blaTEM TEM-type enzymes that need further investigation. With respect to the possible origin of CMY-2, SHV-12, and CTX-M in pets, Italian companion animal practitioners admit to rather diffuse off-label use of expanded-spectrum cephalo- sporins registered for human use in pet therapy that began in the early 1990s, even earlier than in farm animal practice, where their administration is still limited to selected cases, for obvious economic reasons. The results of this study are of public health concern because nonjudicious use or misuse of highly valuable antimicrobial drugs can result in selective pres- sure on bacterial populations of companion animals. This may lead to the spread of pathogens carrying resistance to newer antimicrobials by vertical and horizontal transmission of genes, with the subsequent risk of transfer to humans. In this respect, further population-based epidemiological surveys may provide valuable information about the diffusion of multiresistant E. coli in companion animals. We thank Gabriele Panfili for conducting necropsies and microbio- logical testing of animals included in this study. We thank Carmela Buccella, Cinzia Onorati, Tamara Cerci, Andrea Pietrella, Patrizia Palmieri, and Luigi Sorbara for technical assistance. The results presented in this paper were produced by activities also supported by research grants from the Italian Ministry of Health (Re- search Projects LT RFS 225/99 and IZSLT 01/2002). REFERENCES 1. Bauernfeind, A., I. Stemplinger, R. Jungwirth, and H. Giamarellou. 1996. Characterization of the plasmidic �-lactamases CMY-2, which is responsible for cephamycin resistance. Antimicrob. Agents Chemother. 40:221–224. 2. Bonnet, R., C. Dutour, J. L. M. Sampaio, C. Chanal, D. Sirot, R. Labia, C. De Champs, and J. Sirot. 2001. Novel cefotaxime (CTX-M-16) with in- creased catalytic efficiency due to substitution Asp-2403Gly. Antimicrob. Agents Chemother. 45:2269–2275. 3. Briñas, L., M. A. Moreno, M. Zarazaga, C. Porrero, Y. Sáenz, M. Garcı́a, L. Dominguez, and C. Torres. 2003. Detection of CMY-2, CTX-M-14, and SHV-12 �-lactamases in Escherichia coli fecal-sample isolates from healthy chickens. Antimicrob. Agents Chemother. 47:2056–2058. 4. Coque, T. M., J. Oliver, A. O. Perez-Diaz, F. Baquero, and R. Canton. 2002. Genes encoding TEM-4, SHV-2, and CTX-M-10 extended-spectrum �-lac- tamases are carried by multiple Klebsiella pneumoniae clone in a single hospital (Madrid, 1989 to 2000). Antimicrob. Agents Chemother. 46:500– 510. 5. European Commission. 1999. Opinion of the Scientific Steering Committee on Antimicrobial Resistance, DGXXIV, Consumer Policy and Consumer Health Protection. [Online.] http://europa.eu.int/comm/food/fs/sc/ssc /out50_en.pdf (last accessed 5 April 2004). 6. Hujer, A. M., M. G. P. Page, M. S. Helfand, B. Yeiser, and R. A. Bonomo. 2002. Development of a sensitive and specific enzyme-linked immunosorbent assay for detecting and quantifying CMY-2 and SHV-12 �-lactamases. J. Clin. Microbiol. 40:1947–1957. 7. National Committee for Clinical Laboratory Standards. 2002. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard—second edition. M31A2. Na- tional Committee for Clinical Laboratory Standard, Wayne, Pa. 8. National Committee for Clinical Laboratory Standards. 2003. Performance standards for antimicrobial disk susceptibility test; approved standards—8th edition. M2-A8/M7-A6 and supplemental tables M100-S14. National Com- mittee for Clinical Laboratory Standard, Wayne, Pa. 9. Normand, E. H., N. R. Gibson, D. J. Taylor, S. Carmichael, and S. W. J. Reid. 2000. Trends of antimicrobial resistance in bacterial isolates from a small animal referral hospital. Vet. Rec. 146:151–155. 10. Sanchez, S., M. A. McCrackin Stevenson, C. R. Hudson, M. Maier, T. Buffington, Q. Dam, and J. J. Maurer. 2002. Characterization of multidrug- resistant Escherichia coli isolates associated with nosocomial infections in dogs. J. Clin. Microbiol. 40:3586–3595. 11. Shiraki, Y., N. Shibata, Y. Doi, and Y. Arakawa. 2004. Escherichia coli producing CTX-M-2 �-lactamase in cattle, Japan. Emerg. Infect. Dis. 10: 69–75. 12. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233–2239. 13. Teshager, T., L. Domı́nguez, M. A. Moreno, Y. Saénz, C. Torres, and S. Cardeñosa. 2000. Isolation of an SHV-12 �-lactamase-producing Escherichia coli strain from a dog with recurrent urinary tract infections. Antimicrob. Agents Chemother. 44:3483–3484. VOL. 49, 2005 NOTES 835 o n A p ril 5 , 2 0 2 1 a t C A R N E G IE M E L L O N U N IV L IB R h ttp ://a a c.a sm .o rg / D o w n lo a d e d fro m http://aac.asm.org/