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Antimicrobial Agents and Chemotherapy, February 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 ß-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 Sanità,2 Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Rome, Italy1

Received 23 July 2004/ Returned for modification 6 September 2004/ Accepted 12 October 2004


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ABSTRACT
 
We report expanded-spectrum cephalosporin resistance in Escherichia coli from dogs and cats in Rome, Italy. Three major ß-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 ß-lactamase genes in E. coli from companion animals.


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TEXT
 
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 production 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 expanded-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 Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, 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 testing performed by the agar diffusion method with 16 different antimicrobial drugs. Sensitivity testing for ampicillin, amikacin, amoxicillin-clavulanic acid, cefotaxime, cephazolin, chloramphenicol, enrofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfonamides, tetracycline, and trimethoprim-sulfamethoxazole were interpreted in accordance with the recommendations of the National Committee for Clinical Laboratory 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 cefotaxime (20 [6.7%] of 298) and/or cefoxitin (13 [4.4%] of 298). Twelve strains (4%) also showed resistance to ß-lactamase inhibitors (amoxicillin-clavulanic acid), and all strains showed resistance to several different antimicrobials, including nalidixic acid (20.1%), enrofloxacin (15.1%), aminoglycosides (gentamicin [8.1%], kanamycin [15.4%], streptomycin [37.2%], and amikacin [0.7%]), trimethoprim-sulfamethoxazole (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 ß-lactamase producers might have been underestimated in this collection.

The characteristics of the 21 E. coli strains showing resistance to expanded-spectrum cephalosporins are shown in Table 1.


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  		TABLE 1. Characteristics of E. coli isolates recovered from sick and healthy dogs and cats in Rome, Italy from 2001 to 2003

This wide spectrum of antimicrobial resistance, especially toward extended-spectrum cephalosporins, prompted further characterization of the isolates. To investigate the genetic relationship among the isolates, we analyzed the chromosomal patterns obtained by pulsed-field gel electrophoresis (PFGE) after digestion with the XbaI restriction enzyme. Twelve different 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 chromosomal 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 previously described primer pairs (blaSHV and blaAmpC gene primers in reference 6, CTX-MA and CTX-MB primers in reference 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 ß-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 ß-lactamase. E. coli producing plasmid-mediated CTX-M ß-lactamase have been reported in cattle from Japan (11), but to our knowledge, E. coli isolates carrying the blaCTX-M-1 gene have never been described 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 isolate, 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 carrying genes encoding CMY-2 from pets, although blaCMY-2-positive E. coli strains were previously reported to be associated with nosocomial infections in dogs (10).

A significant extended-spectrum ß-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 ß-lactamase has previously 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 infection, probably favored by the high animal density in the kennel. However, it is of concern that the same blaSHV-12 gene was also 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 ß-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 cephalosporins 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 pressure 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.


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ACKNOWLEDGMENTS
 
We thank Gabriele Panfili for conducting necropsies and microbiological 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 (Research Projects LT RFS 225/99 and IZSLT 01/2002).


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FOOTNOTES
 
* 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{at}rm.izs.it. Back


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Antimicrobial Agents and Chemotherapy, February 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.



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