Abstract
One hundred fifty canine and feline Escherichia coli isolates associated with urinary tract infections were screened for the presence of extended-spectrum β-lactamase (ESBL) genes. Out of 60 isolates suspected to be ESBL positive based on antimicrobial susceptibility testing, 11 ESBLs were identified, including one SHV-12 gene, one CTX-M-14 gene, and nine CTX-M-15 genes. This study provides the first report of CTX-M- and SHV-type ESBLs in dogs and cats in the United States.
The first detection of an extended-spectrum β-lactamase (ESBL) in an organism from an animal was reported in Japan in 1988 in an Escherichia coli isolate from a laboratory dog (13). Since that time, numerous reports of ESBL-positive isolates from dogs and cats, as well as from other animal species (5), have been made worldwide (4, 8, 11, 17, 25, 26). Only one study has identified the presence of an ESBL in isolates from animals in the United States, i.e., in Salmonella enterica serovar Newport from horses (21). We hypothesized that ESBL genes would be present in urinary E. coli isolates from companion animals in the United States. The purpose of this study was to screen a group of 150 E. coli isolates from canine and feline patients that had been diagnosed with a urinary tract infection (UTI) for the presence of ESBL genes.
A convenience sample of 150 E. coli isolates collected from canine and feline patients at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania with clinical signs or evidence on routine urinalysis of a UTI between 1 September 2004 and 31 December 2007 was used in this study. Isolates were frozen in Microbank tubes (ProLab Diagnostics, Austin, TX) and stored at −70°C prior to use.
MICs were determined using a Negative Combo 31 panel on a MicroScan Walkaway 40 (Dade Behring, Siemens Healthcare Diagnostics, Deerfield, IL). Results were interpreted using Clinical and Laboratory Standards Institute (CLSI) breakpoints (7). Isolates collected from the same individual animal within a 45-day period were considered to be the same strain, and only the first isolate collected was analyzed (subsequent isolates were considered redundant). All isolates with a cefpodoxime MIC of ≥4 μg/ml and a ceftazidime MIC of ≥1 μg/ml were identified as an “ESBL alert” on the MicroScan Walkaway. ESBL confirmatory testing was performed via the Etest method using ceftazidime-ceftazidime-clavulanic acid and cefotaxime-cefotaxime-clavulanic acid strips in accordance with CLSI guidelines (7).
Due to the high prevalence of cefoxitin resistance in this population, PCR was performed to detect the presence of a blaAmpC gene, the product of which can mask the effects of clavulanic acid on the ESBL confirmatory test (18, 27). Since the primers used in this study to identify the blaAmpC gene (18) have since been shown to amplify the plasmid-mediated blaCMY gene from Citrobacter freundii (19), it can be inferred that the genes detected were part of the blaCMY lineage. Salmonella Newport strain 0007-33 was used as the positive control (21). PCR was performed for the genes blaTEM, blaSHV, and blaCTX-M as published previously (9, 10, 27). Salmonella Newport strain 0007-33 was also used as the TEM and SHV positive control (21), and E. coli strain MISC 336 (CTX-M-1 positive) was used as the CTX-M positive control.
The blaTEM, blaSHV, and blaCTX-M PCR products were sequenced using both strands of DNA for each PCR product. Protein sequences were aligned using Lasergene software (DNASTAR, Inc., Madison, WI) and included GenBank sequences (http://www.ncbi.nlm.nih.gov/GenBank/index.html) to confirm ESBL genotype. Mutations were evaluated with reference to the Lahey Clinic website (http://www.lahey.org/studies/). GenBank accession numbers used for alignment of protein sequences were AAR25033 for TEM-1 and ABF29674 for SHV-2. CTX-M accession numbers were derived from a list on the Lahey Clinic website. Specific primers for the CTX-M-1 group (M13U and M13L) were used to amplify the entire coding sequences of these blaCTX-M genes (23). Sequencing and analysis were carried out as described above to identify the specific CTX-M subtype.
Seventy of the 150 E. coli isolates had an “ESBL alert” on the MicroScan Walkaway, and after removal of redundant isolates, 60 isolates were tested further. ESBL confirmatory testing was positive for six of these 60 isolates (Table 1, column 3).
TABLE 1.
Distribution of β-lactamase and extended-spectrum β-lactamase genes
| Isolate | Species | ESBL test | blaCMY | blaSHV | blaTEM | blaCTX-M |
|---|---|---|---|---|---|---|
| 1 | Canine | + | − | SHV-12 | TEM-1 | − |
| 3 | Canine | − | + | − | − | − |
| 6 | Canine | − | + | − | TEM-1 | − |
| 11 | Canine | − | + | − | TEM-1 | − |
| 17 | Canine | − | + | − | TEM-1 | − |
| 19 | Canine | − | + | − | − | − |
| 21 | Feline | + | − | − | − | CTX-M-15 |
| 26 | Canine | − | + | − | TEM-1 | − |
| 27 | Canine | − | + | − | TEM-1 | − |
| 31 | Canine | − | + | − | TEM-1 | − |
| 32 | Canine | − | + | − | TEM-1 | − |
| 33 | Canine | − | + | − | − | − |
| 41 | Canine | − | + | − | TEM-1 | − |
| 42 | Canine | − | + | − | − | − |
| 44 | Canine | − | + | − | TEM-1 | − |
| 53 | Canine | − | + | − | − | − |
| 57 | Feline | − | + | − | − | − |
| 62 | Canine | − | + | − | TEM-1 | − |
| 67 | Canine | − | + | − | − | − |
| 74 | Canine | − | + | − | TEM-1 | − |
| 75 | Feline | + | − | − | TEM-1 | CTX-M-15 |
| 82 | Feline | − | + | − | TEM-1 | − |
| 85 | Canine | − | + | − | TEM-1 | − |
| 86 | Canine | − | + | − | TEM-1 | − |
| 87 | Canine | − | + | − | − | − |
| 88 | Feline | − | + | − | − | − |
| 91 | Canine | − | + | − | − | − |
| 98 | Canine | − | + | − | TEM-1 | − |
| 102 | Canine | − | + | − | − | − |
| 104 | Canine | − | + | − | − | − |
| 112 | Canine | − | + | − | TEM-1 | − |
| 119 | Canine | − | + | − | − | − |
| 131 | Canine | − | − | Not done | Not done | Not done |
| 133 | Feline | − | + | − | − | − |
| 138 | Feline | − | + | − | TEM-1 | − |
| 147 | Canine | − | + | − | TEM-1 | − |
| 149 | Feline | − | + | − | TEM-1 | − |
| 157 | Canine | + | − | − | − | CTX-M-15 |
| 165 | Canine | − | + | − | TEM-1 | − |
| 166 | Canine | − | + | − | TEM-1 | − |
| 168 | Canine | − | + | − | − | − |
| 182 | Canine | − | + | − | − | − |
| 183 | Feline | − | + | − | TEM-1 | − |
| 190 | Feline | − | + | − | TEM-1 | − |
| 205 | Canine | − | + | − | TEM-1 | − |
| 209 | Canine | − | + | − | TEM-1 | − |
| 210 | Canine | − | + | − | − | − |
| 219 | Feline | − | + | − | TEM-1 | CTX-M-15 |
| 220 | Canine | + | − | − | TEM-1 | CTX-M-15 |
| 230 | Canine | − | + | − | TEM-1 | − |
| 234 | Canine | − | + | − | TEM-1 | − |
| 236 | Canine | + | − | − | TEM-1 | CTX-M-14 |
| 240 | Canine | − | + | − | TEM-1 | − |
| 242 | Canine | − | + | − | TEM-1 | CTX-M-15 |
| 246 | Feline | − | + | − | − | CTX-M-15 |
| 265 | Canine | − | + | − | TEM-1 | CTX-M-15 |
| 266 | Canine | − | + | − | TEM-1 | − |
| 267 | Canine | − | + | − | − | CTX-M-15 |
| 268 | Canine | − | + | − | − | − |
| 269 | Canine | − | + | − | − | − |
Fifty-three of the 60 isolates were positive for the blaCMY gene (43 canine and 10 feline samples) (Table 1, column 4). Of the seven negative isolates, six were those previously found to be positive for ESBL production via ESBL testing. The remaining canine isolate (isolate 131, negative for both blaCMY PCR and ESBL testing) was not analyzed further.
A total of 40 E. coli isolates were found to carry one or more β-lactamase genes. PCR detected a blaSHV gene in one canine isolate, a blaTEM gene in 29 canine and seven feline isolates, and a blaCTX-M gene in six canine and four feline isolates. The remaining 19 isolates were confirmed as negative for blaSHV, blaTEM, and blaCTX-M genes (Table 1, columns 5 to 7).
Based on sequence analysis (Table 1, column 5), the one SHV-positive strain (isolate 1) was determined to carry SHV-12. All 36 strains positive for a TEM gene were identified as carrying TEM-1 (Table 1, column 6). Nine of 10 strains positive for a CTX-M gene carried genes of the CTX-M-1 group. The final strain (isolate 236) was concluded to carry CTX-M-14, given the presence of mutations in the amplified consensus sequence unique to CTX-M-14. DNA from each of the nine CTX-M-1 group strains was amplified using the CTX-M-1 group-specific primer set (23). Sequence analysis identified all nine strains as carrying CTX-M-15 (Table 1, column 7).
The CTX-M-type ESBLs identified in this study provide evidence for the dissemination of these genes in the United States. The CTX-M-1 group has frequently been reported in animals in countries other than the United States (2, 4, 8, 11, 17). The CTX-M-15 gene has not been identified in any bacterial isolate from animals in the United States. Animal sources of this gene have been identified only in E. coli isolated from the cloacae of Belgian poultry and an E. coli isolate from the urine of a cow in France (14, 24). The presence of CTX-M-14 genes in the Enterobacteriaceae has also been documented across the globe (22), including in six E. coli strains isolated from the feces of dogs in Chile (17). The CTX-M-14 gene identified in this study is the first identified from an animal in the United States and the first linked to a clinical case of UTI in a dog. The SHV-12 gene has been detected in bacterial isolates from animals in several countries (1, 4, 6, 26), including, in 2005, a Salmonella enterica serovar Newport strain from a horse in the United States (21).
Interestingly, of the 11 ESBL-positive isolates identified by sequence analysis, only six were positive by ESBL confirmatory testing, likely due to the concurrent presence of a blaCMY gene in the other five isolates. The product of this gene is known to mask the effects of clavulanic acid on the ESBL confirmatory test (28). By relying on ESBL confirmatory testing alone, it is likely that the prevalence of ESBLs is being underestimated, particularly in populations with a high frequency of blaAmpC, such as in the current study. During the study period, E. coli was isolated from samples submitted to our laboratory from 1,318 individual animals, and of these, 257 met the criteria for ESBL confirmatory testing. Of the 257 isolates tested, 14 (5%) were identified as being ESBL producers based on the Etest method, including the six reported in this study. The overall prevalence of E. coli isolates that were positive for an ESBL during this period was 1% (S. Rankin, unpublished observations). It is possible that this is an underestimation, based on the high frequency of blaCMY detected in this study.
Though, historically, the most common ESBLs in the United States have been TEM and SHV types (16, 20), more recent studies have identified CTX-M genes, first reported in 2003 (15). CTX-M ESBLs now predominate in some U.S. health care systems (12). The findings from the current study are in agreement with current trends in the United States and other parts of the world (3). This study is the first report of E. coli strains that encode SHV-12, CTX-M-15, and CTX-M-14 ESBL genes in companion animals in the United States.
Acknowledgments
This study was supported by a Merck-Merial grant, as well as by awards T32RR007063 and T35RR07065 from the National Center for Research Resources.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.
We thank Estela M. Galván for her technical assistance with this project and Irving Nachamkin for his provision of a CTX-M positive-control strain (E. coli strain MISC 336).
Footnotes
Published ahead of print on 17 May 2010.
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