Characterization of ESBL-producing Escherichia coli recovered from companion dogs in Tai'an, China

Introduction: Animals are considered to be reservoirs of extended-spectrum beta-lactamase (ESBL)-producing bacteria, but few epidemiological data on ESBL-producing Escherichia coli urinary tract isolates in pet dogs are available in China. Methodology: This study was conducted to describe the prevalence and characterization of ESBL producers among E. coli urinary tract isolates from pet dogs in Tai'an, China. Results: A total of 118 E. coli were obtained from urinary samples of 80 companion dogs suffering from acute or chronic cystitis, of which three isolates from different dogs were ESBL producers. One isolate from dog A was of phylogroup A/ST410/CTX-M-15/TEM-1; one from dog B was of phylogroup B1/ST533/CTX-M-15/TEM-1; one from dog C was of phylogroup D/ST648/CTX-M-15. All ESBL producers were resistant to ampicillin, cephalexin, cefalotin, cefpodoxime, ceftiofur, enrofloxacin, marbofloxacin, and trimethoprim/sulfamethoxazole, but were susceptible to imipenem and amoxicillin/clavulanic acid. E. coli of ST533 carrying blaCTX-M-15 were first detected in pet dogs in China. Conclusions: Collectively, the findings could expand our knowledge about the prevalence and characterization of ESBL-producing E. coli urinary tract isolates in pet dogs in China.


Introduction
Since the introduction of third-generation cephalosporins in the early 1980s, extended-spectrum beta-lactamase (ESBL)-producing bacteria have rapidly emerged in human and veterinary practices [1]. The main resistance mechanism of these bacteria is the production of ESBLs, but the enzymes can be inhibited by clavulanic acid, sulbactam, and tazobactam [2]. ESBL producers, apart from being resistant to β-lactam antibiotics, can also be resistant to other classes of antibiotics such as tetracyclines, fluoroquinolones, sulfmethoxazole/trimethoprim, and aminoglycosides [3,4]. There is no doubt that the pan-resistance of ESBL producers limits clinical therapy option and increases medical costs.
ESBL producers were initially detected in human medical practice, but recent investigations have shown that ESBL producers have been found in farm animals and wild animals [5][6][7][8][9][10]. The increasing number of ESBL-producing isolates found in animals has led to the hypothesis that animals might become infection sources or even reservoirs contributing to the spread of these bacteria [11]. As humans often live in close contact with pets, companion animals could become potential sources of ESBL-producing isolates causing community-acquired infections.
ESBL-producing isolates from companion animals mainly include E. coli and Klebsiella pneumoniae. ESBL-producing E. coli not only are the intestinal pathogen, but also the common causative bacterium for urinary tract infections (UTIs) [11]. To date, numerous investigations about prevalence and characterization of ESBL-producing E. coli from humans and companion animals have been reported [12][13][14][15][16][17]; however, information about characteristics of ESBL-producing E. coli from pet animals in China is very limited. To fill the literature gap, the present study was designed to describe the prevalence and characterization of ESBL producers among E. coli urinary tract isolates from pet dogs in Tai'an, China.

Ethics statement
The study was approved by the ethics committee of Taishan Medical University (permit ECTSMU2011-009).

Bacterial isolates
Between January 2011 and November 2013, urine samples of 80 pet dogs suffering from acute or chronic cystitis were collected by cystocentesis in 6 animal hospitals in Tai'an, China. The collected samples were spread onto blood agar plates and cultured at 37°C for 24 hours. E. coli isolates were identified using traditional biochemical methods and the Vitek2 system (bioMérieux, Hazelwood, USA). The identified isolates were stored at -20°C in cryoprotective media prior to use.

Phenotypic ESBL detection and antimicrobial susceptibility testing
According to the manufacturer's protocols, Etest ESBL strips (bioMérieux, Marcy l'Etoile, France) were used to determine ESBL production of E. coli isolates. According to the Clinical and Laboratory Standards Institute guidelines [18], agar dilution method was used to test susceptibility of ESBL-producing E. coli isolates against 12 antimicrobial agents. The tested drugs included ampicillin, cephalexin, cefalotin, cefpodoxime, ceftiofur, enrofloxacin, marbofloxacin, imipenem, tetracycline, amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, and amikacin (Tianhe, Hangzhou, China). E. coli ATCC 25922 was used as a quality control strain.
If ESBL-producing E. coli isolates obtained from the same individual companion dog showed the same drug-resistant phenotype, ESBL gene, and multilocus sequence type (ST), these isolates were considered to be the same strain and only one was included in this study. An E. coli isolate was considered to be multidrug-resistant (MDR) when it exhibited resistance to antimicrobials of at least three different classes [19].

Detection of beta-lactamase genes
Polymerase chain reaction (PCR) was used in this study to amplify beta-lactamase resistance genes (bla CTX-M , bla TEM , and bla SHV ) for all ESBL-producing isolates, and the corresponding primers and reaction conditions were used as previously described [16]. The amplified products were either directly sequenced from both ends or cloned in pMD18-T and then sequenced. The deduced amino acid sequences were aligned using Lasergene software (DNASTAR, Madison, USA), and compared with sequences available at GenBank (http://www.ncbi.nlm.nih.gov/GenBank/index.html) to determine ESBL genotype. Mutations were also analyzed with reference to the Lahey Clinic website (http://www.lahey.org/studies/).

Determination of E. coli phylogroups in ESBL producers
E. coli has four main phylogroups (A, B1, B2, and D), among which groups A and B1 typically contain commensal isolates and strains of groups B2 and D are more likely to carry pathogenicity-associated genes [20,21]. The analysis of phylogenetic groups was carried out using multiplex PCR, according to the method described previously [22].

Results
A total of 118 E. coli isolates were obtained from urinary samples of 80 pet dogs suffering from acute or chronic cystitis in 6 animal hospitals in Tai'an city between January 2011 and November 2013. In total, 3 ESBL-producing E. coli isolates were isolated from urinary samples of 3 pet dogs (A, B, and C): 1 isolate from dog A (a male Pekingese), 1 from dog B (a female Pekingese), and 1 from dog C (a male Golden Retriever) ( Table 1).
A total of 3 ESBL-producing E. coli isolates in this study were all MDR and were all susceptible to amoxicillin/clavulanic acid and imipenem. In addition, 2 isolates from dogs B and C were resistant to amikacin ( Table 2). Among 3 ESBL-producing E. coli isolates, 1 strain from dog A belonged to phylogroup A, carried bla CTX-M-15 and bla TEM-1 genes, and was of ST410; 1 strain from dog C was of ST648, carried bla CTX-M-15 , and belonged to phylogroup D; and 1 isolate from dog B belonged to phylogroup B1, contained bla CTX-M-15 and bla TEM-1 genes, and was of ST533 (Table 1).

Discussion
All ESBL-producing E. coli isolates in this study were resistant to ampicillin, cephalexin, cefalotin, cefpodoxime, ceftiofur, enrofloxacin, marbofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. The result may be related to the fact that plasmids containing bla CTX-M often carry resistance genes, such as fluoroquinolones and aminoglycosides [4,24]. However, ESBL-producing E. coli isolates were all susceptible for amoxicillin/clavulanic acid and imipenem. In addition, only one ESBL-producing E. coli isolate from dog A was susceptible to amikacin.
E. coli of ST410 carrying the bla CTX-M-15 gene has been detected in dog urinary samples and human samples in China and other countries [16,[25][26][27]. E. coli of ST648 carrying bla CTX-M-15 gene has been frequently found in clinical ESBL-producing E. coli isolates from humans and animals worldwide [15,16,24,28], and therefore the ESBL-producing E. coli of ST648 is regarded as the potential extended-host spectrum genotype. ESBL-producing E. coli of ST533 is sparse and was only detected twice in humans with UTIs in Brazil and Germany [29], once in manure samples of gulls in France [30], and once in urinary samples of pet dogs in Switzerland [16]. To our best knowledge, E. coli of ST533 carrying bla CTX-M-15 was detected in companion dog for the first time in China.
In this study, all three ESBL-producing E. coli isolated from urinary samples of companion dogs carried the bla CTX-M-15 gene, and two of three ESBLproducing E. coli carried the bla TEM-1 gene. bla CTX-M+TEM was the dominant bla gene type, which is consistent with the results of other studies detecting these genes in ESBL producers from companion animals in China and other counties [11,16,31]. CTX-M-15-producing E. coli of ST131 was not found in the present study, which is regarded as an emerging human pandemic clone [32]. However, ESBL-producing E. coli has been detected in urinary samples of dogs in Europe [12]. Additionally, no bla SHV gene was found in this study, which is in agreement with the results of other studies about ESBLproducing E. coli from dogs in China [33,34]. However, in the United States, bla SHV-12 was detected from urinary samples of companion animals [13].

Conclusions
In summary, the limitation of this study was the relatively small number of ESBL-producing E. coli from pet dogs. The findings of this study, however, could improve our knowledge about the prevalence and characterization of ESBL-producing E. coli urinary tract isolates in pet dogs in China.