Detection of the Pandemic O25-ST131 Human Virulent Escherichia coli CTX-M-15-Producing Clone Harboring the qnrB2 and aac(6′)-Ib-cr Genes in a Dog

The recent worldwide emergence of plasmid-mediated quinolone resistance due to qnr and aac(6′)-Ib-cr genes is a concerning fact among human and animal gram-negative pathogens (9, 14). The association of plasmid-mediated quinolone resistance with the production of extended-spectrum β-lactamases (ESBLs), including the widespread human CTX-M-15 enzyme, has been reported (8, 9). Very recently, an internationally disseminated Escherichia coli clone (O25:H4-ST131) with the CTX-M-15 ESBL was described (12). Furthermore, representative isolates of this globally diffusing clone were characterized as highly virulent and belonging to the B2 phylogenetic group (3). The aim of this study was to determine the prevalence of plasmid-mediated qnr genes among 61 consecutive nonrepetitive clinical strains of E. coli and to subsequently characterize positive isolates. These strains were isolated from dogs (n = 41) and cats (n = 20) with urinary tract infections at the Faculty of Veterinary Medicine in Lisbon, Portugal, from 2004 to 2006. Thirty-four percent and 26% of the strains were resistant to nalidixic acid and ciprofloxacin, respectively. Two were CMY-2 producers, and only one was an ESBL-producing strain. The presence of the qnrA, qnrB, and qnrS genes was investigated by dot blot DNA hybridization assays (ECL direct nucleic acid labeling and detection system 3000). The qnrA and qnrS genes were not found. The qnrB gene was detected in one (1.6%) of 61 E. coli isolates. The full qnrB2 gene nucleotide sequence was then determined. The QnrB2-positive strain, FMV5825, was isolated in Portugal from a 15-year-old female dog with chronic cystitis, after prolonged antimicrobial therapy, and was considered resistant to different antibiotics. These included ampicillin and cephalosporins (except 7-α-methoxy-cephalosporins), aztreonam, piperacillin, ticarcillin, trimethoprim-sulfamethoxazole, aminoglycosides (amikacin, gentamicin, and tobramycin), nalidixic acid, and fluoroquinolones (ciprofloxacin, norfloxacin, and levofloxacin) (Table ​(Table1).1). The MICs were determined by Dade MicroScan or by standard broth microdilution assays, performed and interpreted according to a Clinical and Laboratory Standards Institute guideline (5). Quinolone chromosome-encoded resistance of the FMV5825 strain was attributed to the Ser83→Ile and Asp87→Asn substitutions in GyrA and the Ser80→Ile and Glu84→Val substitutions in ParC, both previously associated with high-level fluoroquinolone resistance in E. coli (7). TABLE 1. Antimicrobial susceptibilities of the E. coli FMV5825 uropathogenic canine clinical isolate and E. coli J53 transconjugant and recipient strains The transferability of the qnrB2 gene was initially assessed by transformation. Plasmid DNA, extracted with a Qiagen plasmid mini kit, was transferred by electroporation into E. coli DH5α cells. Transformants were selected with 10 μg/ml ceftazidime. To avoid the nalidixic acid chromosome-encoded resistance of DH5α cells, further conjugation experiments were performed at 35°C using the E. coli DH5α-5825 transformant as the donor and E. coli J53 Azir as the recipient (11). Resistance to β-lactams, amikacin, tobramycin, ciprofloxacin, and norfloxacin but not to levofloxacin was cotransferred (Table ​(Table1).1). This phenotype raised the possibility of the association of the aminoglycoside acetyltransferase variant, AAC(6′)-Ib-cr, capable of acetylating ciprofloxacin and norfloxacin, as well as the presence of an ESBL enzyme. Indeed, we identified the following cotransferred genes: qnrB2, aac(6′)-Ib-cr, blaCTX-M-15, blaTEM-1B, and blaOXA-1. The insertion sequence ISEcp1 was found upstream of the blaCTX-M-15 gene. These genes and ISEcp1, except qnrB2, have also been detected in E. coli strains from humans with urinary tract infections in Portugal (10). To our knowledge, this is the first description of a canine uropathogenic E. coli CTX-M-15-producing strain containing the qnrB2 and aac(6′)-Ib-cr fluoroquinolone resistance genes. In this study, the plasmid encoding the multidrug resistance of the FMV5825 strain belonged to the narrow-host-range incompatibility group IncFII and had the FII replicon and the FIA replicon, according to a PCR-based replicon-typing scheme (2). The association of the blaCTX-M-15 gene with IncFII plasmids also containing blaTEM-1B, blaOXA-1, and aac(6′)-Ib-cr from human E. coli strains has been reported for different continents, suggesting a common origin for all of them (1, 6). A comparable multidrug resistance region was found in our animal IncFII plasmid except for the presence of qnrB2. Our E. coli strain of animal origin shares features identical to those of an intercontinental human clone (12), also belonging to the B2 phylogenetic group, O25 serogroup, and ST131 (determined by phylogenetic group multiplex PCR assay [4], O typing by the traditional antiserum method, and the MLST scheme [www.mlst.net] [15], respectively). Representative isolates of this globally diffusing clone constitute the B2 phylogenetic subgroup I, characterized by the abilities of its members to produce biofilm and to be highly virulent in mice despite lacking classical extraintestinal pathogenicity islands and their adhesion factors (3). The absence of pap, sfa, afa fimbriae, and hly operons was also noted for our E. coli strain. The presence of the iutD gene (representative of the siderophore aerobactin operon), the biofilm-producing ability (evaluated using a fluorescent in situ hybridization method [13]), and the similarity with the O25:H4-ST131 TNN (TE2) French E. coli isolate (3, 17) raise the possibility that the E. coli strain isolated from our canine patient belongs to the same phylogenetic B2 subgroup. This is the first report of the presence of the O25-ST131 human virulent E. coli CTX-M-15-producing clone harboring the disseminated incompatibility group IncFII plasmid with a multidrug resistance region containing the blaCTX-M-15, blaTEM-1B, blaOXA-1, aac(6′)-Ib-cr, and qnrB2 genes in animals. It is possible that human-to-animal or animal-to-human transmission of the O25-ST131 clone has occurred.