c Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum ␤-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried bla CTX-M-15 and bla TEM-1 , while K. pneumoniae subsp. pneumoniae isolates carried bla SHV-12 and bla TEM-1 . Conjugation experiments demonstrated that bla CTX-M-15 and bla TEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with bla CTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the bla CTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 ␤-lactamases in the United Kingdom. We also describe the genetic environment of bla CTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.

Escherichia coli are opportunistic pathogens with the potential to cause a variety of infections in both humans and animals and in many cases have developed antimicrobial resistance. In this study, we characterized extended-spectrum cephalosporin resistant (ESCR) E. coli isolates from diseased companion animals (dogs, cats, and horses) and related the results to clinical findings. ESCR E. coli clinical isolates obtained over a 6-year period were screened for extended-spectrum β-lactamase (ESBL) and/or plasmid mediated AmpC (pAmpC) and virulence markers likely to be associated with extraintestinal pathogenic E. coli (ExPEC). ESBL and/or pAmpC genetic determinants were identified in 79.9% of the ESCR E. coli isolates with bla CTX-M genes being the most common ESBL genotype of which bla CTX-M-15 , bla CTX-M-14 , and bla CTX-M-55 were the most prevalent. In addition, bla CMY -2 was the most common genotype identified amongst pAmpC producing isolates. Phylogenetic group typing showed that B2 was the most prevalent phylogroup among the ESCR E. coli isolates, followed by the closely related phylogroups D and F which are also associated with extra-intestinal infections. ESCR was also identified in phylogroups commonly regarded as commensals (B1, A, and C). Virulence factor (VF) scores >2 were mostly present amongst isolates in phylogroup B2. Higher virulence scores were found in isolates lacking ESBL/pAmpC resistance genes compared with those carrying both genes ( p < 0.05). Five of phylogroup B2 isolates, were typed to the pandemic virulent O25b-ST131 clone and three ST131 isolates carrying bla CTX-M-15 belonged to the subclade C2/H30Rx whilst one isolate carrying bla CTX-M-27 typed to the recently described sub-clade C1-M27. MLST typing also identified other sequence types commonly associated with infections in humans (ST410, ST10, and ST648). Most ESCR E. coli isolates obtained in pure growth were cultured from normally sterile body sites (mostly from urinary tract infections, UTIs) whilst only a small proportion were obtained from body sites populated with commensal flora ( p < 0.0001). Our study has shown that ExPEC ESBL/pAmpC producing E. coli isolates are common amongst companion animal isolates and are associated with colonization and infection. In addition, their isolation from a normally sterile site is likely to be clinically significant and warrants antimicrobial treatment.

Use of antibiotics in food animals may contribute to development and spread of resistant organisms, particularly so in some countries. The aim of this study was two-fold; first, to establish the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in chicken production in a region within Romania. Second, to study the relatedness of ESBL-producing E. coli isolates recovered from broilers, abattoir workers where the chickens were slaughtered and from the human clinical specimens from two regional hospitals. The results indicated a very high (69%) rate of carriage of ESBL and AmpC-producing E. coli in chickens with 36% CTX-M producers. Sequencing showed that chickens in Romania have the highest worldwide prevalence (53%) of blaCTX-M-15 reported in poultry E. coli isolates. The majority (53%) of the extended-spectrum cephalosporin-resistant E. coli carried plasmid-mediated blaampC genes, mostly blaCMY-2 type, one of the highest prevalences reported in Europe. The predominant CTX-M type found in the human clinical E. coli isolates was blaCTX-M-15 and most isolates coharbored blaOXA-1, blaTEM, and aac(6')-ib-cr. The majority (60%) of the human clinical isolates belonged to the pandemic virulent clone B2-ST131. The clonal relationship between broiler and the human CTX-M-producing E. coli isolates was assessed by macrorestriction pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), which indicated strain diversity with no common STs found between human and poultry isolates. Moreover, IncI1 was the most prevalent replicon found in broiler ESBL-producing E. coli isolates and also in transconjugants, indicating that plasmids and not clonal spread may play a role in the transfer of blaCTX-M genes. This study identifies a high prevalence of ESBL-producing E. coli from broiler chickens in Romania with a high occurrence incidence of blaCTX-M-15, which reflects the main ESBL type found in human E. coli infections in this country.

We characterized extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) in 32 Escherichia coli extended spectrum cephalosporin (ESC)-resistant clinical isolates from UK companion animals from several clinics. In addition, to investigate the possible dissemination of ESBL clinical isolates within a veterinary hospital, two ESBL-producing E. coli isolates from a dog with septic peritonitis and a cluster of environmental ESC-resistant E. coli isolates obtained from the same clinic and during the same time period, as these two particular ESBL-positive clinical isolates, were also included in the study. Molecular characterization identified blaCTX-M to be the most prevalent gene in ESC-resistant isolates, where 66% and 27% of clinical isolates carried blaCTX-M-15 and blaCTX-M-14, respectively. The only PMQR gene detected was aac(6')-Ib-cr, being found in 34% of the ESC E. coli isolates and was associated with the carriage of blaCTX-M-15. The clinical and environmental isolates investigated for hospital dissemination had a common ESBL/AmpC phenotype, carried blaCTX-M-15, and co-harbored blaOXA-1, blaTEM-1, blaCMY-2, and aac(6')-Ib-cr. Multilocus sequence typing identified them all as ST410, while pulse-field gel electrophoresis demonstrated 100% homology of clinical and environmental isolates, suggesting hospital environmental dissemination of CTX-M-15–producing E. coli ST410.