This study used a PCR-based approach targeting 16S rRNA gene fragments to determine the occurrence and association of the three bovine digital dermatitis (BDD) treponeme phylogroups within lesions found in cattle from the United Kingdom. Examination of 51 BDD lesions collected from infected cattle across the United Kingdom revealed that BDD treponeme group 1 (Treponema medium/Treponema vincentii-like), group 2 (Treponema phagedenis-like), and group 3 (Treponema putidum/Treponema denticola-like) were present in 96.1%, 98%, and 76.5% of BDD lesions, respectively. The three phylogroups were present together in 74.5% of lesions. The PCR assays enabled the isolation of further treponeme strains from previously mixed primary BDD lesion cultures. Here a representative from each of the three distinct treponeme phylogroups was isolated from a single BDD lesion for the first time. These data highlight the extent to which this disease is polytreponemal. Immunohistochemistry and electron microscopy were used to investigate lesional hoof tissues, resulting in treponemes being identified copiously in hair follicles and sebaceous glands, suggesting a potential route of exit and/or entry for these pathogens. This study gives further evidence for the importance of the three treponeme groups in BDD pathogenesis and reiterates the value of molecular genetic approaches for isolating and identifying fastidious anaerobes.

BackgroundThere is a growing concern regarding the increase of antimicrobial resistant bacteria in companion animals. Yet, there are no studies comparing the resistance levels of these organisms in European countries. The aim of this study was to investigate geographical and temporal trends of antimicrobial resistant bacteria causing urinary tract infection (UTI) in companion animals in Europe. The antimicrobial susceptibility of 22 256 bacteria isolated from dogs and cats with UTI was determined. Samples were collected between 2008 and 2013 from 16 laboratories of 14 European countries. The prevalence of antimicrobial resistance of the most common bacteria was determined for each country individually in the years 2012–2013 and temporal trends of bacteria resistance were established by logistic regression.ResultsThe aetiology of uropathogenic bacteria differed between dogs and cats. For all bacterial species, Southern countries generally presented higher levels of antimicrobial resistance compared to Northern countries. Multidrug-resistant Escherichia coli were found to be more prevalent in Southern countries. During the study period, the level of fluoroquinolone-resistant E. coli isolated in Belgium, Denmark, France and the Netherlands decreased significantly. A temporal increase in resistance to amoxicillin-clavulanate and gentamicin was observed among E. coli isolates from the Netherlands and Switzerland, respectively. Other country-specific temporal increases were observed for fluoroquinolone-resistant Proteus spp. isolated from companion animals from Belgium.ConclusionsThis work brings new insights into the current status of antimicrobial resistance in bacteria isolated from companion animals with UTI in Europe and reinforces the need for strategies aiming to reduce resistance.

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This study describes the association between bovine digital dermatitis (BDD) treponemes and three 'non-healing' bovine hoof horn lesions, namely, 'toe necrosis' (TN), 'non-healing white line disease' (nhWLD) and 'non-healing sole ulcer' (nhSU), which are disorders that involve penetration through the horn capsule to involve the corium. In this study, these non-healing disorders (n=44) were identified as foot lesions that exhibited a topical granular appearance, exuded a typical pungent smell, were severely painful to the animal involved, and typically originated from farms where BDD is endemic. Given the similarities between these 'non-healing' lesions and BDD, the authors subjected samples of diseased tissue to PCR assays to detect the presence of DNA of BDD treponemes. All the three characterised BDD treponeme groups were identified as present together in 84.2, 81.3 and 55.6 per cent of samples of TN (n=19), nhWLD (n=16) and nhSU (n=9), respectively. In contrast, healthy control horn samples from similar sites (n=16) were PCR-negative for the BDD treponemes. Hence, these non-healing hoof lesions were strongly associated with BDD treponemes. Samples from typical heel horn erosions (n=9) were also subjected to BDD treponeme PCR assays and no association could be identified between the BDD treponemes and this horn manifestation.

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.

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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.