Amy L. Wedley

Cross-sectional study of antimicrobial-resistant bacteria in horses. Part 1: Prevalence of antimicrobial-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus

REASONS FOR PERFORMING STUDY The increasing prevalence of antimicrobial-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and antimicrobial-resistant Escherichia coli represents a significant problem. However, the carriage of such bacteria by horses in the UK has not been well characterised. OBJECTIVES To estimate the prevalence of nasal carriage of MRSA and faecal carriage of antimicrobial-resistant E. coli amongst horses in the general equine community of the mainland UK. METHODS A cross-sectional study of horses recruited by 65 randomly selected equine veterinary practices was conducted, with nasal swabs and faecal samples collected. Faecal samples were cultured for antimicrobial-resistant E. coli. Nasal swabs were cultured for staphylococcal species; methicillin-resistant isolates identified as S. aureus were characterised by SCCmec and spa gene typing. Multilevel logistic regression models were used to calculate prevalence estimates with adjustment for clustering at practice and premises levels. Spatial variation in risk of antimicrobial resistance was also examined. RESULTS In total, 650 faecal samples and 678 nasal swabs were collected from 692 horses located on 525 premises. The prevalence of faecal carriage of E. coli with resistance to any antimicrobial was 69.5% (95% CI 65.9-73.1%) and the prevalence of extended-spectrum β-lactamase (ESBL)-producing E. coli was 6.3% (95% CI 4.1-9.6%). The prevalence of nasal carriage of MRSA was 0.6% (95% CI 0.2-1.5%). Spatial analysis indicated variation across the UK for risk of carriage of resistant and multidrug-resistant (resistant to more than 3 antimicrobial classes) E. coli. CONCLUSIONS AND POTENTIAL RELEVANCE Carriage of MRSA by horses in the community appears rare, but the prevalence of antimicrobial-resistant E. coli (including ESBL-producing E. coli) is higher. A high prevalence of antimicrobial-resistant bacteria could have significant health implications for the horse population of the UK.

Prevalence of antimicrobial-resistant Escherichia coli in dogs in a cross-sectional, community-based study

The prevalence of carriage of antimicrobial-resistant (AMR) and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli was determined in 183 healthy dogs from a semi-rural community in Cheshire. Isolates were tested against a panel of antimicrobials and by PCR to detect resistance genes. In the suspected ESBL-producing isolates, the presence of blaSHV, blaTEM, blaCTX-M and blaAmpC genes was determined by PCR and sequencing. A total of 53 (29 per cent, 95 per cent confidence interval [CI] 22.4 to 35.5 per cent) dogs carried at least one AMR E coli isolate. Twenty-four per cent (95 per cent CI 17.9 to 30.2 per cent) of dogs carried isolates resistant to ampicillin, 19.7 per cent (95 per cent CI 13.9 to 25.4 per cent) to tetracycline and 16.9 per cent (95 per cent CI 11.5 to 22.4 per cent) to trimethoprim. A blaTEM gene was detected in 39 of 54 ampicillin-resistant isolates, a tet(B) gene in 12 of 45 tetracycline-resistant isolates, and a dfr gene in 22 of 33 trimethoprim-resistant isolates. Multidrug-resistant isolates were demonstrated in 15 per cent (28 of 183; 95 per cent CI 10.1 to 20.5 per cent) of dogs. Nine suspected ESBL-producing E coli were isolated, of which only one was confirmed by double disc diffusion testing. Two of these isolates carried the blaTEM-1 gene and seven carried the blaCMY-2 gene.

Cross-sectional study of antimicrobial-resistant bacteria in horses. Part 2: Risk factors for faecal carriage of antimicrobial-resistant Escherichia coli in horses.

REASONS FOR PERFORMING STUDY The increasing prevalence of antimicrobial resistant bacteria such as antimicrobial-resistant and extended spectrum β-lactamase (ESBL)-producing Escherichia coli represents a significant problem for human and veterinary medicine. Despite this, the risk factors for faecal carriage of such bacteria by horses in the UK, particularly those in the wider community, have not been well described. OBJECTIVES To characterise the risk factors for faecal carriage of antimicrobial-resistant E. coli amongst horses in the mainland UK. METHODS A cross-sectional study of horses recruited by 65 randomly selected equine veterinary practices was conducted, with a faecal sample collected and self-administered questionnaire completed by the horse owner. Faecal samples were cultured for antimicrobial-resistant E. coli, with isolates confirmed as E. coli having their antimicrobial resistance profile determined. Multilevel, multivariable logistic regression models were used to investigate risk factors for the carriage of antimicrobial-resistant E. coli in the sample population. RESULTS Faecal samples and completed questionnaires were obtained for 627 horses located on 475 premises. Recent hospitalisation, contact with specific types of nonequid animals, the type of premises, the surrounding land use, the reason for veterinary treatment received in the last 6 months and antimicrobial treatment in the previous 10 days were identified as risk factors for many of the antimicrobial-resistance outcomes considered. Being stabled on the same yard as a recently hospitalised horse was identified as a risk factor for increased risk of carriage of ESBL-producing E. coli. CONCLUSIONS AND POTENTIAL RELEVANCE Increasing antimicrobial resistance may have significant health implications for the horse population of Great Britain. This form of epidemiological investigation highlights potential risk factors that may be controlled to limit the extent of the problem.

Carriage of Staphylococcus species in the veterinary visiting dog population in mainland UK: Molecular characterisation of resistance and virulence

This study investigated the prevalence of nasal carriage of staphylococci in dogs and determined the characteristics of the isolates. A total of 724 dogs from 87 veterinary practices across the mainland UK were screened for carriage of Staphylococcus spp. All isolates were examined for meticillin resistance (MR) and the presence of the mecA gene investigated in those isolates showing resistance. All coagulase-positive staphylococci and MR coagulase-negative staphylococci (MRCoNS) were subjected to antimicrobial susceptibility testing. Spa typing and DNA microarray analysis of resistance and virulence genes was carried out on all MR S. aureus (MRSA) and a subset of meticillin susceptible S. aureus (MSSA). Staphylococci were isolated from 399 (55.1%) of the dogs; only seven (1%) carried MRSA, all of which were identified as the dominant UK healthcare-associated strain (EMRSA-15, ST22). MSSA was identified in 47 (6.5%) dogs, the sequence types of which have been suggested as precursors to successful MRSA clones. Forty (5.5%) dogs carried MRCoNS, while no dogs carried MR S. pseudintermedius, although this is increasingly reported in mainland Europe. Resistance to antimicrobials among the isolates varied between species, with multidrug resistance (MDR) in 87.5% of MRCoNS and 21.8% of coagulase positive staphylococci. Microarray analysis of MRSA and a subset of MSSA isolates identified numerous virulence genes associated with pathogenesis, which are commonly identified in isolates of human origin. However, no isolates carried Panton-Valentine leukocidin (PVL) genes. This study suggests that MRSA carriage is low in the vet visiting dog population, but there is a diverse range of virulence and resistance determinants in canine S. aureus and MRCoNS isolates.

Carriage of antimicrobial resistant Escherichia coli in dogs: Prevalence, associated risk factors and molecular characteristics

Resistance to antimicrobials, in particular that mediated by extended spectrum β-lactamases (ESBL) and AmpC β-lactamases are frequently reported in bacteria causing canine disease as well as in commensal bacteria, which could be a potential health risk for humans they come into contact with. This cross-sectional study aimed to estimate the prevalence and investigate the molecular characteristics of ESBL and plasmid encoded AmpC (pAmpC)-producing E. coli in the mainland UK vet-visiting canine population and, using responses from detailed questionnaires identify factors associated with their carriage. Faecal samples were cultured for antimicrobial resistant (AMR), ESBL and pAmpC-producing E. coli. A subset of ESBL and pAmpC-producing isolates were subjected to multi-locus sequence typing and DNA microarray analyses. Multivariable logistic regression analysis was used to construct models to identify risk factors associated with multidrug resistant (MDR, resistance to three or more antimicrobial classes), fluoroquinolone resistant, ESBL and AmpC-producing E. coli. AMR E.coli were isolated from 44.8% (n=260) of samples, with 1.9% and 7.1% of samples carrying ESBL and pAmpC-producing E. coli, respectively. MDR E. coli were identified in 18.3% of samples. Recent use of antimicrobials and being fed raw poultry were both identified as risk factors in the outcomes investigated. A number of virulence and resistance genes were identified, including genes associated with extra-intestinal and enteropathogenic E. coli genotypes. Considering the close contact that people have with dogs, the high levels of AMR E. coli in canine faeces may be a potential reservoir of AMR bacteria or resistance determinants.

Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and –poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.

A role for flies (Diptera) in the transmission of Campylobacter to broilers

SUMMARY Campylobacter is the leading cause of bacterial diarrhoeal disease worldwide, with raw and undercooked poultry meat and products the primary source of infection. Colonization of broiler chicken flocks with Campylobacter has proved difficult to prevent, even with high levels of biosecurity. Dipteran flies are proven carriers of Campylobacter and their ingress into broiler houses may contribute to its transmission to broiler chickens. However, this has not been investigated in the UK. Campylobacter was cultured from 2195 flies collected from four UK broiler farms. Of flies cultured individually, 0·22% [2/902, 95% confidence interval (CI) 0–0·53] were positive by culture for Campylobacter spp. Additionally, 1293 flies were grouped by family and cultured in 127 batches: 4/127 (3·15%, 95% CI 0·11-6·19) from three broiler farms were positive for Campylobacter. Multilocus sequence typing of isolates demonstrated that the flies were carrying broiler-associated sequence types, responsible for human enteric illness. Malaise traps were used to survey the dipteran species diversity on study farms and also revealed up to 612 flies present around broiler-house ventilation inlets over a 2-h period. Therefore, despite the low prevalence of Campylobacter cultured from flies, the risk of transmission by this route may be high, particularly during summer when fly populations are greatest.

Characterization of a novel EAST-negative enteropathogenic E. coli strain implicated in a food-borne outbreak of diarrhoea in adults

Enteropathogenic Escherichia coli (EPEC) is usually associated with outbreaks and sporadic cases of severe infantile diarrhoea in the developing world, and less commonly with sporadic cases in developed countries. Very little evidence indicates that EPEC is a food‐borne pathogen for adults. In a previous study, two groups of adult travellers became ill, and eae+ E. coli of serogroup O111 was isolated from affected individuals and epidemiologically linked to food consumption. Here the strain responsible was further investigated and characterized as an unusual atypical EPEC. PCR analysis of the designated type isolate showed the presence of the rorf1 and espB genes of the LEE pathogenicity island, which was inserted at the chromosomal selC locus. The isolate was negative for the enteroaggregative E. coli EAST‐1 toxin present in other strains of EPEC associated with food‐borne outbreaks. The strain adhered sparsely to HEp‐2 cell monolayers in a diffuse manner, but fluorescent actin staining demonstrated that it was capable of inducing polymerization of actin at the sites of bacterial attachment. Strain P2583 is the first EAST‐negative EPEC to be confirmed as a cause of outbreaks of infection in adults following the consumption of contaminated food or water.

Identification and initial characterisation of a protein involved in Campylobacter jejuni cell shape.

Campylobacter jejuni is the leading cause of bacterial food borne illness. While helical cell shape is considered important for C. jejuni pathogenesis, this bacterium is capable of adopting other morphologies. To better understand how helical-shaped C. jejuni maintain their shape and thus any associated colonisation, pathogenicity or other advantage, it is first important to identify the genes and proteins involved. So far, two peptidoglycan modifying enzymes Pgp1 and Pgp2 have been shown to be required for C. jejuni helical cell shape. We performed a visual screen of ∼2000 transposon mutants of C. jejuni for cell shape mutants. Whole genome sequence data of the mutants with altered cell shape, directed mutants, wild type stocks and isolated helical and rod-shaped ‘wild type’ C. jejuni, identified a number of different mutations in pgp1 and pgp2, which result in a change in helical to rod bacterial cell shape. We also identified an isolate with a loss of curvature. In this study, we have identified the genomic change in this isolate, and found that targeted deletion of the gene with the change resulted in bacteria with loss of curvature. Helical cell shape was restored by supplying the gene in trans. We examined the effect of loss of the gene on bacterial motility, adhesion and invasion of tissue culture cells and chicken colonisation, as well as the effect on the muropeptide profile of the peptidoglycan sacculus. Our work identifies another factor involved in helical cell shape.