Carriage of antimicrobial-resistant Escherichia coli and staphylococci in dogs in the community: molecular mechanisms

While previous studies have determined the prevalence of meticillin resistant Staphylococcus aureus (MRSA) and antimicrobial resistant (AMR) Escherichia coli in canine populations, few have included sufficiently large sample sizes and fewer still have characterised the isolates or investigated risk factors that might be associated with their carriage. The main aims of the work presented in this thesis were; to determine the nasal prevalence of MRSA and other AMR staphylococci and the faecal prevalence of AMR E. coli in faeces in the canine population of mainland UK. The study also aimed to characterise the bacteria isolated using molecular techniques in order for comparisons to be made with isolates of human origin, and to determine the presence of potential risk factors associated with faecal carriage of AMR E. coli. These objectives were achieved by carrying out two studies. The first study used frozen canine faecal samples collected during a cross sectional study of a semi-rural community in Cheshire to determine the prevalence of AMR E. coli. The second study collected faecal and nasal swabs from dogs visiting veterinary practices across mainland UK. Antimicrobial resistance of the isolates obtained from both studies were characterised using disc diffusion methods and PCR assays. In addition, isolates collected during the second study were subjected to multi-locus sequence typing and DNA micro array analysis of resistance and virulence genes. For antimicrobial resistant E. coli, risk factors associated with carriage were investigated. The prevalence of MRSA in the canine population was found to be low at 1% and all isolates were identical to EMRSA-15, the main human endemic strain in many UK hospitals. The overall S. aureus prevalence was 7.5%, with a higher prevalence of 11.0% of S. pseudintermedius, in which no meticillin resistance was found. Meticillin resistant coagulase negative Staphylococcus spp. was found in 5.5% of dogs. AMR in the isolates varied between species; however resistance to fusidic acid was consistently high. AMR E. coli was common in both studies (29.0% in community study and 44.8% in nationwide study). Resistance to ampicillin (24.0% and 37.2%), tetracycline (19.7% and 30.0%), trimethoprim (16.9% and 23.8%) and resistance to three or more antimicrobial classes (15.3% and 18.1%) was found to be high in both studies (community and nationwide respectively), while resistance to augmentin, chloramphenicol, ciprofloxacin and nalidixic acid was below 10% in both studies. A variety of genes responsible for resistance to expanded spectrum β-lactams was identified; including blaCTX-M-15 and blacmy2, both of which have previously been identified in humans and dogs. A number of variables were found to be associated with resistance to antimicrobials, with previous prescription of antimicrobials and consumption of raw poultry meat remaining in the final model of more than one resistance outcome. The carriage of MRSA and antimicrobial resistant E. coli could pose a potential problem both in terms of the welfare of the dogs carrying such bacteria as well as the zoonotic potential of the bacteria and resistance determinants

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

YesEnteropathogenic 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

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

<i>Campylobacter jejuni</i>transmission and colonisation in broiler chickens is inhibited by Faecal Microbiota Transplantation

ABSTRACT BACKGROUND Campylobacter jejuni, the most frequent cause of foodborne bacterial infection, is found on around 70% of retail chicken. As such there is a need for effective controls in chicken production. Microbial-based controls such as probiotics are attractive to the poultry industry, but of limited efficacy. Furthermore, as commercially-produced chickens have no maternal contact, their pioneer microbiome is likely to come from the hatchery environment. Early delivery of microbials that lead to a more ‘natural avian’ microbiome may, therefore, improve bird health and reduce susceptibility to C.jejuni colonisation. A faecal microbiota transplant (FMT) was used to transfer a mature cecal microbiome to newly-hatched broiler chicks and its effects on C.jejuni challenge assessed. We used both a seeder-bird infection model that mimics natural bird-to-bird infection alongside a direct-challenge model. We used a 16S rRNA gene sequencing-based approach to characterize the transplant material itself alongside changes to the chicken microbiome following FMT. RESULTS FMT changes the composition of the chicken intestinal microbiome. We observed little change in species richness following FMT compared to untreated samples, but there is an increase in phylogenetic diversity within those species. The most significant difference in the ceca is an increase in Lactobacilli, although not a major component of the transplant material, suggesting the FMT results in a change in the intestinal milieu as much as a direct change to the microbiome. Upon direct challenge, FMT resulted in lower initial intestinal colonisation with C.jejuni. More significantly, in a seeder-bird challenge of infection transmission, FMT reduced transmission and intestinal colonisation until common UK retail age of slaughter. In a repeat experiment, transmission was completely blocked following FMT treatment. Delayed FMT administration at 7 days of-age had limited effect on colonisation and transmission. CONCLUSIONS We show that transfer of a whole mature microbiome to newly-hatched chicks reduces transmission and colonisation of C.jejuni. This indicates that modification of the broiler chick microbiome can reduce intestinal colonisation of C.jejuni to levels projected to lead to lower the human infection rate. We believe these findings offer a way to identify key taxa or consortia that are effective in reducing C.jejuni colonisation and improving broiler gut health

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.This work was funded by Biotechnology and Biological Sciences Research Council (http://www.bbsrc.ac.uk) grant BB/K004514/1. D.P.W. was funded by a Wellcome Trust (https://wellcome.ac.uk) Infection and Immunity PhD rotation studentship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Use of Galleria mellonella as a Model for Insect Vector Transmission of the Foodborne Pathogen Campylobacter jejuni in Broiler Chickens: A Pilot Study

There is growing pressure to find a way to eradicate or reduce the levels of foodborne pathogens such as Campylobacter in broiler chickens, whilst limiting the use of antimicrobials. For Campylobacter, there is currently no vaccine and on-farm biosecurity alone is insufficient to prevent colonization of broiler chicken flocks. Dipteran flies are proven carriers of Campylobacter and their entry into broiler houses may contribute to its transmission to broiler chickens. As there is currently no experimental vector transmission model for Campylobacter and chickens, we decided to examine experimentally whether Galleria mellonella could be used as vector to transmit Campylobacter to broiler chickens. More recently, the use of live insect feed has been proposed both for its nutritional qualities and improving bird welfare through the encouragement of natural foraging behaviours and it is unclear any risk this poses in terms of pathogen transmission. In this study, day-old chicks (n = 29) were obtained from a commercial hatchery. At three weeks of age, birds were split into 4 This groups; Group 1 was infected via oral gavage with 106 cells of C. jejuni-M1, Group 2 was fed Galleria mellonella infected with 106 cells of C. jejuni-M1, Group 3 was fed uninfected Galleria mellonella, whilst the remaining group was unchallenged. Cloacal swabs were taken at 2, 4, and 6 days post-infection (dpi) to follow transmission and at 8 dpi birds culled and C. jejuni load quantified in the caeca and liver. At 8 dpi, all birds in both the Campylobacter gavage group and those in the group fed the Campylobacter infected Galleria mellonella were Campylobacter positive, whereas those fed uninfected Galleria mellonella and the control group were all Campylobacter negative. The mean caecal Campylobacter load in the Campylobacter gavage group was 1.7 &times; 1010 per gram compared with 8.6 &times; 109 in the group fed the Campylobacter-infected Galleria mellonella. No liver positives were found in any of the groups. Our findings indicate that feeding broiler chickens with the vector Galleria mellonella infected with C. jejuni-M1 is sufficient to establish colonisation with C. jejuni. We propose that Galleria can be used as an easy and flexible model for vector transmission of foodborne pathogens in chicken

Use of Galleria mellonella as a Model for Insect Vector Transmission of the Foodborne Pathogen Campylobacter jejuni in Broiler Chickens: A Pilot Study

There is growing pressure to find a way to eradicate or reduce the levels of foodborne pathogens such as Campylobacter in broiler chickens, whilst limiting the use of antimicrobials. For Campylobacter, there is currently no vaccine and on-farm biosecurity alone is insufficient to prevent colonization of broiler chicken flocks. Dipteran flies are proven carriers of Campylobacter and their entry into broiler houses may contribute to its transmission to broiler chickens. As there is currently no experimental vector transmission model for Campylobacter and chickens, we decided to examine experimentally whether Galleria mellonella could be used as vector to transmit Campylobacter to broiler chickens. More recently, the use of live insect feed has been proposed both for its nutritional qualities and improving bird welfare through the encouragement of natural foraging behaviours and it is unclear any risk this poses in terms of pathogen transmission. In this study, day-old chicks (n = 29) were obtained from a commercial hatchery. At three weeks of age, birds were split into 4 This groups; Group 1 was infected via oral gavage with 106 cells of C. jejuni-M1, Group 2 was fed Galleria mellonella infected with 106 cells of C. jejuni-M1, Group 3 was fed uninfected Galleria mellonella, whilst the remaining group was unchallenged. Cloacal swabs were taken at 2, 4, and 6 days post-infection (dpi) to follow transmission and at 8 dpi birds culled and C. jejuni load quantified in the caeca and liver. At 8 dpi, all birds in both the Campylobacter gavage group and those in the group fed the Campylobacter infected Galleria mellonella were Campylobacter positive, whereas those fed uninfected Galleria mellonella and the control group were all Campylobacter negative. The mean caecal Campylobacter load in the Campylobacter gavage group was 1.7 × 1010 per gram compared with 8.6 × 109 in the group fed the Campylobacter-infected Galleria mellonella. No liver positives were found in any of the groups. Our findings indicate that feeding broiler chickens with the vector Galleria mellonella infected with C. jejuni-M1 is sufficient to establish colonisation with C. jejuni. We propose that Galleria can be used as an easy and flexible model for vector transmission of foodborne pathogens in chicken.</jats:p

Timing and delivery route effects of cecal microbiome transplants on Salmonella Typhimurium infections in chickens: potential for in-hatchery delivery of microbial interventions.

BackgroundExposure to microbes early in life has long-lasting effects on microbial community structure and function of the microbiome. However, in commercial poultry settings chicks are reared as a single-age cohort with no exposure to adult birds which can have profound effects on microbiota development and subsequent pathogen challenge. Microbiota manipulation is a proven and promising strategy to help reduce pathogen load and transmission within broiler flocks. However, administration of microbiota transplant products in a hatchery setting may prove challenging. Effective administration strategies are dependent on key factors, such as; the age of chicks receiving interventions and mode of delivery. This study aimed to assess these two aspects to provide supporting evidence towards microbiome manipulation strategies for use in commercial hatcheries.ResultsManipulation of the microbiota between 4 and 72 h of hatch markedly reduced faecal shedding and colonisation with the foodborne pathogen Salmonella enterica serovar Typhimurium (ST4/74). Administration of transplant material via spray or gel drop delivery systems had minimal effect on the protection conferred with fewer birds in transplant groups shown to shed ST4/74 in the faeces compared to PBS-gavaged control birds. Analysis of the microbiome following transplantation demonstrated that all transplant groups had higher diversity and species richness than non-transplant groups during the first week of life and the early stages of infection with ST47/4.The relative abundance of the bacterium Faecalibacterium prausnitzii was significantly higher in CMT groups compared to PBS controls. The presence of F. prausnitzii was also shown to increase in PBS-challenged birds compared to unchallenged birds potentially indicating a role of this bacterium in limiting Salmonella infections.ConclusionsThis study demonstrated that administration of microbiome transplants, using methods that would align with hatchery practices, effectively reduced colonisation and shedding of Salmonella in chickens. Age of chicks at microbiome administration had limited effect on the diversity and composition of the microbiome and conferred protection against Salmonella infections. Traditional hatchery delivery systems, such as spray or gel-drop, are sufficient to transfer donor material, alter the microbiome and confer protection against Salmonella. This study helps highlight the opportunity for use of microbiome modification methods within the hatchery

Carriage of antimicrobial-resistant Escherichia coli and Staphylococci in dogs in the community : molecular mechanisms

While previous studies have determined the prevalence of meticillin resistant Staphylococcus aureus (MRSA) and antimicrobial resistant (AMR) Escherichia coli in canine populations, few have included sufficiently large sample sizes and fewer still have characterised the isolates or investigated risk factors that might be associated with their carriage. The main aims of the work presented in this thesis were; to determine the nasal prevalence of MRSA and other AMR staphylococci and the faecal prevalence of AMR E. coli in faeces in the canine population of mainland UK. The study also aimed to characterise the bacteria isolated using molecular techniques in order for comparisons to be made with isolates of human origin, and to determine the presence of potential risk factors associated with faecal carriage of AMR E. coli. These objectives were achieved by carrying out two studies. The first study used frozen canine faecal samples collected during a cross sectional study of a semi-rural community in Cheshire to determine the prevalence of AMR E. coli. The second study collected faecal and nasal swabs from dogs visiting veterinary practices across mainland UK. Antimicrobial resistance of the isolates obtained from both studies were characterised using disc diffusion methods and PCR assays. In addition, isolates collected during the second study were subjected to multi-locus sequence typing and DNA micro array analysis of resistance and virulence genes. For antimicrobial resistant E. coli, risk factors associated with carriage were investigated. The prevalence of MRSA in the canine population was found to be low at 1% and all isolates were identical to EMRSA-15, the main human endemic strain in many UK hospitals. The overall S. aureus prevalence was 7.5%, with a higher prevalence of 11.0% of S. pseudintermedius, in which no meticillin resistance was found. Meticillin resistant coagulase negative Staphylococcus spp. was found in 5.5% of dogs. AMR in the isolates varied between species; however resistance to fusidic acid was consistently high. AMR E. coli was common in both studies (29.0% in community study and 44.8% in nationwide study). Resistance to ampicillin (24.0% and 37.2%), tetracycline (19.7% and 30.0%), trimethoprim (16.9% and 23.8%) and resistance to three or more antimicrobial classes (15.3% and 18.1%) was found to be high in both studies (community and nationwide respectively), while resistance to augmentin, chloramphenicol, ciprofloxacin and nalidixic acid was below 10% in both studies. A variety of genes responsible for resistance to expanded spectrum β-lactams was identified; including blaCTX-M-15 and blacmy2, both of which have previously been identified in humans and dogs. A number of variables were found to be associated with resistance to antimicrobials, with previous prescription of antimicrobials and consumption of raw poultry meat remaining in the final model of more than one resistance outcome. The carriage of MRSA and antimicrobial resistant E. coli could pose a potential problem both in terms of the welfare of the dogs carrying such bacteria as well as the zoonotic potential of the bacteria and resistance determinants.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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