An investigation of the prevalence, shedding dynamics and molecular chatracteristics of Shiga toxin-producing Escherichia coli in Irish sheep using a OneHealth Approach

Shiga toxin-producing Escherichia coli (STEC) are zoonotic foodborne bacteria capable of causing serious human illness. Ovine ruminants, including sheep, are recognized as an important STEC reservoir and a notable contributor to contamination within the food production chain. Sheep shed the pathogen via faecal excretion and humans may ingest the organism by direct or indirect means, such as direct animal contact or ingestion of faecally contaminated meat or ovine produce. The primary virulence determinant of STEC are the bacteriophage-encoded Shiga toxins (Stx). While all STEC are capable of causing disease, the severity of infection is determined by the array of auxiliary virulence determinants harboured by each individual strain. Ireland has the highest prevalence rates of STEC infection in Europe with 17.3 cases per 100,000 population despite the implementation of numerous intervention strategies and control measures. However, to date, no investigation into the prevalence, shedding dynamics, and characteristics of STEC circulating within the Irish sheep herd has been conducted. This doctoral study applied a OneHealth framework to carry out the surveillance of STEC in Irish sheep and a thorough assessment of their zoonotic potential. During this study, the prevalence of STEC within this ruminant population and the factors, both intrinsic and extrinsic, that effect the shedding dynamics of individual animals was investigated. Further, a whole-genome based approach was employed to characterise a selection of the isolated STEC strains and a detailed molecular evaluation of the virulence-associated genes and mobile genomic islands present within these strains was conducted. In this study, 840 recto-anal junction swab samples were collected from Irish slaughter-age sheep. In total, 704/840 (83.8%) swab samples were Shiga toxin positive following an initial screen, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were found to be shedding STEC O157 and three of these were identified as super-shedders using a novel, quantitative real-time polymerase chain reaction protocol. No STEC O26 was isolated. Post-hoc statistical analysis showed that younger animals are more likely to harbour STEC and carriage rates of the bacterium were highest during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, fifteen of which were novel in sheep, and serotype O91:H14 was the most frequently reported. Eight Shiga toxin gene variants were identified, two stx1 and six stx2, and three novel Shiga toxin subunit combinations were observed. An investigation into the phylogenetic relationship of the Irish STEC strains to other STEC genomes isolated from environmental, ruminant, and clinical cases of infection revealed that strains tended to cluster by phylogroup and serogroup, regardless of geographic location or isolation source. Furthermore, some of the Irish sheep STEC strains shared a high degree of genetic relatedness to clinical strains. Interestingly, many of the sheep strains are members of phylogroup B1 and A and lacked many of the traditional virulence determinants frequently uncovered in Enterohaemorrhagic E. coli strains. This suggests that the zoonotic potential of E. coli is not limited by source and that strains of each phylogroup, even those historically considered as commensal, are capable of causing human illness regardless of origin.
This study also highlights the significance and important utility of whole-genome sequencing to better describe the virulence potential of STEC isolates cultured from sheep based on analysis of the associated virulence factor content. Further these data can be harnessed to better understand the transmission dynamics of this organism within sheep. Data provided in this study can support refined improvement in food safety controls, designed to protect public health.