Prevalence and Whole-Genome Sequence-Based Analysis of Shiga Toxin-Producing Escherichia coli Isolates from the Recto-Anal Junction of Slaughter-Age Irish Sheep

Abstract

Shiga toxin-producing Escherichia coli (STEC) organisms are a diverse group of pathogenic bacteria capable of causing serious human illness, and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir, and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes, and shedding dynamics of STEC, including the supershedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (n = 840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR (RT-PCR) for Shiga toxin prevalence and serogroup. A subset of STEC isolates (n = 199) were selected for whole-genome sequencing and analyzed in silico. In total, 704/840 (83.8%) swab samples were Shiga toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157, and three of these were identified as supershedders. No STEC O26 was isolated. Post hoc statistical analysis showed that younger animals are more likely to harbor STEC and that STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, 15 of which were not yet reported for sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga toxin gene variants were reported, two stx₁ and six stx₂, and three novel Shiga-toxin subunit combinations were observed. Variant stx_1c was the most prevalent, while many strains also harbored stx_2b. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) bacteria are foodborne, zoonotic pathogens of significant public health concern. All STEC organisms harbor stx, a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via fecal excretion and are increasingly recognized as important contributors to the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and that prevalence is related to animal age and seasonality. Further, sheep harbor a variety of non-O157 STEC, whose prevalence and contribution to human disease have been underinvestigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.

Keywords: Shiga toxin-producing Escherichia coli; non-O157 STEC; sheep; supershedding; whole-genome sequencing.

FIG 1

Heat map plotting the distribution of Shiga toxin variants. Strains are labeled by serogroup and clustered based on their Shiga toxin profile. The rows are annotated with the respective MLST profile of each strain. Strains are designated an integer value between 0 and 2 to indicate whether they harbor a one toxin subunit (0.5), both subunits of a toxin variant (1), or multiples of each.

FIG 2

Diagram describing the microbiological analysis of samples during the study. All samples were assessed following 5 h of incubation to determine O157/O26 supershedding status, followed by the primary analysis of a sample’s STEC status following 24 h of enrichment. Samples which were both STEC and serogroup positive for O157 or O26 underwent an extended isolation protocol to ensure serogroup confirmation. Samples often presented with multiple virulence profiles, and one representative colony for every profile was stored per sample.