Wastewater surveillance is a cost-effective tool for monitoring SARS-CoV-2 transmission in a community. However, challenges remain with regard to interpretating such studies, not least in how to compare SARS-CoV-2 levels between different-sized wastewater treatment plants. Viral faecal indicators, including crAssphage and pepper mild mottle virus, have been proposed as population biomarkers to normalise SARS-CoV-2 levels in wastewater. However, as these indicators exhibit variability between individuals and may not be excreted by everyone, their utility as population biomarkers may be limited. Coprostanol, meanwhile, is a bacterial metabolite of cholesterol which is excreted by all individuals. In this study, composite influent samples were collected from a large- and medium-sized wastewater treatment plant in Dublin, Ireland and SARS-CoV-2 N1, crAssphage, pepper mild mottle virus, HF183 and coprostanol levels were determined. SARS-CoV-2 N1 RNA was detected and quantified in all samples from both treatment plants. Regardless of treatment plant size, coprostanol levels exhibited the lowest variation in composite influent samples, while crAssphage exhibited the greatest variation. Moreover, the strongest correlations were observed between SARS-CoV-2 levels and national and Dublin COVID-19 cases when levels were normalised to coprostanol. This work demonstrates the usefulness of coprostanol as a population biomarker for wastewater surveillance studies.

Introduction. Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria that cause severe bloody diarrhoea and haemolytic uraemic syndrome and are associated with neurological complications and potentially life-threatening infections. Children under five and elderly individuals are particularly vulnerable to STEC infections. The majority of STEC infections have been traced back directly or indirectly to ruminants, and there is an unmet need for a vaccine to protect these vulnerable cohorts. Gap Statement. We previously identified a series of STEC proteins that are involved in attachment to human gastrointestinal epithelial cells, some of which are protective in a murine challenge model. However, the prevalence of the genes in dairy herds was unknown. Aim. Due to zoonotic transmission, the development of a successful human vaccine relies on any protective vaccine antigens being prevalent in ruminant herds in different geographical locations. We aimed to examine the potential geographical differences in antigen gene prevalence. Methodology. Faecal isolates collected from a dairy herd in CA, USA, and isolated from dairy cows in Ireland were analysed by quantitative PCR. Bioinformatic analysis of previously sequenced isolates was also performed. Results. Four antigen genes were highly prevalent among STEC shed from cattle in a Californian dairy herd (>95.7%). Genes encoding FkpA, YiaF and GlnH were also highly prevalent in faecal samples from cows sampled in Ireland (93%). The point prevalence of terD among cattle in the Irish cohort appeared low relative to Californian isolates, but this is most likely due to the absence of tellurite in isolation media used in the former. Expansion of the study to examine Irish bovine and ovine Whole genome sequencing datasets increased the detection of terD in Irish isolates. Conclusion. Overall, three antigen genes are prevalent in both geographically distinct cohorts. Discrepancies in terD gene prevalence are likely due to the use of selective agars in isolation protocols.