Tracing the origin of SARS-CoV-2 omicron-like spike sequences detected in an urban sewershed: a targeted, longitudinal surveillance study of a cryptic wastewater lineage.

BACKGROUND: The origin of novel SARS-CoV-2 spike sequences found in wastewater, without corresponding detection in clinical specimens, remains unclear. We sought to determine the origin of one such cryptic wastewater lineage by tracking and characterising its persistence and genomic evolution over time. METHODS: We first detected a cryptic lineage, WI-CL-001, in municipal wastewater in Wisconsin, USA, in January, 2022. To determine the source of WI-CL-001, we systematically sampled wastewater from targeted sub-sewershed lines and maintenance holes using compositing autosamplers. Viral concentrations in wastewater samples over time were measured by RT digital PCR. In addition to using metagenomic 12s rRNA sequencing to determine the virus's host species, we also sequenced SARS-CoV-2 spike receptor binding domains, and, where possible, whole viral genomes to identify and characterise the evolution of this lineage. FINDINGS: We traced WI-CL-001 to its source at a single commercial building. There we detected the cryptic lineage at concentrations as high as 2·7 × 109 genome copies per L. The majority of 12s rRNA sequences detected in wastewater leaving the identified source building were human. Additionally, we generated over 100 viral receptor binding domain and whole-genome sequences from wastewater samples containing the cryptic lineage collected over the 13 consecutive months this virus was detectable (January, 2022, to January, 2023). These sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in humans in Wisconsin at low levels from October, 2020, to February, 2021. Despite this, mutations in the spike gene and elsewhere resembled those subsequently found in omicron variants. INTERPRETATION: We propose that prolonged detection of WI-CL-001 in wastewater indicates persistent shedding of SARS-CoV-2 from a single human initially infected by an ancestral B.1.234 virus. The accumulation of convergent omicron-like mutations in WI-CL-001's ancestral B.1.234 genome probably reflects persistent infection and extensive within-host evolution. People who shed cryptic lineages could be an important source of highly divergent viruses that sporadically emerge and spread. FUNDING: The Rockefeller Foundation, Wisconsin Department of Health Services, Centers for Disease Control and Prevention, National Institute on Drug Abuse, and the Center for Research on Influenza Pathogenesis and Transmission.

Survival of bovine-associated serotypes of Salmonella enterica in bedding sand.

Cattle persistently infected with bovine-adapted serotypes of Salmonella enterica are an important animal health and food safety issue. One possible mechanism by which infection is sustained in a dairy herd is by survival of Salmonella in sand used as bedding material. In this study we assessed the survival of 107 to 108 cfu bovine-associated serotypes of Salmonella enterica (sv. Cerro, Dublin, and Heidelberg) in sterile sand, recycled bedding sand, and gray water collected from a Wisconsin dairy farm. All 3 serotypes persisted at relatively high numbers (>106 cfu/g) for at least 28 d in sterile sand, with Salmonella sv. Dublin decreasing less than 1 log10 over 70 d. To our surprise, when low numbers of Salmonella sv. Dublin (103 cfu) were inoculated into sterile sand, the organism multiplied within 3 d to approximately 106 cfu/g sand and persisted at that level for 28 d. When we inoculated Salmonella sv. Dublin into recycled bedding sand or sand taken directly from cow pens, we observed a significant decrease in colony-forming units by d 7. In contrast, we observed a significant increase in colony-forming units when Salmonella sv. Dublin was inoculated into gray water from the sand recycling system. These data demonstrate that Salmonella can persist for extended periods of time in bedding sand, although this is limited to some extent by the native microbiota in recycled bedding sand.

Assessing the microbiota of recycled bedding sand on a Wisconsin dairy farm.

BACKGROUND: Sand is often considered the preferred bedding material for dairy cows as it is thought to have lower bacterial counts than organic bedding materials and cows bedded on sand experience fewer cases of lameness and disease. Sand can also be efficiently recycled and reused, making it cost-effective. However, some studies have suggested that the residual organic material present in recycled sand can serve as a reservoir for commensal and pathogenic bacteria, although no studies have yet characterized the total bacterial community composition. Here we sought to characterize the bacterial community composition of a Wisconsin dairy farm bedding sand recycling system and its dynamics across several stages of the recycling process during both summer and winter using 16S rRNA gene amplicon sequencing. RESULTS: Bacterial community compositions of the sand recycling system differed by both seasons and stage. Summer samples had higher richness and distinct community compositions, relative to winter samples. In both summer and winter samples, the diversity of recycled sand decreased with time drying in the recycling room. Compositionally, summer sand 14 d post-recycling was enriched in operational taxonomic units (OTUs) belonging to the genera Acinetobacter and Pseudomonas, relative to freshly washed sand and sand from cow pens. In contrast, no OTUs were found to be enriched in winter sand. The sand recycling system contained an overall core microbiota of 141 OTUs representing 68.45% ± 10.33% SD of the total bacterial relative abundance at each sampled stage. The 4 most abundant genera in this core microbiota included Acinetobacter, Psychrobacter, Corynebacterium, and Pseudomonas. Acinetobacter was present in greater abundance in summer samples, whereas Psychrobacter and Corynebacterium had higher relative abundances in winter samples. Pseudomonas had consistent relative abundances across both seasons. CONCLUSIONS: These findings highlight the potential of recycled bedding sand as a bacterial reservoir that warrants further study.