RNA Viromics of Southern California Wastewater and Detection of SARS-CoV-2 Single-Nucleotide Variants.

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the coronavirus disease 2019 (COVID-19) pandemic to track disease burden through analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the health care system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven wastewater treatment plants (WTPs), collected from August 2020 to January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single-nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater, and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically important SARS-CoV-2 variants along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. IMPORTANCE Wastewater-based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the coronavirus disease 2019 (COVID-19) pandemic to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without adding burden to health care systems. In this study, we used metatranscriptomics and reverse transcription-droplet digital PCR (RT-ddPCR) to assay RNA viruses across Southern California wastewater from August 2020 to January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single-nucleotide variants through sequencing. Likewise, wastewater treatment plants (WTPs) harbored different viromes, and we detected other human pathogens, such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

A Combined Digital PCR and Next Generation DNA-Sequencing Based Approach for Tracking Nearshore Pollutant Dynamics Along the Southwest United States/Mexico Border.

Ocean currents, multiple fecal bacteria input sources, and jurisdictional boundaries can complicate pollution source tracking and associated mitigation and management efforts within the nearshore coastal environment. In this study, multiple microbial source tracking tools were employed to characterize the impact and reach of an ocean wastewater treatment facility discharge in Mexico northward along the coast and across the Southwest United States- Mexico Border. Water samples were evaluated for fecal indicator bacteria (FIB), Enterococcus by culture-based methods, and human-associated genetic marker (HF183) and Enterococcus by droplet digital polymerase chain reaction (ddPCR). In addition, 16S rRNA gene sequence analysis was performed and the SourceTracker algorithm was used to characterize the bacterial community of the wastewater treatment plume and its contribution to beach waters. Sampling dates were chosen based on ocean conditions associated with northern currents. Evidence of a gradient in human fecal pollution that extended north from the wastewater discharge across the United States/Mexico border from the point source was observed using human-associated genetic markers and microbial community analysis. The spatial extent of fecal contamination observed was largely dependent on swell and ocean conditions. These findings demonstrate the utility of a combination of molecular tools for understanding and tracking specific pollutant sources in dynamic coastal water environments.

Metagenomics of Wastewater Influent from Southern California Wastewater Treatment Facilities in the Era of COVID-19.

Sequencing wastewater may be useful for detecting pathogens and assaying microbial water quality. We concentrated, extracted, and sequenced nucleic acids from 17 composite influent wastewater samples spanning seven southern California wastewater treatment facilities in May 2020. Bacteria were the most proportionally abundant taxonomic group present, followed by viruses and archaea.