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 COVID-19 pandemic to track disease burden through analyses of 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 healthcare 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 treatment plants (WTPs), collected August 2020 – 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 COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 – 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, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

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 COVID-19 pandemic to track disease burden through analyses of 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 healthcare 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 treatment plants (WTPs), collected August 2020 - 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 COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 - 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, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

Antibiotic therapy is expected to impact host microbial communities considerably, yet many studies focused on microbiome and health are often confounded by limited information about antibiotic exposure. Given that antibiotics have diverse pharmacokinetic and antimicrobial properties, investigating the type and concentration of these agents in specific host specimens would provide much needed insight into their impact on the microbes therein. Here, we developed liquid chromatography mass spectrometry (LC-MS) methods to detect 18 antibiotic agents in sputum from persons with cystic fibrosis. Antibiotic spike-in control samples were used to compare three liquid extraction methods on the Waters Acquity Quattro Premier XE. Extraction with dithiothreitol captured the most antibiotics and was used to detect antibiotics in sputum samples from 11 people with cystic fibrosis, with results being compared to the individuals' self-reported antibiotic use. For the sputum samples, two LC-MS assays were used; the Quattro Premier detected nanomolar or micromolar concentrations of 16 antibiotics, whereas the Xevo TQ-XS detected all 18 antibiotics, most at sub-nanomolar levels. In 45% of tested sputum samples (71/158), at least one antibiotic that was not reported by the subject was detected by both LC-MS methods, a discordance largely explained by the thrice weekly administration and long half-life of azithromycin. For ∼37% of samples, antibiotics reported as taken by the individual were not detected by either instrument. Our results provide an approach for detecting a variety of antibiotics at the site of infection, thereby providing a means to include antibiotic usage data into microbiome studies.

From this protocol, readers will be able to extract volatile organic compounds from a biological sample with the vacuum assisted sorbent extraction method, run samples on a GC-MS with the use of the Entech autosampler, and analyze data.

UBXD1 is a recently identified adaptor for p97, a highly abundant and conserved member of the AAA family of ATPase that plays pivotal roles in a multitude of cellular processes involving the ubiquitin-proteasome pathway. Very little is known about the biochemical, cellular, and molecular functions of UBXD1. Here we report the generation of two mouse monoclonal antibodies, 5C3-1 and 2F8-24, that recognize UBXD1 using Western blotting, immunoprecipitation, and immunofluorescence.