Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes

Abstract

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR).Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 – August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed.To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population’s wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.

Sequencing and variant detection of eight abundant plant-infecting tobamoviruses across Southern California wastewater

“…We applied similar molecular and bioinformatic methods to eight agriculturally-relevant tobamoviruses sequenced from influent wastewater, representing a sewer shed of approximately 16 million Southern Californians across eight wastewater treatment plants (3,10). These tobamoviruses were abundant and widespread throughout our wastewater samples, comprising eight of the top 10 viruses in our dataset (10).…”

“…Wastewater-based epidemiology (WBE) has been used to characterize pathogen abundances and genomics for a variety of diseases and is often employed to detect antibiotic resistance or diseases relevant to public health (5). We applied similar molecular and bioinformatic methods to eight agriculturally-relevant tobamoviruses sequenced from influent wastewater, representing a sewer shed of approximately 16 million Southern Californians across eight wastewater treatment plants (3, 10). These tobamoviruses were abundant and widespread throughout our wastewater samples, comprising eight of the top 10 viruses in our dataset (10).…”

“…We applied similar molecular and bioinformatic methods to eight agriculturally-relevant tobamoviruses sequenced from influent wastewater, representing a sewer shed of approximately 16 million Southern Californians across eight wastewater treatment plants (3, 10). These tobamoviruses were abundant and widespread throughout our wastewater samples, comprising eight of the top 10 viruses in our dataset (10). PMMoV may be the best known and is often regarded as the most abundant virus in fecal and wastewater samples (7, 9), however, we were surprised to find that ToBRFV was much more abundant, mirroring the results of a recent study from Maryland (5).…”

“…Also, as many tobamoviruses may serve as water quality indicators and are impactful diseases to agriculture, studies should be conducted to understand the genomics of these viruses (2,9). We obtained raw sequencing data as FASTQ files from the NCBI Sequence Read Archive under BioProject PRJNA729801, and we refer to Rothman, et al 2021 (3) and Rothman et al 2022 (10) for all sampling, RNA extraction, and sequencing methods. We used BBTools (11) "bbduk" to remove sequencing adapters, primers, and low-quality bases from the reads, BBTools "dedupe" to remove optical duplicates, and removed human genome reads (hg38) with Bowtie2 (12).…”

“…As part of a large WBE effort across Southern California, we used metatranscriptomic sequencing to investigate the genomics and single nucleotide variants (SNVs) of eight tobamoviruses sourced from 275 samples across eight wastewater treatment plants from July 2020 – August 2021 (3, 10). These viruses were Bell Pepper Mottle Virus (BPeMV), Cucumber Green Mottle Mosaic Virus (CGMMV), Pepper Mild Mottle Virus (PMMoV) Tobacco Mild Green Mosaic Virus (TMGMV), Tomato Brown Rugose Fruit Virus (ToBRFV), Tomato Mosaic Virus (ToMV), Tomato Mottle Mosaic Virus (ToMMV), and Tropical Soda Apple Mosaic Virus (TSAMV).…”