Wastewater, which contains everything from pathogens to pollutants, is a geospatially- and temporally-linked microbial fingerprint of a given population. As a result, it can be leveraged for monitoring multiple dimensions of public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (n=1,419 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County from 2020-2022. First, we used targeted amplicon sequencing (n=966) to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with clinical caseloads from University students (N = 1,503) and Miami-Dade County hospital patients (N = 3,939 patients), as well as an 8-day earlier detection of the Delta variant in wastewater vs. in patients. Additionally, in 453 metatranscriptomic samples, we demonstrate that different wastewater sampling locations have clinically and public-health-relevant microbiota that vary as a function of the size of the human population they represent. Through assembly, alignment-based, and phylogenetic approaches, we also detect multiple clinically important viruses (e.g., norovirus) and describe geospatial and temporal variation in microbial functional genes that indicate the presence of pollutants. Moreover, we found distinct profiles of antimicrobial resistance (AMR) genes and virulence factors across campus buildings, dorms, and hospitals, with hospital wastewater containing a significant increase in AMR abundance. Overall, this effort lays the groundwork for systematic characterization of wastewater to improve public health decision making and a broad platform to detect emerging pathogens.

BTT is compensated for consulting with Seed Health and Enzymetrics Biosciences on microbiome study design and holds an ownership stake in the former. CEM is a co-Founder of Onegevity and Biotia. No entity listed here was involved in funding or advising the contents of this study.

This study was financially supported by the National Institute on Drug Abuse of the National Institutes of Health (NIH) under Award Number U01DA053941. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The IRB of Weill Cornell Medicine gave ethical approval for this work.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

In compliance with the NIH RADx-rad Data Coordination Center (DCC) requirements, the raw sequencing data was submitted to the Sequence Read Archive (SRA). The wastewater samples were annotated with the rich metadata and the sequencing information (fastq files) was included in the submission. The submitted data can be found in the SRA under the accession PRJNA946141. Furthermore, the metadata associated with the wastewater samples' sequencing data was extracted from the Illumina operational files, validated, organized, and submitted to the NIH data hub via DCC [https://radx-hub.nih.gov/home], where the SF-RAD data is associated with the dbGaP study accession phs002525.v1.p1. The metadata standards specifications used to describe the data were developed in collaboration with the SF-RAD members and the DCC and formally defined and registered at FAIRsharing.org.