Metagenomic Sequencing and Quantitative Real-Time PCR for Fecal Pollution Assessment in an Urban Watershed

Kyle D Brumfield; Joseph A Cotruvo; Orin C Shanks; Mano Sivaganesan; Jessica Hey; Nur A Hasan; Anwar Huq; Rita R Colwell; Menu B Leddy

doi:10.3389/frwa.2021.626849

Metagenomic Sequencing and Quantitative Real-Time PCR for Fecal Pollution Assessment in an Urban Watershed

Front Water. 2021 Feb:3:626849. doi: 10.3389/frwa.2021.626849. Epub 2021 Feb 15.

Authors

Kyle D Brumfield^{1

2}, Joseph A Cotruvo³, Orin C Shanks⁴, Mano Sivaganesan⁴, Jessica Hey⁴, Nur A Hasan², Anwar Huq¹, Rita R Colwell^{1

2

5}, Menu B Leddy⁶

Affiliations

¹ Maryland Pathogen Research Institute, University of Maryland, College Park, MD, United States.
² University of Maryland Institute for Advanced Computer Studies, University of Maryland, College Park, MD, United States.
³ Joseph Cotruvo and Associates LLC, Washington, DC, United States.
⁴ U.S. Environmental Protection Agency, Office of Research and Development, Cincin nati, OH, United States.
⁵ CosmosID Inc., Rockville, MD, United States.
⁶ Essential Environmental and Engineering Systems, Huntington Beach, CA, United States.

Abstract

Microbial contamination of recreation waters is a major concern globally, with pollutants originating from many sources, including human and other animal wastes often introduced during storm events. Fecal contamination is traditionally monitored by employing culture methods targeting fecal indicator bacteria (FIB), namely E. coli and enterococci, which provides only limited information of a few microbial taxa and no information on their sources. Host-associated qPCR and metagenomic DNA sequencing are complementary methods for FIB monitoring that can provide enhanced understanding of microbial communities and sources of fecal pollution. Whole metagenome sequencing (WMS), quantitative real-time PCR (qPCR), and culture-based FIB tests were performed in an urban watershed before and after a rainfall event to determine the feasibility and application of employing a multi-assay approach for examining microbial content of ambient source waters. Cultivated E. coli and enterococci enumeration confirmed presence of fecal contamination in all samples exceeding local single sample recreational water quality thresholds (E. coli, 410 MPN/100 mL; enterococci, 107 MPN/100 mL) following a rainfall. Test results obtained with qPCR showed concentrations of E. coli, enterococci, and human-associated genetic markers increased after rainfall by 1.52-, 1.26-, and 1.11-fold log₁₀ copies per 100 mL, respectively. Taxonomic analysis of the surface water microbiome and detection of antibiotic resistance genes, general FIB, and human-associated microorganisms were also employed. Results showed that fecal contamination from multiple sources (human, avian, dog, and ruminant), as well as FIB, enteric microorganisms, and antibiotic resistance genes increased demonstrably after a storm event. In summary, the addition of qPCR and WMS to traditional surrogate techniques may provide enhanced characterization and improved understanding of microbial pollution sources in ambient waters.

Keywords: culture; fecal indicator bacteria; metagenomic analysis; microbial source tracking; quantitative real-time PCR; rainfall–runoff; whole metagenome sequence.