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

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.

Microbiome Analysis for Wastewater Surveillance during COVID-19

“…Metagenomic sequencing has been used to explore AMR trends in anthropogenically impacted environments ( 41 ) and to detect wastewater associated enteric pathogens ( 36 , 95 ). However, viability or infectious potential of detected microorganisms requires additional analysis of metabolic activity.…”

“…Detection of wastewater associated pathogens by employing molecular techniques has been done at WWTPs ( 36 , 38 , 124 ) and used for source tracking of fecal pollution ( 126 , 95 , 54 ). However, a major hurdle for WS is communication of the results and their interpretation to public health officials.…”

“…Presenting metagenomic sequencing results as cell numbers provides more precise microbial load estimates and permits normalization of studies. To present the utility of this application, the cell number of microorganisms detected in wastewater samples, e.g., pathogens and bacteria commonly employed as indicators of human fecal contamination ( 95 , 59 ), is presented in Table 1 , and can be used as a comparative baseline for metagenomic investigations employing this technique.…”

Environmental parameters associated with incidence and transmission of pathogenic Vibrio spp.

“…DNA metagenome sequencing (metagenomics) is a valuable technology for detection of all Vibrio spp. in the environment, including VBNC Vibrio, and is particularly useful because it simultaneously profiles virulence and antimicrobial resistance associated genes (Roy et al, 2018;Acharya et al, 2020;Brumfield et al, 2020Brumfield et al, , 2021. Most of the HTS studies reported to date have focused on clinical isolates obtained by culture previously from patient samples.…”

“…Intense rainfall will not only alter the salinity profile but also nutrient availability in coastal rivers, with increasing inflow of freshwater. Stormwater runoff can also promote transmission of antimicrobial resistance and virulence associated genes among bacteria (Brumfield et al ., 2021). The evidence is clear that Vibrio spp .…”