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Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement.

ABSTRACT: Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal-indicator bacteria are used; however, they cannot distinguish human fecal waste from other animal pollution sources. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based on initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against an animal fecal reference library, and crAssphage genetic markers were highly abundant in raw sewage and sewage-impacted water samples. In addition, CPQ_056 and CPQ_064 performance was compared to HF183/BacR287 and HumM2 assays in paired experiments. Findings confirm that viral crAssphage qPCR assays perform at a similar level to well-established bacterial human-associated fecal-source-identification approaches. These new viral-based assays could become important water quality management and research tools.

SUBMITTER: Stachler E

PROVIDER: S-EPMC7350147 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement.

Stachler Elyse E Kelty Catherine C Sivaganesan Mano M Li Xiang X Bibby Kyle K Shanks Orin C OC

Environmental science & technology 20170725 16

Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal-indicator bacteria are used; however, they cannot distinguish human fecal waste from other animal pollution sources. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of ...[more]

PMID: 28700235

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Project description: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.

Dataset Information

Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement.

Publications

Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement.

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