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Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences
Citation:
Paar, J., M. Doolittle, M. Varma, S. Siefring, K. Oshima, AND Rich Haugland. Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences. JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 112(2):28-35, (2015).
Impact/Purpose:
Beach closures from elevated levels of fecal pollution are commonplace in many metropolitan communities throughout the United States. Urban beach-goers face potentialhealth risks from exposure to microbial pollution from the fecal waste of warm-blooded organisms. Fecal material containing pathogenic viruses, bacteria, and protozoa is deposited directly on and/or transported to beaches by groundwater, storm-water, and surface water. Measures taken to reduce, remediate, and/or eliminate these pollution sources are only effective when sources of fecal contamination can be accurately and consistently identified. Current culture-based recreational water quality test methods for general microbial indicators cannot identify whether fecal pollution originates from humans or nonhumans. The use of multiple genetic markers for identifying specific fecal sources, such as human, in environmental water is a well-accepted, if not frequently employed, concept. While a number of bacterial and other markers have been developed for the detection of fecal contamination from human, as well as other animal sources, these targets may differ in their persistence to enteric viral pathogens which have been reported to be the most common agents of human illnesses resulting from exposures to surface waters. In theory, bacteriophage such as the FRNA coliphages might be expected to more closely mimic the environmental fate of viral pathogens although several challenges have been raised about their potential as indicators of these pathogens. Considerable effort has been expended in the assessment of these phages both as general indicators of fecal pollution and also as indicators of fecal sources. A complete method, incorporating recently improved reverse transcriptase-qPCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed at the U.S. EPA, New England Regional Laboratory for the direct, culture-independent detection of genetic markers from FRNA coliphage Genogroups I-IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA coliphage genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods at the EPA, ORD laboratory in Cincinnati. Strong associations were observed between the occurrence of the putatively human related FRNA coliphage Genogroup II marker and the densities of general and humanbacterial markers in the stormwater drain and outfall samples. FRNA coliphage markers were detected in fewer river and stream samples than general and human bacterial markers. The results indicate that the FRNA coliphage method shows promise as a tool for the assessment of impacts by storm waters on the microbiological quality of surface waters and the determination of human and non-human sources of fecal contamination.
Description:
A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct, culture-independent detection of genetic markers from FRNA coliphage genogroups I, II& IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods. Strong associations were observed between the occurrence of the putatively human related FRNA genogroup II marker and the densities of the bacterial markers in the stormwater drain and outfall samples. However fewer samples were positive for FRNA coliphage compared to either the general bacterial fecal indicator or the human-related bacterial fecal indicator markers particularly for ambient water samples. Together, these methods show promise as complementary tools for the identification of contaminated storm water drainage systems as well as the determination of human and non-human sources of contamination.
