Fecal Bacteroidales detection and persistence of viable cells and their DNA in the aqueous environment

Quantitative microbial source tracking (MST) approaches using fecal Bacteroidales and quantitative PCR (qPCR) assays to measure gene copies of host-specific 16S rRNA genetic markers are promising because they can allow for identifying and quantifying fecal loadings from a particular animal host and understanding the fate and transport of host-specific Bacteroidales over a range of conditions in water bodies.;A successful approach presented here is to remove the qPCR signal derived from free DNA and dead host-specific Bacteroidales cells by selectively binding the DNA and consequently inhibiting PCR amplification using light-activated propidium monoazide (PMA). Optimal PMA-qPCR conditions as 100 microM of PMA concentration and a 10-min light exposure time at different solids concentrations resulted in the selective exclusion of DNA from heat-treated cells of non-culturable Bacteroidales in human feces and wastewater influent and effluent samples.;The persistence of feces-derived Bacteroidales DNA and their cells (determined by universal, human-, cow- and dog-specific Bacteroidales qPCR assays) in seawater was investigated in microcosms at environmental conditions. The average T99 (two log reduction) value for viable Bacteroidales cells was 28 h, whereas that for total Bacteroidales DNA was 177 h in seawater microcosms. Natural sunlight did not have a strong influence on the fate of fecal Bacteroidales cells and their DNA in seawater, presumably because the presence of oxygen significantly affected the viability and persistence of these obligate anaerobes.;The survival of Bacteroidales cells and their DNA relative to fecal indicator bacteria and their relationship with pathogens such as Campylobacter jejuni, Salmonella enterica, and adenovirus DNA were examined in freshwater microcosms. The marked difference in decay of the host-specific Bacteroidales cells were observed between sunlight exposure and dark conditions, whereas the persistence of Bacteroidales DNA under dark conditions was comparable to that of Bacteroidales DNA exposed to sunlight. The persistence of Bacteroidales cells and Campylobacter cells in sunlight exposure was similar and Bacteroidales DNA and waterborne pathogen DNA were degraded at a similar rate. The total Enterococcus DNA increased during the early periods (23 h) under sunlight exposure and persisted during the entire period.;In conclusion, measuring Bacteroidales DNA in viable cells is recommended in applied MST studies because extracellular Bacteroidales DNA persists longer in the environment. Therefore, the application of both PMA-qPCR and qPCR methods within the same matrix may yield more realistic information about recent sources of fecal contamination and results in an improved predictive relationship with the presence of waterborne pathogens and assessment of health risk in ambient waters.