Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r 2 ‫؍‬ 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.

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Laboratory microcosm experiments were used to assess the eff ects of environmental parameters on the persistence of the Bacteroides 16S rRNA genes derived from equine fecal samples in stream water to investigate the utility of Bacteroides spp. as fecal indicator organisms. Quantitative real-time polymerase chain reaction (qPCR) was used to measure gene concentrations over time with treatments designed to compare fi ltered vs. unfi ltered stream water, fecal aggregate size, initial fecal concentrations, and water temperatures. Comparison of Bacteroides16S rRNA genes/mL in microcosms constructed with unfi ltered stream water and fi ltered stream water indicated that stream water fi ltration to remove indigenous microorganisms followed by temperature had the largest eff ects on gene persistence. Firstorder exponential decay functions were fi tted to the data from each microcosm constructed using unfi ltered stream water, and the decay constants (k) ranged from 0.0071 h -1 in the microcosms incubated at 5°C to 0.0336 h -1 in a set of microcosms incubated at 25°C. Analysis of k calculated from the 10 experimental treatments indicated that k is more highly correlated to temperature than initial Bacteroides 16S rRNA gene starting concentrations. Th e equation resulting from graphing k (as the dependent variable) vs. temperature (as the independent variable) best fi t a peak, Gaussian, 3 parameter function with a maximum decay at 30°C, a r 2 of 0.83 and all parameters were signifi cant (P < 0.0015). Th us this data suggest that factors that reduce biological activity, such as physical removal of stream microorganisms by fi ltration and low temperature, result in slower Bacteroides 16S rRNA gene decay.