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A New Sampling Strategy for the Detection of Fecal Bacteria Integrated with USEPA Method 1622/1623
Citation:
RYU, H., B. Iker, J. W. SANTO-DOMINGO, M. W. WARE, E. VILLEGAS, AND T. TRAN. A New Sampling Strategy for the Detection of Fecal Bacteria Integrated with USEPA Method 1622/1623 . Presented at American Society for Microbiology 110th General Meeting, San Diego, CA, May 23 - 27, 2010.
Impact/Purpose:
To inform the public.
Description:
USEPA Method 1622/1623 requires the concentration of Cryptosporidium and Giardia from 10 liters of water samples prior to detection. During this process the supernatant is discarded because it is assumed that most protozoa are retained in the filtration and centrifugation steps. In this study, we tested the discarded materials for the presence of bacterial markers used to detect sources of fecal pollution in environmental waters. Two river water samples as well as reagent water amended with sludge were each processed in triplicate by Method 1622/1623. From these samples, three subsamples were collected from the raw water (100 mL), the eluate supernatant, and the solution remaining after the immunomagnetic separation (IMS) step. Most Probable Number-PCR and quantitative PCR targeting Bacteroidales 16S rRNA genes were used to determine the bacterial DNA yields from each step. Based on data from both PCR assays, the DNA yields for eluate supernatant ranged from 59 to 71%, while an additional 1-10% of the targeted Bacteroidales was recovered in the IMS supernatant. Bacteroidales 16S rRNA gene clone libraries were constructed for the eluate and IMS supernatant (165 clones each) and compared using the Naïve Bayesian rRNA Classifier. The distribution of clones from subsamples was not statistically different, suggesting that there were no recovery biases introduced in these steps. Additional studies showed the detection of other relevant fecal populations such as human-specific Bacteroidales and enterococci in the discarded material. These results showed that although potentially up to 40% of bacterial DNA was not recovered from the two supernatant subsamples, the equivalent sample volumes was approximately an order of magnitude higher than raw water, which from a detection standpoint may compensate for any significant loss of microbial DNA. Overall, the results from this study show the potential value of using the discarded fraction for source tracking purposes. These finding are also relevant as it enables us to compare source tracking results with the presence of protozoan genotypes in the same water samples collected using Method 1622/1623.
