Microbial Source Tracking: Library Independent Host-Specific Bacteroidales 16S rRNA gene PCR Assay in a Mixed Use Watershed.

The goal of Microbial Source Tracking (MST) is to identify the source of microbial contamination in natural waters. Transport of human and animal waste into aquatic systems can result in contamination with pathogenic microorganisms, which has become an increasingly serious health concern. The Upper Sugar Creek watershed located in northeast Ohio was selected as our study area. This mixed use watershed was suitable to examine the source of microbial contamination from human and agricultural activity and/or wildlife. Our methods included a PCR based, library independent and host-specific Bacteroidales 16S rRNA gene assay. One of the advantages of this molecular approach is that there is no need to prepare a library of cultured reference isolates due to the specificity and sensitivity of PCR. Therefore, rapid microbial contamination source diagnosis is possible. The purpose of our study was to determine the applicability of this MST method in a specific geological area. Samples were collected from 21 sites throughout the study watershed beginning July 2006 and terminating September 2008.   In addition, intensive sample collection, including storm event sampling, was conducted at suspected hotspots for close investigation of transport characteristics of microbial contaminants. We tested host-specific Bacteroidales assays (human, ruminant, horse, pig. and dog) to determine potential host sources, and we also tested a quantitative PCR (qPCR) assay for general Bacteroidales to investigate the magnitude of fecal contamination. Viable counts of E.Coli were determined for statistical comparison with the Bacteroidales PCR assay. These fecal contamination data were analyzed along with land management data. In addition to the water samples, fecal samples of potential hosts from the study sites were tested for host specificity of the method. We observed frequent human specific positive signals at residential land use areas and also observed high magnitude of general Bacteroidales qPCR signals at concentrated livestock operation area and at residential area. The results indicate the potential application of the MST method for land management decisions to control microbial contamination at the watershed scale.