Microbial Source Tracking of Human and Animal Waste Pollution of Diverse Watersheds and of Urban Drainage Systems using Molecular Methods

The central hypothesis of microbial source tracking (MST) is that there exists a distinguishable relationship between microbes excreted in feces and their particular hosts. These associations could be due to adaptations of microbes to their host's gut environment and may involve coevolution between microbes and their hosts. In this study, we performed phylogenetic analyses on 16S rRNA gene sequences associated with different host groups to determine if Bacteroides dorei and Enterococcus faecium show co-evolutionary characteristics, thus, host specificity. Bioinformatic analyses were also conducted on E. faecium surface protein gene (espfm) for host specificity. The results of our analyses provide good support for B. dorei human host specificity, and very little support for such specificity in E. faecium. .;Given these findings, a redesigned primer and probe set was developed for a probe based qPCR assay that specifically targets bacterial sequences from a human-specific B. dorei lineage (HF68-HR183rc) and another targeting the E. faecium espfm gene. The results from comparative assays of HF68-HR183rc against a HF183 protocol obtained from the literature showed similar sensitivity, but an improvement in the specificity of our newly designed protocol. However, assays of rabbit samples showed a high number of positive assays with both the redesigned (58%) and the HF183 (67%) protocol. The newly designed HF68-HR183rc assay could be of considerable use to screen a watershed for human fecal sources of pollution if it is known not to be impacted by rabbits. Assays of the redesigned espfm protocol were not as successful as a quantitative assay, requiring an enrichment process.;The HF68-HR183rc qPCR MST protocol was used to help characterize the sources of pollution of sub-watersheds of the temperate environs of Philadelphia/Delaware watershed; watersheds of the tropical island of Puerto Rico; and different types of green infrastructures in Philadelphia and New York.;Keywords: microbial source tracking; Bacteroides dorei; Enterococcus faecium; qPCR; molecular (genetic) markers; 16S rRNA gene; bioinformatics; phylogenetic.