Microbial Source Tracking Project Summary
The purpose of this project is to use rapid molecular techniques to evaluate the sources of fecal contamination in a mixed-use watershed. We plan to quantify the total amount of contamination as well as the relative contributions from humans, ruminants and other animals. In order to do this we will perform a quantitative PCR (qPCR) assay targeted to host-specific markers for different members of the bacterial genus Bacteroides.
The Bacteroides are a genus of obligately anaerobic organisms that represent the dominant bacteria of the large intestine. Their abundance in feces makes them an excellent indicator organism for direct detection by molecular methods. In addition, there is a substantial amount of genetic diversity among different members of the group. This diversity has allowed researchers to identify host-specific markers that can be used to distinguish between different sources of fecal contamination. A number of different markers have been identified for several host species (Dick et al., 2005; Layton et al., 2006)
We plan to use three sets of PCR primers (targets) to quantify Bacteroides from 1) all sources 2) human sources, and 3) bovine sources. By subtracting the bovine and human contributions from the total, we will infer the amount of contamination caused by all other animals. This assay is based on published results from a study sponsored by the Tennessee Department of Environmental Conservation (Layton et al., 2006).
Layton, A., L. McKay, D. Williams, V. Garrett, R. Gentry and G. Sayler. 2006. Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for estimation of Total, Human, and Bovine Fecal Pollution in Water. Appl. Environ. Microbiol. 72(6):4214-4224.
Dick, L.K., A.E. Bernhard, T.J. Brodeur, J.W. Santo-Domingo, J.M. Simpson, S.P. Walters and K.G. Field. 2005. Host Distributions of Uncultivated Fecal Bacteroidales Bacteria Reveal Genetic Markers for Fecal Source Identification. Appl. Environ. Microbiol. 71(6):3184-3191.