Factors Influencing Persistence of Fecal Bacteroides in Stream Water

Fecal contamination leads to increased risk of exposure to enteric pathogens in aquatic environments used for drinking water, recreation, and commercial shellfishing. Current indicators of fecal contamination recommended by the EPA such as E. coli and enterococcus can fall short of meeting ideal indicator criteria by having widely-varying persistence in the environment, reproducing in the environment, occurring in the gut in low and variable concentrations, and requiring time-consuming assays. Furthermore, both these indicators lack the degree of host specificity needed for use in identifying sources of fecal contamination, which is an important tool for identifying and reducing fecal inputs to the aquatic environment. Preliminary studies have suggested bacteria belonging to the genus Bacteroides can be an alternative to current fecal indicators. This thesis assessed the utility of Bacteroides as a fecal indicator first through a review of the literature investigating the use of Bacteroides and highlighting current research needs. Next, a series of microcosm experiments investigating the effects of varying environmental parameters on the persistence of the Bacteroides 16S rRNA marker in stream water were performed. These studies used real-time PCR detection to develop decay curves for Bacteroides concentrations under different conditions of water filtration, initial fecal aggregate size, initial fecal concentration, temperature, and fecal source organism. Statistical models were used to determine the significance of the decay curves and identify those variables having the greatest significance to the rate of decline of Bacteroides. The results of this study indicated that filtration, temperature, and the initial fecal aggregate size had significant effects on the rate of removal of the Bacteroides genetic marker from stream water. The decline was significantly less in filtered (0.25 log removal/day) versus unfiltered stream water (0.67 log removal/day), 5 °C (0.32 log removal/day) versus 25 °C (1.62 log removal/day), and coarse (0.62 log removal/day) versus fine aggregate size (1.41 log removal/day). The initial concentration of fecal matter and the host organism did not have significant affects on removal. This suggests that the primary mechanism behind decline of the Bacteroides marker in stream water is biologic removal, such as grazing by protozoa or infection by bacteriophage. Finally, this thesis presents the development of the EqBac assay, a new quantitative PCR assay using the Bacteroides 16S rRNA gene from horses to detect equine fecal contamination.