Survival and persistence of Bacteroidales human and ruminant specific fecal markers and occurrence with fecal pathogens

• Main Author: Walters, Sarah P
• Format: Dissertation
• Language: English
• Subjects: biology, microbiology; biology, molecular; engineering, environmental; health sciences, public health

Aquatic fecal contamination from non-point sources impairs environmental health and serves as a vehicle for transmission of waterborne disease, resulting in economic losses worldwide. Accurate methods of diagnosing fecal pollution and its source are needed to prevent human exposure, remediate pollution, and reduce economic impacts. In order to obtain this goal, fecal indicator organisms should demonstrate persistence and survival profiles similar to fecal pathogens and be detected when pathogens are present. Molecular markers designed from Bacteroidales anaerobic fecal bacteria make good alternative indicator candidates because they identify fecal pollution sources and are not expected to grow in oxygenated environments. To further investigate the utility of these markers as indicators of fecal contamination and predictors of pathogen exposure, we evaluated co-occurrence of pathogenic E. coli O157:H7, Salmonella spp., and Campylobacter spp. with general, human, ruminant, and porcine-specific Bacteroidales molecular markers. We detected host-specific markers in samples where pathogens were present and found a significant correlation between presence of the ruminant markers and E. coli O157:H7, and the human markers and pathogenic Campylobacter spp. We examined growth and persistence of Bacteroidales organisms in aerobically incubated sewage influent using bromodeoxyuridine (BrdU) labeling and immunocapture. We identified growing Bacteroidales bacteria in BrdU-labeled DNA fractions after immunocapture with fluorescent fragment detection following a low, quantitative number of PCR cycles. We consistently detected growth of Bacteroidales organisms but were unable to detect growth of the human-specific Bacteroidales organisms using this method. We constructed microcosms to investigate how ambient light exposure affects survival and persistence of ruminant and human-specific Bacteroidales markers, relative to standard indicators. The ruminant-specific Bacteroidales markers displayed differential persistence and survival profiles in both light and dark incubations; this was not observed among the human-specific markers. In each microcosm, the standard indicators persisted and survived longer than any of the Bacteroidales host-specific markers. Host-specific Bacteroidales genetic markers show promise as an accurate, rapid, and reliable tool in health risk based analyses of fecal pollution. The molecular markers are detected when pathogens are present, persist less than 14 days in natural river water at 13°C, and the marker organisms do not proliferate in the environment.