Microbial water quality is generally assessed using fecal indictor organisms; however host-specific microbial source tracking (MST) methodologies can be employed to differentiate sources of fecal pollution. The central goal of this research was to develop and validate a QPCR assay for the quantification of two human-specific polyomaviruses (HPyVs) in environmental water samples. These viruses are prevalent worldwide and produce lifelong, asymptomatic viruria in immunocompetent individuals. A Taqman® quantitative PCR (QPCR) assay based on the conserved T-antigen of two HPyVs (JCV and BKV) was developed and optimized (Chapter 2). HPyVs were detected in a high proportion of human-associated waste samples (e.g. sewage) and were not detected in animal excrement samples (Chapter 2). The effects of ultraviolet radiation, temperature, and salinity on the persistence of HPyVs in water were reported in Chapter 3. Laboratory studies analyzing the effects of various UV doses, temperatures, and/or salinities demonstrated high doses of UV were required to significantly decrease the detection of HPyVs DNA and salinity stabilized pure cultures of HPyVs virus particles at high temperatures (25°C and 35°C). Solar radiation as well as potential predation from microorganisms in sewage significantly reduced the persistence of HPyVs DNA in outdoor mesocosm studies (Chapter 3). An improved method to extract human polyomavirus (HPyVs) DNA from environmental water samples was developed, and the recoveries were larger and more consistent over a range of DNA concentrations as compared to the standard protocol (Chapter 4). In the California beaches study (Chapter 4), the presence of HPyVs by either QPCR or PCR had a high degree of matching results with the adenoviruses (83-91%), Methanobrevibacter smithii marker (82-92%) and moderate degree of matching results with the human-associated Bacteroidales spp. marker (57-80%) (Chapter 4). HPyVs were detected in the presence of various pathogens including: Giardia spp., Cryptosporidium spp., Vibrio spp., enteroviruses, and noroviruses (Chapter 5). The presence of HPyVs in relatively high concentrations of sewage and the specificity of HPyVs combined with the relatively conservative persistence of HPyVs when exposed to environmental conditions and the correlation of HPyVs with pathogens demonstrates that this assay is a useful MST method to detect human sewage.

Urban waterways are highly vulnerable to bacterial contamination, which presents significant risks to public health and water quality. Common methodologies typically measure the total concentration of fecal indicator bacteria (FIB) but are unable to address the complex sources of contamination contributing to the overall bacterial load. This study established chip-based digital polymerase chain reaction (cdPCR) techniques for microbial source tracking to unveil the origins of Escherichia coli (E. coli). Along with a simultaneous analysis of physicochemical water quality indicators, an assessment was conducted using host-associated genetic markers that indicate fecal sources from humans (HF183/BacR287), ruminants (Rum2Bac), dogs (DG3), and birds (GFD) in the lower portion of Rock Creek River (RCR) in the District of Columbia, United States. Stream samples were collected twice a month (n = 24) and after rain events (n = 6) from three sites along the RCR in the district area that feature a mix of highly developed urban areas and park surface regions. Approximately 50% of the stream samples (n = 96) were found to be impaired, exceeding the district's single sample maximum assessment level (410 MPN/100 ml) for E. coli. Herein, we adopted a multi-scale characterization of the relationship of cultural E. coli with host-associated markers, the regression with in-stream physiochemical constituents, the distinction between sampling sites, and the correlation with sizeable land cover categories. In Chapter 1, a comprehensive overview of MST methods is presented. This chapter summarizes the development of MST, categorizes common MST techniques into library-dependent versus library-independent and culture-dependent versus culture-independent groups, and provides a brief history of the advancements in molecular instrumentation used for culture-independent methods. In Chapter 2, consistently elevated E. coli levels were observed at all sites during wet weather, highlighting the substantial impact of storm runoff on water quality deterioration. Among the four molecular markers tested, HF183/BacR287, which indicates human-associated contamination, was particularly prevalent, with the highest frequency found in one of the tributaries. The second marker, derived from avian sources (GFD), showed a moderate to low frequency across the sites. Detection of the ruminant- and dog-specific markers was sporadic at all three sites. Correlation and regression analyses involving E. coli, molecular markers, and physicochemical constituents revealed significant statistical relationships. Notably, turbidity and flow were useful indicators for quickly assessing bacterial contamination. These findings emphasize the importance of reducing microbial contributions from runoff in watershed areas to urban streams during wet weather. The methods and findings of this study are expected to assist stormwater management and regulatory agencies in developing best management practices (BMP) to protect the water quality of urban streams. In Chapter 3, a strong association of E. coli with low-intensity developed land was established, but this association to forested areas at smaller spatial scales. The HF183/BacR287 marker exhibited similar trends, reinforcing its role as a reliable indicator of E. coli contamination sources. This study highlights the value of MST markers in identifying sources of microbial contamination. It provides important insights for managing water quality across various land cover types and changing weather conditions. In Chapter 4, the scalability of cdPCR to cell equivalents was investigated. By transforming scaled cdPCR DNA copies, the study revealed that 3,153 DNA copies per 100 mL of human-associated HF183BacR287 corresponded to the same regulatory threshold as cultured E. coli, enabling direct comparison between cdPCR and Colilert methods for contamination detection. This approach highlights the potential of cdPCR as a complementary tool to traditional methods in MST studies, offering a more detailed and efficient approach for water quality monitoring and management. In Chapter 5, a summary of the results is presented, and a perspective of future research direction is proposed. / Doctor of Philosophy / Urban waterways are highly susceptible to bacterial contamination, which can pose serious risks to public health and water quality. Traditional methods for monitoring bacterial contamination typically focus on measuring total levels of fecal indicator bacteria (FIB), but they are incapable of distinguishing the bacterial sources. This study aimed to fill that gap by using advanced techniques to trace the origins of Escherichia coli (E. coli) contamination in an urban creek in Washington, D.C. This study utilized a chip-based digital PCR (cdPCR) system to identify fecal pollution from various sources, including humans, animals, and wildlife. Throughout the study, water samples were collected from three sites along the creek within the district area during both dry weather and after rainstorms. The results indicated that approximately half of the samples had E. coli levels exceeding safe limits, particularly following rainfall, highlighting the significant impact of stormwater runoff on water quality. Among the identified sources of contamination, human-associated pollution was the most prevalent, followed by contamination from birds. The study also combined analysis of water quality indicators, such as turbidity and flow. Strong correlations between E. coli levels, molecular markers, and certain water quality indicators were observed, which can help in quickly assessing water contamination. This research underscores the potential of molecular techniques, like cdPCR, to accurately identify the sources of bacterial contamination in urban waterways. The findings can aid in better management of stormwater runoff and inform efforts to improve water quality in urban streams. Additionally, the study provides valuable insights into how land use, such as urban development and forest cover, can influence bacterial contamination, offering a more comprehensive understanding of the environmental factors contributing to water pollution.