Investigation of Reservoirs of Fecal Indicator Bacteria and Water Quality on the Presence of Allochthonous Pathogens and the Ecology and Virulence of Vibrio vulnificus

The quality of recreational and shellfishing waters has historically been monitored using commensal, allochthonous bacteria shed in feces (fecal indicator bacteria, FIB). The fate of FIB in the environment should mimic that of bacterial, protozoan, and viral human pathogens, which may also be allochthonous (e.g. Salmonella, Cryptosporidium, or enteric viruses) or autochthonous (e.g. Vibrio spp.) to aquatic environments. FIB are contributed to water from human and animal sources; however, pollution source cannot be determined by conventional FIB measurements. Because fecal source determination is important for pollution remediation and assessment of human health risks, microbial source tracking (MST) methods are increasingly used in water quality studies. The host-specific genes (markers) used for MST include the 16S rRNA of Bacteroides HF183 and the T-antigen of human polyomaviruses (HPyVs). In my work, correlations among FIB, MST markers, and autochthonous pathogens were explored in the context of factors that may influence these relationships. Specifically, the effects of stormwater runoff, sediment resuspension, and survival/persistence of FIB on submerged aquatic vegetation were investigated in a recreational lake. Furthermore, the relationship between FIB and concentrations of the autochthonous pathogen, V. vulnificus, was investigated at water bodies surrounding Tampa Bay. I hypothesized that degraded water quality would influence the concentration and/or population structure of V. vulnificus, a potentially lethal human pathogen. Finally, I hypothesized that the gene encoding a sodium-phosphate transporter (nptA) would be differentially expressed in V. vulnificus strains under varying conditions of salinity and phosphate concentration. I hypothesized that stormwater infrastructure/runoff, SAV, and sediments would serve as reservoirs for FIB, human-associated microbes (HF183 and HPyVs), and allochthonous pathogens (Salmonella, Cryptosporidium, Giardia, and enteric viruses). FIB concentrations in the water were positively associated with those in the sediment, SAV, and with 24hr antecedent rainfall. At least one MST marker or pathogen was found in 35% of samples following rain events. These data were incorporated into a Bayesian model, which predicted pathogen absence when fecal coliform concentrations were low. Stormwater was also shown to be an important reservoir/conveyance system for FIB, human-associated microbes, and pathogens. I hypothesized that polluted estuarine waters in Tampa Bay, and oysters harvested from them, would contain higher V. vulnificus concentrations, and that the population structure would be altered compared to unpolluted waters. Enumeration included direct plating, enrichment followed by plating, and quantitative PCR (qPCR). V. vulnificus colonies isolated directly on mCPC agar were rarely PCR-confirmed, although enrichment and qPCR methods yielded a higher confirmation frequency. Unconfirmed colonies resembling V. vulnificus were identified as V. sinaloensis via 16S rRNA sequence analysis and were more frequently detected in less polluted waters. Comparison of growth rates among V. vulnificus and V. sinaloensis strains in enrichment media and seawater showed that V. vulnificus had faster growth rates (µ) in enrichment media, but that µ of V. sinaloensis strains was greater in seawater. V. sinaloensis presence can therefore lead to overestimation of V. vulnificus concentrations when samples are directly plated. These results highlight a need for better understanding of the ecology and virulence potential of this newly-described species. Finally, I hypothesized that V. vulnificus strains with varying virulence potential would differentially express the nptA gene in response to changes in environmental conditions. Expression studies were performed on biotype 1, 2, and 3 strains, and strains more closely associated with environmental reservoirs (water or oysters) showed up to 100-fold greater nptA expression than strains isolated from clinical cases. Gene expression in environmentally-associated, but not clinically-isolated, strains was highest in media at pH 6.0 vs. those at pH ≥ 7.0 and at 10 / salinity. In contrast, expression was highest among clinical strains at 10 / salinity, pH 8.0 media. Sequence analysis of the nptA gene also divided strains into environmentally- and clinically-isolated groups. These results suggest that differences in gene expression may be related to host preference and may be associated with differential virulence of strains in humans. These studies demonstrate a relationship between water quality (determined by FIB concentrations) and the prevalence of allochthonous and autochthonous human pathogens, and reveal that many environmental habitats may serve as reservoirs for FIB and pathogens. Differences in water quality were further demonstrated to impact the community structure of Vibrio spp. and may affect the relative abundance of strains with greater virulence potential