Tangential flow ultrafiltration and molecular detection of surrogates and pathogens in large-volume environmental water samples

Emerging human and animal microbial pathogens associated with waterborne transmission are of paramount concern to the public health community. One limiting factor in evaluating microbial water quality is the lack of an effective method for the simultaneous collection and recovery of bacteria, viruses, and protozoa and the inability of these microbes to be rapidly identified and quantified. This dissertation addresses the inadequacies of current methods and standards used for determining microbial water quality within various water sources.;A tangential flow ultrafiltration (UF) method was optimized to simultaneously concentrate and recover each class of microorganism from 100L water samples. Filter type, surfactant and dispersant addition, and elution steps were evaluated. For these evaluations, lab-based microbial surrogates were added to either drinking water (DW) or surface water (SW). Recovery efficiency across microbes and water types was 40 to 80%. Real time polymerase chain reaction (qPCR) and reverse transcription PCR (qRT-PCR) assays were employed for the detection of select microbial surrogates as well as host-specific enteric viruses. An internal standard using Hepatitis G virus RNA was also optimized and then analyzed by qRT-PCR along with processed UF concentrates to systematically identify sample inhibition.;The combined UF-qPCR methodology was then applied to 100L environmental water samples collected from Lower Yakima Valley, WA (n=21) and Ghana (n=19). Each sample was analyzed for bacterial indicators and human and bovine (not applicable to Ghana) enteric viruses using culture and qPCR and qRT-PCR assays, respectively. Lower Yakima Valley samples revealed human enteric viruses in 4 of 10 private groundwater (GW) wells sampled and human and bovine enteric viruses in 4 of 11 SW samples. In Ghana, each type of water source (GW, SW, DW) was positive for at least one human enteric virus. Source water (n=6) and DW (n=6) samples from two drinking water treatment plants were also processed using the OF method and analyzed for human enteric viruses---no viruses were detected. This research also demonstrated that indicator bacteria are poorly correlated with the presence of human enteric viruses. Overal, by developing a universal method for the recovery of microorganisms, a more comprehensive understanding of the microbial contamination of various water sources is possible.