The goal of this study was to assess the effect of residence time on the chemical and microbial quality of reclaimed water in two distribution systems located in southern Arizona. Utility A produced Class A water and utilized chlorine as a means of disinfection whereas Utility B produced Class A+ water and utilized UV radiation as a means of disinfection. Water-based pathogens were consistently detected in both distribution systems beyond the point of compliance, while microbial indicators like Escherichia coli was only detected in Utility B suggesting that treatment eliminated waterborne pathogens. Heterotrophic plate concentrations in samples from both utilities initially increased rapidly with increased distance from the point-of-compliance and were as high as 10⁹ CFU/100ml. Regardless of the initial level of treatment, the microbial quality deteriorated with increased residence time in the distribution systems. The second study was designed to evaluate the effect of reclaimed water storage on microbial and chemical quality of two classes of reclaimed water (Class A and Class A+). In Class A water, nitrification was observed during both field scale trials resulting in concentrations greater than 10mg/L while nitrification was not observed in Class A+. Chlorine residuals rapidly decreased within 48hours of storage. HPC concentration were as high as 10⁷ - 10⁸ /100ml. In both field scale trials, there was no observed growth of HPC during storage and waterborne indicator bacteria were rarely detected, and if detected, only at low concentrations. Based on this data, deterioration of microbial water quality during storage is minimal.

This study presents the spatial and temporal variability of Salmonella enterica in urban and rural streams in a model watershed (Grand River watershed, Ontario, Canada), and examines the antimicrobial resistance (AMR) and genetic diversity of various serotypes. Using a swab collection method and various media types, Salmonella were detected in 78.4% of samples between November 2003 and July 2005. A diverse range of Salmonella serotypes (n=38) were isolated from water. Predominant serotypes and phagetypes (PT), including S. Typhimurium PT 104 and S. Heidelberg PT 19, and the proportion of isolates demonstrating AMR (33%), was similar to those for humans and farm animals locally and across Canada, a trend not commonly reported. There was a greater diversity of serotypes and AMR profiles in isolates from the urban stream compared to the rural/agricultural streams. Plasmid-borne resistance was observed in 28.6% of AMR isolates, with two different plasmids responsible for resistance; the TEM-1 plasmid (8.1Kb plasmids carrying blaTEM-1, responsible for ampicillin resistance) and CMY-2 plasmid (95.5Kb plasmids carrying blaCMY-2, responsible for 3rd generation cephalosporin resistance). CMY-2 plasmids were only found in the urban stream and did not create a biological burden under non-selective conditions, indicating the long-term permanence of these plasmids. Seasonal differences in the overall diversity of serotypes and predominance of serotypes of human health significance (S. Typhimurium and S. Heidelberg) were observed. The lower occurrence of S. Typhimurium and S. Heidelberg in February and March was not the result of lower survival of these serotypes at low temperatures. Peaks in occurrence of S. Typhimurium and S. Heidelberg in the summer and spring, respectively, were pronounced in the rural/agricultural streams, as opposed to the urban stream. Pulsed-field gel electrophoresis and plasmid-typing revealed diversity within multiple drug resistant S. Typhimurium PT 104 isolates, indicating genetic differences among tributaries. The ubiquitous nature of Salmonella in water and the predominance of serotypes/phagetypes of human or veterinary health significance suggest that environmental exposure through consumption or contact with contaminated water is plausible. These streams may act as a vehicle for the dissemination of these organisms and their resistance genes between different hosts or environments.

This study presents the spatial and temporal variability of Salmonella enterica in urban and rural streams in a model watershed (Grand River watershed, Ontario, Canada), and examines the antimicrobial resistance (AMR) and genetic diversity of various serotypes. Using a swab collection method and various media types, Salmonella were detected in 78.4% of samples between November 2003 and July 2005. A diverse range of Salmonella serotypes (n=38) were isolated from water. Predominant serotypes and phagetypes (PT), including S. Typhimurium PT 104 and S. Heidelberg PT 19, and the proportion of isolates demonstrating AMR (33%), was similar to those for humans and farm animals locally and across Canada, a trend not commonly reported. There was a greater diversity of serotypes and AMR profiles in isolates from the urban stream compared to the rural/agricultural streams. Plasmid-borne resistance was observed in 28.6% of AMR isolates, with two different plasmids responsible for resistance; the TEM-1 plasmid (8.1Kb plasmids carrying blaTEM-1, responsible for ampicillin resistance) and CMY-2 plasmid (95.5Kb plasmids carrying blaCMY-2, responsible for 3rd generation cephalosporin resistance). CMY-2 plasmids were only found in the urban stream and did not create a biological burden under non-selective conditions, indicating the long-term permanence of these plasmids. Seasonal differences in the overall diversity of serotypes and predominance of serotypes of human health significance (S. Typhimurium and S. Heidelberg) were observed. The lower occurrence of S. Typhimurium and S. Heidelberg in February and March was not the result of lower survival of these serotypes at low temperatures. Peaks in occurrence of S. Typhimurium and S. Heidelberg in the summer and spring, respectively, were pronounced in the rural/agricultural streams, as opposed to the urban stream. Pulsed-field gel electrophoresis and plasmid-typing revealed diversity within multiple drug resistant S. Typhimurium PT 104 isolates, indicating genetic differences among tributaries. The ubiquitous nature of Salmonella in water and the predominance of serotypes/phagetypes of human or veterinary health significance suggest that environmental exposure through consumption or contact with contaminated water is plausible. These streams may act as a vehicle for the dissemination of these organisms and their resistance genes between different hosts or environments.

In January 2010, a magnitude 7.0 earthquake devastated Haiti, one of the poorest countries in the Western Hemisphere. Haiti’s weak sanitation infrastructure and limitations in the public health system made the country susceptible to the spread of waterborne diseases. Following the earthquake, cholera rapidly spread through Haiti, killing 4,672 people in 5 months and leaving thousands hospitalized (MSNBC, 2010). Cholera is an infectious diarrheal disease caused by the pathogen, Vibrio cholerae, which results in severe dehydration with a high mortality risk. The source of the epidemic was traced to the Artibonite River, the island’s longest and most essential drinking water source (Encyclopaedia Britannica. n.d.). The origin of the contamination was later discovered to be unsanitary conditions left from United Nations peacekeepers from Nepal. Eight years later, cholera cases are still prevalent, although numbers have declined recently due to aid from private organizations (Dowell, S.F. et al 2011, Katz, J.M. 2013). However, with climate-related increases in ocean water temperatures, scientists expect hurricanes to intensify and increase damage to developing countries (Center for Climate and Energy Solutions. n.d.). Natural disasters promote the spread of waterborne illness by isolating people from safe drinking water and destroying public health infrastructure such as happened with the cholera outbreak in Haiti (Funari, E. et al 2013). To prevent future waterborne disease epidemics in such areas with limited resources, it would be beneficial to improve environmental surveillance through development of rapid, reliable, and portable detection methods for waterborne pathogens. The advent of high-throughput sequencing technologies has enabled the detection and characterization of microbial communities in their natural environments, an approach known as metagenomics. Metagenomic sequencing, unlike more traditional PCR methodologies, is capable of sequencing thousands of organisms in a sample. This metagenomic shotgun sequencing approach detects the abundance of microbes and bacterial diversity in the environment (Illumina, n.d.). The Oxford Nanopore MinION is a shotgun sequencing device that is optimal for portable, rapid detection of the microbial diversity in an environmental sample (Oxford MinION, n.d.). This handheld device has enormous potential for field use in emergency preparedness and disease response, particularly in developing countries where more advanced analytical equipment may be inaccessible due to lack of facilities or damaged infrastructure. Having access to quick, infield assessment technology for rapidly emerging outbreaks would be beneficial to a disease-specific public health response. Current protocols recommend that DNA is extracted from environmental samples as rapidly as possible after collection. If cooling is available with an insulated ice chest, samples may be transported/stored for periods ranging from 6 to 24 hours. The shorter timeframes minimize unwanted shifts in microbial structure (U.S. Geological Survey, 1997, WHO, n.d.). Access to cold storage in remote areas is unlikely, and the use of liquid preservation methods could assist in maintaining quality of DNA, and hence produce more accurate data in metagenomic analyses. In the absence of cold storage facilities, infield filtration coupled with preservation techniques are necessary to maintain samples integrity for transport to laboratory facilities. This thesis aimed to develop an infield filtration and sequencing protocol, coupled with the Oxford Nanopore MinION sequencing platform, to identify the potential bacteria, viruses, protozoa, fungi, antimicrobial resistance (AMR), pathogenic strains, and virulence associated genes for use in remote locations. Five locations across Lake Warner, Massachusetts were used for method development, coupled with Millipore Sterivex filters for field filtration to determine the most effective method for sample preparation in remote locations. Additionally, a chemical preservation method was assessed using dimethyl sulfoxide, disodium EDTA, and saturated NaCl (DESS). A study by Gray et al, found that liquid preservation methods (DNAgard, RNAlater, and DESS) outperformed the card-based preservatives (FTA cards and FTA Elute cards) in terms of bacterial recovery (Gray, M.A., et al 2013). DESS was selected for investigation in this thesis because of the low cost compared to the other liquid-based preservatives. Lake Warner in Hadley, Massachusetts, which is heavily used for fishing and boating activities, flows into the Connecticut River via the Mill River. Historically, the lake experienced high Escherichia coli (E. coli) levels due to pollution from primary effluent released in the 1950s from the Amherst Wastewater Treatment Plant (Johnson, J., 2015). Similar to Vibrio cholerae, E.coli spp is a waterborne bacteria caused by fecal contamination. Although most E.coli are natural inhabitants of the gastrointestinal tract, pathogenic serotypes can result in severe complications in vulnerable populations such as kidney failure in children and the elderly adults. (Todar, K., 2012). Lake Warner was chosen for the method development because of its history of E. coli pollution and recreational traffic as well as its general accessibility for study. Designing a methodology for rapid detection of pathogenic bacteria using a metagenomic approach could help improve surveillance for environmental pathogens that pose future epidemic risk. These tools are becoming increasingly important for prediction and response to waterborne diseases as climate impacts increase the frequency, intensity, and duration of extreme weather events that damage critical infrastructure for vulnerable populations (van Aalst, M.K. 2006).

During the 1980’s millions of households in Bangladesh switched from drinking surface water to private groundwater wells to reduce their exposure to fecal microorganisms. Sadly, this switch to shallow groundwater resulted in the largest example of drinking water poisoning in history, with approximately 100 million people exposed to high concentrations of naturally occurring Arsenic in the groundwater. Spatial distribution of Arsenic in the shallow aquifers tends to be patchy, so the most economical mitigation option has been lateral switching from high Arsenic wells to nearby low Arsenic wells. The recently developed Arsenic flushing conceptual model, which explains the spatial distribution of Arsenic throughout the shallow aquifers in Bangladesh, suggests however, that low Arsenic zones are recharged via coarse-grained, rapid flow pathways and therefore represent a higher risk for waterborne pathogens. The objectives of this dissertation are to evaluate new methods for sampling and detection of waterborne pathogens, while also identifying sources of fecal contamination and transport pathway(s) to private wells emplaced within the shallow aquifers. It was demonstrated that private wells are broadly contaminated with E. coli, with prevalence ranging from 30 to 70%. The fact that E. coli was detected more frequently in private wells than sealed monitoring wells (p<0.05) suggests that well construction and/or daily pumping contribute to fecal contamination of the private wells. Using DNA-based molecular fecal source tracking, contamination was demonstrated to originate from human fecal waste. Unsanitary latrines, which spill effluent onto the open ground, were demonstrated to cause elevated levels of fecal bacteria in ponds, found in every village. These ponds were demonstrated to have an influence on concentrations of fecal bacteria to at least distances of 12m into the adjacent aquifer. In a culture where latrines, private wells and ponds are frequently clustered closely together, these findings suggest that improvements in the management of human fecal waste changes in placement and construction of private wells could substantially reduce exposure of people to fecal pathogens. Fecal contamination was found to be pervasive in low Arsenic, unconfined, shallow aquifers, and therefore gains from well switching to avoid Arsenic need to be balanced with the risk of consuming waterborne pathogens.

Waterborne pathogen presence caused by fecal pollution is a leading cause of morbidity and mortality worldwide. In developed countries, this problem can result in waterborne outbreaks. Research suggests that there is a need for better fecal indicators because current methods (total coliforms and E. coli) are insufficient. This study investigated Bacteroidales 16S rRNA markers as a microbial source tracking tool in an agricultural watershed. Correlations between pathogens and markers were also investigated. Water quality monitoring was conducted following assay validation of ruminant-, bovine-, human-specific, and universal Bacteroidales markers. Results revealed a positive relationship between E. coli and the universal marker. Ruminant- and bovine-specific marker detection was associated with increased runoff due to precipitation; however, the human associated marker was not detected. Furthermore, no correlations between Campylobacter, Salmonella, or E. coli O157:H7 could be made. Consequently, these techniques have potential to become a powerful tool; however, further research is needed

The analysis of potentially harmful biological particles is imperative for the mitigation of disease. As a result, there is a growing need for tools which can characterize, detect, and separate biological particles for the alleviation of a multitude of disease. One powerful technique for the analysis of cells is the use of dielectrophoresis (DEP) forces for the manipulation of particle movement. DEP is a particle transport phenomenon, induced by the presence of non-uniform electric fields. The dependence on intrinsic electrical properties of cells, have enabled DEP force to be utilized for numerous biological analyses. This thesis presents the investigation of breast cancer, pathogen, neuronal and glial cells and their DEP profiles. The drug response of various breast cancer cell lines when exposed to a variety of chemical stimuli were analyzed using shifts in their DEP profiles in relation to control groups. These results were supplemented with gene expression analysis to identify biophysical changes which could contribute to the DEP shifts. Additional experiments were conducted for the monitoring of pathogens. Live/dead bacteria mixtures were evaluated using an integrated system with DEP enrichment and impedance spectroscopy. Another application of DEP which was investigated was the separation of heterogeneous mixtures. Through the use of a novel microfluidic channel design, the separation of simulated circulating tumor cells (CTCs) from diluted blood and neuron cells from glial cells was demonstrated. The wide range of applications examined in this thesis highlights the versatility of DEP and the flexibility of the reported devices. / MS / Microscale technology can be utilized for the identification, characterization and sorting of biological material in a plethora of biomedical applications. One promising technique which is capable of cell manipulation is dielectrophoresis (DEP). DEP is a microscale force which causes particles to be attracted or repelled by specific geometries in microchannels. The DEP force is produced by the application of electric fields and can be utilized to analysis biological cell populations. This is because biological particles have unique electrical properties based on their cell morphology. Distinctions in their external protrusions and internal structures contribute to their electrical properties and can be identified in their DEP profiles. Based on this concept a variety of biomedical applications of DEP was explored. Chapter 2 and 3 describe the investigation of cells when exposed to various drugs. Drug induced responses were characterized based on their shifts in their DEP profiles. Chapter 4 presents the a rapid and low-cost live/dead assay for bacteria in aqueous samples through DEP and impedance spectroscopy. In chapter 5, the development of a DEP platform for cell sorting is reported. The wide range of biomedical applications which were explored demonstrate the useful nature of the DEP phenomenon.

Water quality is an essential component of vibrant societies and ecosystems. For decades, researchers, managers, and policymakers around the world have struggled to accelerate societal progress while preserving and enhancing water quality and human health. This thesis consists of two studies that I hope will contribute to better understanding, policy, and management. In the first study, I evaluated spatial and temporal patterns in global water quality and their relationship to gross domestic product (GDP) per capita, as a metric of socioeconomic development status. Using global water quality datasets containing over 2.7 million observations, I tested the Environmental Kuznet Curve (EKC) hypothesis, which predicts that environmental degradation is highest at intermediate levels of socioeconomic development. I found that 46% of pollutants persisted at elevated concentrations despite GDP per capita. Because of this, high income countries experience a false sense of water security as water regulation violations are common on a global scale. In the second study, I measured waterborne pathogens in Guayaquil, the largest city in Ecuador. With a population of over 3 million and distinct hydrology from monsoonal rains and estuarine flooding, the Guayaquil metropolitan area faces drinking water and sanitation challenges similar to much of the developing world. I found that 100% of the samples we collected had unsafe total coliform counts. Water pollution is widespread and is a result of careless action. Moving forward, chronic pollution can be prevented with proper legislation that holds governments, companies, and individuals accountable.

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