Opportunistic premise (i.e., building) plumbing pathogens (OPPPs) and antibiotic resistant bacteria are emerging microbial concerns in drinking water. OPPPs, such as Legionella pneumophila, are the leading cause of drinking water disease in many developed countries. Contributing factors include the relative success in controlling fecal pathogens, the presence of complex building plumbing systems that create habitats for OPPPs, and the relative resistance of OPPPs to disinfectants, and aging populations that are susceptible to infection. Concurrently, drinking water is increasingly being scrutinized as a potential environment that is conducive to horizontal gene transfer of antibiotic resistance genes (ARGs), selection pressure for enhanced survival of resistant bacteria, and a route of transmission of antibiotic resistant pathogens. While maintaining a disinfectant residual is an established approach to controlling OPPPs in premise plumbing, some studies have indicated that co-resistance and cross-resistance to disinfectants can increase the relative abundances of resistant bacteria and ARGs. Thus, there may be trade-offs to controlling both OPPPs and antibiotic resistance in premise plumbing that call for controlled study aimed at optimizing residual disinfection application for this purpose. A critical review of the scientific literature in Chapter 2 revealed that premise plumbing is a biologically and chemically complex environment, in which the choice of pipe material has cascading effects on water chemistry and the corresponding premise plumbing microbiome. This, in turn, has broad implications for the control of OPPPs, which need to be elucidated through controlled experiments in which worst case premise plumbing conditions are held constant (e.g., warm temperature), while other variables are manipulated. Chapter 3 introduces the convectively-mixed pipe reactors (CMPRs) as a novel low-cost, small footprint approach to replicably conduct such experiments. The CMPRs were demonstrated to effectively simulate key chemical and biological phenomena that occur in distal reaches of premise plumbing. In Chapter 4, the CMPRs were leveraged to study the interactive effects of four disinfectants (chlorine, monochloramine, chlorine dioxide, and copper-silver ionization) and three pipe materials (PVC copper, and iron). The CMPRs were inoculated with two antibiotic-resistant OPPPs: Pseudomonas aeruginosa and Acinetobacter baumannii. It was found that pipe-material (PVC or PVC combined with iron or copper) profoundly impacted the water chemistry in a manner that dictated disinfection efficacy. In Chapter 5, we applied shotgun metagenomic shotgun sequencing to evaluate effects of the combination of pipe material and disinfectant type on the wider microbial community, especially their ability to select for or reduce ARGs. In Chapter 6, we used CMPRs and metagenomic sequencing in a study comparing Dutch drinking water practices to our prior testing in an American system. Dutch drinking water is of interest because of lack of historical use of disinfectants was hypothesized to result in a microbial community that is relatively depleted of ARGs or mobile genetic elements, which can enhance spread of ARGs as disinfectants are applied. Generally, it was found that OPPPs required higher doses of disinfectants for inactivation than the general microbial community, sometimes concentrations approaching the regulatory limits in the US (e.g., 4 mg/L of total chlorine). Even successful reductions were modest, typically ~1-log, and failed to eliminate either P. aeruginosa or A. baumannii. Moreover P. aeruginosa, A. baumannii, and non-tuberculous mycobacteria varied substantially in their preference for pipe material and susceptibility to disinfectants. We found that disinfectants tended to increase the relative abundance of OPPPs, ARGs, and mobile genetic elements. Disinfectants were sometimes associated with net increases in levels of these pathogens and genes when applied at low levels (e.g., 0.1 mg/L of monochloramine), which effectively acted to reduce competition from less resistant and non-pathogenic taxa. When a low dose of monochloramine was applied to PVC CMPRs in the US, we estimated from metagenomic sequencing data that this water contained roughly 100,000 cells per milliliter of taxa known to contain pathogenic members. The Dutch drinking water exhibited more diverse microbial communities and lower relative abundances of taxa containing pathogens. ARGs were two times proportionally more abundant in CMPRs operated in the US without disinfectant than in the corresponding CMPRs operated in the Netherlands. The findings of this dissertation can help to optimize the application of in-building disinfectant addition for addressing concerns related both to OPPPs and antibiotic resistance. The studies herein highlight the necessity of developing comprehensive OPPP and antibiotic resistance control strategies that emphasize not just disinfectant dose, but other key control parameters such as contact time, hydraulics, and temperature. The functional diversity of OPPPs, antibiotic resistant bacteria, and the background premise plumbing microbiome further necessitates broad, holistic programs for monitoring and control. / Doctor of Philosophy / Efforts to provide safe drinking water face two emerging threats: the rise of pathogens that thrive in the plumbing environment that delivers water to the tap and the rise of antibiotic resistance. In the US and many other parts of the world, opportunistic pathogens are the predominant agents responsible for disease spread by tap water. Opportunistic pathogens tend to infect aged or immunocompromised individuals (hence, 'opportunistic') and grow well in in-building plumbing. Globally, antibiotic resistance is on the rise and becoming a fundamental threat to modern medicine. Pathogenic bacteria become resistant to antibiotics used to treat infections when they acquire antibiotic resistance genes (ARGs), which can happen either by mutation or from other resistant bacteria sharing ARGs. Overuse or misuse of antibiotics can impose selection pressure that stimulates horizontal gene transfer and enhance survival of bacteria that are resistant. Prior studies have suggested that under some circumstances, disinfectants used to control pathogens in drinking water can also select for antibiotic resistant bacteria. Thus, the overarching goal of this research was to optimize the type and dose of disinfectant used, depending on building-level factors such as pipe material, for effectively controlling proliferation of both opportunistic pathogens and antibiotic resistance. This dissertation largely focuses on in-building plumbing systems, which are home to potentially tens of thousands of bacterial cells per milliliter of water or per square centimeter of internal pipe surfaces. These bacteria interact not only with each other and other microbes, but also with features of the plumbing environment, such as the water chemistry or the pipe materials. Building plumbing systems are highly intricate ecosystems that can undermine the effectiveness of disinfectants provided by utilities. One major contribution of this research is the development of the convectively-mixed pipe reactors (CMPRs) as a simple and easy-to-use test system that recreates combinations of features of interest encountered in in-building plumbing. We applied the CMPRs to study two common residual disinfectants (chlorine and monochloramine) supplied by water utilities, and two other disinfectants (chlorine dioxide and copper-silver ionization) which are commonly dosed by building operators, especially in hospitals and other buildings housing individuals susceptible to infection. These four disinfectants were applied to CMPRs consisting of PVC, copper, and iron pipe. Chemical, culture, and DNA methods were used to understand how these disinfectants affected the microbes and their ecology. We then took the opportunity to set up CMPRs in the Netherlands, where there has been no historical exposure to chlorine because their water quality regulations emphasize limiting nutrients in the water and elevating the hot water line temperatures as means to control microbial growth. The CMPRs effectively produced worst-case plumbing scenarios, where opportunistic pathogens were especially difficult to control through residual disinfection. Dosed disinfectants tended to be no longer measurable in the water after five hours. The CMPRs also showed that the disinfectant most effective for one pathogen could be the least effective for another. If doses were applied near regulatory limits, the concentrations of pathogens and antibiotic resistance genes decreased. However, opportunistic pathogens tended to survive better than background populations of bacteria. Bacteria carrying ARGs also survived some disinfectant conditions better as well. Thus, if doses were applied at levels that could inactivate some microbes, but not the opportunistic pathogens, pathogen abundances sometimes increased. These results were largely confirmed in the experiment with Dutch drinking water. Here, chlorine appeared to be more problematic than monochloramine in terms of enriching pathogens and antibiotic resistance. We also noted that Dutch waters garnered more diverse microbial communities, with fewer DNA markers for pathogens and antibiotic resistance. In general, this research takes a key step towards optimizing application of residual disinfectants for control of both opportunistic pathogens and antibiotic resistance. Because disinfectants can have negative impacts on drinking water microbial communities when supplied insufficiently, it is important that the other features of in-building plumbing, such as the selection of pipe material or the hydraulics, facilitate disinfectants reaching all portions of plumbing and at the necessary concentrations. It is recommended that the selection process for disinfectant type and dose considers the plumbing materials and other conditions such that disinfection can be aimed towards controlling multiple opportunistic pathogens, which can vary in their susceptibility, and antibiotic resistance.

As climate change exacerbates water scarcity and alters available water and fertilizer resources, it is vital that take appropriate measures to ensure sustainable treatment of water, wastewater, and other waste streams that are protective of public health and support recovery and reuse of water and nutrients. The overarching theme of this dissertation is the advancement of next-generation DNA sequencing (NGS) and computational tools for achieving these goals. A suite of relevant fecal and environmental opportunistic pathogens are examined using both culture-based and NGS-based methods. Of particular concern to this research was not only the attenuation and inactivation of pathogens, but also ensuring that optimal treatment approaches reduce antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Key systems that were the focus of this effort included nutrient reuse (wastewater-derived biosolids and cattle-derived manure), water reuse, and drinking water systems disrupted by a major hurricane. A field study was carried out to survey a suite of pathogens from source-to tap in six small drinking water systems in Puerto Rico six months after Hurricane Maria. The study revealed that pathogenic Leptospira DNA was detected in all systems that were reliant on surface water. On the other hand, Salmonella spp. was detected in surface and groundwater sources and some distribution system waters both by culture and PCR. The study provided comparison of molecular-, microscopic-, and culture-based analysis for pathogen detection and highlighted the need for disaster preparedness for small water systems, including back-up power supply and access to chlorination as soon as possible after a natural disaster. A second field-study examined wastewater derived solids across an international transect of wastewater treatment plants in order to gain insight into the range of ARG concentrations encountered. It was found that, while total ARGs did not vary between treatment or continent of origin, clinically-relevant ARGs (i.e., ARGs encoding resistance to important classes of antibiotics used in humans) were significantly higher in solids derived from Asian wastewater treatment plants. Estimated loading rates of ARGs to soil under a scenario of land application were determined, highlighting in all cases that they are orders of magnitude higher than in the aqueous effluent. Livestock manure, derived from control cattle and cattle undergoing typical antibiotic treatment, and corresponding composts were also evaluated as common soil amendments in a separate study. In this study, the amendments were applied to two soil types in a greenhouse setting, in order to compare the resulting carriage of ARGs on a root (radish) versus leafy (lettuce) vegetable. Remarkably, radishes were found to harbor the highest relative abundance of total ARGs enumerated by metagenomics, even higher than corresponding soils or manures. Although the total microbial load will be lower on a harvested vegetable, the results suggest that the vegetable surface environment can differentially favor the survival of ARBs. The role of wastewater and water reuse treatment processes in reducing ARB and ARGs was also investigated at field-scale. Two independent wastewater treatment plants both substantially reduced total ARG relative and absolute abundance through biological treatment and settling according to metagenomic analysis. The subsequent water reuse treatment train of one system produced water for non- potable purposes and found further reduction in ARGs after chlorination, but a five hundred percent increase in the relative abundance of ARGs in the subsequent distribution system. In the second plant, which employed a membrane-free ozone-biologically-activated carbon-granular activated carbon treatment train for indirect potable reuse, there were notable increases in total ARG relative abundance following ozonation and chlorination. However, these numbers attenuated below background aquifer levels before recharge. Culture-based analysis of these systems targeting resistant ESKAPE pathogens (Escherichia coil, Staphylococcus aureus, Klebsiella spp., Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus spp.) indicated similar trends as the metagenomic ARG analysis for both systems, but was challenged by sub-optimal media for wastewater samples and low confirmation rates, limiting statistical analysis. In order to advance the application of NGS, molecular, and associated bioinformatic tools for monitoring pathogens and antibiotic resistance in environmental systems, newly emerging methods and field standards for antibiotic resistance assessment were also evaluated. Hybrid assembly, the assembly for both short and long metagenomic sequencing reads, were assessed with an in silico framework in order to determine which available assemblers produced the most accurate and long contigs. Hybrid assembly was found to produce longer and more accurate assemblies at all coverages by reducing error as compared to short read assembly, though the outputs differed in composition from long read assembly. Where it is possible, it is beneficial to sequence using both long- and short-read NGS technologies and employ hybrid assembly, but further validation is recommended. Genome resolved metagenomics has also emerged as a strategy to recover individual bacterial genomes from the mixed metagenomic samples though this is often not well validated. In order to address this, genomes were assembled from reclaimed water systems and were compared against whole-genome sequences of antibiotic resistant E.coli isolates. Metagenome-derived genomes were found to produce similar profiles in wastewater treatment plant influents. A final theme to this dissertation addresses the need to standardize targets, methodologies, and reporting of antibiotic resistance in the environment. A systematic literature review was conducted on assays for the enumeration of key ARGs across aquatic environments and recommendations are summarized for the production of comparable data. In sum, this dissertation advances knowledge about the occurrence of pathogens, ARB, and ARGs across aquatic and agricultural systems and across several countries. Advances are made in the application of NGS tools for environmental monitoring of antibiotic resistance and other targets and a path forward is recommended for continued improvement as both DNA sequencing technologies and computational methodologies continue to rapidly advance. / Doctor of Philosophy / Understanding bacteria in our engineered systems is critical to ensuring drinking water, recycled water, and manure-derived soil amendments are safe for downstream applications. As novel approaches for assessing bacteria are developed, standardized methods and evaluations much be developed to ensure that sound conclusions are made that can appropriately inform policy and practice for the protection of public health. This dissertation focuses on combining bacterial culture and DNA sequencing methods for the study of pathogens (i.e., disease-causing organisms) and antibiotic resistance (i.e., ability of some bacteria to survive antibiotic treatments) in agricultural manure management, water reuse, and drinking water systems. Additionally, this work sought to advance emergent metagenomic analysis tools, which provides a new and potentially powerful pathogen and antibiotic resistance monitoring approach through direct extraction and sequencing of DNA from environmental samples. Antibiotic resistance is a global health challenge and it has been widely recognized that wastewater and agriculture are key control points. When antibiotics are ingested by people or livestock, they select for resistant bacteria in the gut. Mitigation efforts are needed, particularly at wastewater treatment plants and on farms, to ensure that excreted antibiotics and resistant bacteria do not further propagate and pose a risk. However, additional challenges such as climate change have spurred the need for more efficient use of our water and nutrient resources. In this work I examined how nutrient and water reuse treatment methods affect antibiotic resistant bacteria and antibiotic resistance genes using DNA sequencing as well as culture-based methods. In order to assess agricultural practices, a systems approach was conducted at the greenhouse scale to identify key control points to stem the spread of antibiotic resistance when vegetables are grown in soils amended with cattle-derived manure fertilizers. Along the food production chain, vegetables (i.e., radish and lettuce) were found to harbor higher proportions of bacteria carrying antibiotic resistance genes, although the estimated numbers of these bacteria were lower. Solids from an international transect of wastewater treatment plants (Sweden, Switzerland, USA, India, Hong Kong, Phillippenes) were examined because they are also foten used as soil amendments. DNA sequencing of these solids revealed that total measured antibiotic resistance genes did not vary between treatment or continent of origin. Calculations were made to determine the range of total hypothetical outputs of ARGs if the biosolids are land applied. Wastewater reuse systems were also examined using culture and metagenomic DNA analysis so that living pathogens could be compared alongside the total (dead and alive) antibiotic resistance genes. While standard wastewater and subsequent water reuse treatments were found to reduce the absolute numbers of antibiotic resistance genes and bacteria in a treatment plant producing water for non-potable reuse (i.e., irrigation), increases in culturable resistant pathogens and antibiotic resistance genes were apparent in the distribution system (i.e., in the pipes conveying treated water to the point of use). Similar reductions in antibiotic resistant bacteria and resistance genes were also seen in a plant using more advanced treatment (ozonation paired with biofiltration) to produce water suitable for indirect potable reuse via aquifer recharge, but there were indications that ozone and chlorine can increase the proportion of antibiotic resistant bacteria. Finally, genomes were recovered from the metagenomic sequencing analysis and were compared to sequenced culture isolates to validate the capabilities of metagenomic analysis to re-assemble genomes at the strain level, which is often required for pathogen confirmation. Pathogens were also assessed in disrupted drinking water systems in Puerto Rico after Hurricane Maria. Small scale systems that were disrupted by the storm were sampled to identify if pathogens were measurable six months after the hurricane. This work revealed that genes attributed to pathogenic Leptospira were detected in all surface water reliant systems while Salmonella spp. were detected by culture and DNA methods, but only in the source surface and groundwaters, not in the distribution systems delivering water to from the treatment site to the tap. This research also contributed to the advancement of big data analysis pipelines as well as to the standardization of methods to ensure that data produced across studies are comparable. Hybrid assembly, an emergent method that combines both short and long metagenomic DNA sequences generated by different technologies to more accurately recover genomes, was found to improve reliability and accuracy of algorithms aimed at reassembling DNA fragments. Antibiotic resistance is a global challenge, but without standardized methodologies for environmental monitoring, it will be difficult to compare measurements across countries and treatment processes in order to identify effective mitigation strategies. A critical literature review was conducted on assays for the enumeration of key antibiotic resistance genes across aquatic environments so that comparable data can be generated. This will be critical to tap into the tremendous volumes of antibiotic resistance monitoring data being generated around the globe to help identify trends and inform solutions. Collectively, this dissertation advances knowledge about the occurrence of pathogens, antibiotic resistant bacteria and antibiotic resistance genes across aquatic and agricultural systems while also critically evaluating emerging methods for the detection of antibiotic resistance in the environment.

The building plumbing microbiome has important implications, especially in terms of its role as a reservoir and conduit for the spread of opportunistic pathogens (OPs), such as Legionella pneumophila. This dissertation applied next-generation DNA sequencing tools to survey the composition of building plumbing microbiomes and assessed hypothetical factors shaping them. A challenge to identifying key factors shaping building plumbing microbiomes is untangling the relative contributions of influent water quality, provided by drinking water utilities, and those of building-level features, such as pipe materials. To this end, standardized pipe rigs were deployed at the treatment plants and in distal portions of the water distribution system at five water utilities across the eastern U.S. Source water and treatment practices appeared to be the overarching factors shaping the microbial taxonomic composition at the tap, with five key water chemistry parameters identified (total chlorine, pH, P, SO42- and Mg2+). Hot water plumbing is of particular interest because OPs tend to proliferate in warm water environments and can be inhaled in aerosols when showering. Two identical lab-scale recirculating hot water rigs were operated in parallel to examine the combined effects of water heater temperature set point, pipe orientation, and water use frequency on the hot water plumbing microbiome. Our results revealed distinct microbial taxonomic compositions between the biofilm and water phases. Importantly, above a threshold of 51 °C, water heater temperature, pipe orientation, and water use frequency together incurred a prominent shift in microbiome composition and L. pneumophila occurrence. While heat shock is a popular means of remediating L. pneumophila contamination in plumbing, its broader effects on the microbiome are unknown. Here, heat shock was applied to acclimated lab-scale hot water rigs. Comparison of pre- versus post- heat shock samples indicated little to no change in either the microbial composition or L. pneumophila levels at the tap, where both water heater temperature and water use frequency had the most dominant effect. Overall, this dissertation contributes to advancing guidance regarding where to most effectively target controls for OPs and also advances research towards identifying the features of a 'healthy' built environment microbiome. / PHD

Private wells are understudied potential sources of opportunistic pathogen (OP) infections. OPs, including Legionella and Mycobacterium, are of particular concern for immunocompromised individuals and are known to proliferate in drinking water systems. Much of our knowledge surrounding OP occurrence and growth in drinking water relates to municipal drinking water systems, which primarily use surface water sources and are always treated with primary and secondary disinfection in United States. However, OP occurrence and growth in private wells is not well understood and it is unclear how the knowledge developed in municipal systems will translate to private well systems with rare and infrequent exposure to chemical disinfectants. In addition, because private wells are more susceptible to microbial contamination than municipal systems, the impact of flooding on OP occurrence is of particular concern. Two private well field surveys were conducted to document the incidence of OPs in private well systems. One survey conducted in North Carolina private wells with no history of recent flooding was focused on molecular and culture-based detection of Legionella spp. and Legionella pneumophila. The other survey was a broader molecular (i.e., DNA-based) characterization of the incidence of Legionella spp., L. pneumophila, Mycobacterium spp., Mycobacterium avium (the most commonly nontuberculous mycobacteria associated with disease), and Naegleria fowleri in private wells with recent history of flooding (i.e., Hurricanes Harvey and Irma in 2017, or the Great Louisiana Floods in 2016, extending to Texas, Florida, and Louisiana). All samples in both studies were analyzed for total bacterial 16S rRNA genes, indicator bacteria (e.g., total coliform and Escherichia coli) and inorganic constituents. Information about well system characteristics were obtained through questionnaires sent to participating residents. Widespread detection of OP DNA markers were noted in the flooded well survey. Legionella spp. (detectable in 50-100% of well waters, depending on the flood event) and Mycobacterium spp. (detectable in 13.2-45.0% of well waters) were the most commonly detected among the OPs targets. At the genus level, L. pneumophila (7.9-65.5%) and M. avium (7.9-32.5%) were less commonly detected, but still highly variable. It is not possible to judge whether these OP levels were elevated as a result of the flooding because the sampling was carried out as an emergency response and background levels were not previously tested. Also of interest was whether well characteristics could predict OP levels, including well depth, well type, or treatment. However, none of these emerged as significant predictors of OP detection frequency or levels. Similarly, these OP DNA markers were not elevated in homes reporting submerged wellheads or system damage, suggesting that detection of these OPs is more dependent on the groundwater that supplies these private wells than influx of contaminated surface water. The incidence of DNA markers pertaining to N. fowleri, the "brain eating amoeba" that causes rare incidences of primary amebic meningoencephalitis (PAM), tended to be lower (5.0-12.7%) than that of other OPs targeted, but was more frequently detected in wells reporting submerged well heads, suggesting its occurrence was related to contamination from flood water. A positive association between total bacteria and occurrence of both Legionella spp. and Mycobacterium spp., was observed in private wells of all surveyed areas, contrary to observations in municipal drinking water systems with secondary disinfectant residuals. On the other hand, Legionella reportedly has an optimal growth range of 20-42 °C in municipal systems and recent surveys of municipal systems reported a strong association between Legionella spp. and temperature that was not observed for private wells in this study. We speculate that the essentially "infinite" water age and lack of disinfectant for well water, may contribute to these differences relative to municipal water supplies. The results presented in this work are likely an overestimation of OPs numbers in private wells, as molecular detection of OPs does not distinguish between live and dead cells. In addition, sample sizes were limited by laboratory throughput and budget. Identifying key variables impacting the occurrence of OPs in private wells, given that our study shows that these pathogens are relatively common, might someday help limit the risk of infections. / Master of Science / Non-fecal pathogens that are capable of growth in drinking water systems and causing illness primarily in individuals with compromised immune systems are referred to as opportunistic pathogens (OPs). OPs of concern in drinking water systems in the United States include members of the Legionella genus (i.e., Legionella spp.) and Mycobacterium genus (i.e., Mycobacterium spp.), as well as Naegleria fowleri (also known as “the brain-eating amoeba”). Much of our knowledge surrounding OP occurrence and growth in drinking water relates to municipal drinking water systems. Under the right conditions, OPs have the ability to grow in municipal drinking water systems and in building plumbing systems despite the use of disinfectants. However, OP occurrence and growth in private wells is not well understood and it is unclear how the knowledge developed in municipal systems translates to private well systems that rarely utilize chemical disinfectants. In addition, because private wells are more susceptible to microbial contamination than municipal systems, the impact of flooding on OP occurrence is of particular concern. Two private well surveys were conducted to document the incidence of OP DNA markers and culturable OPs in private well systems. The first survey was conducted in North Carolina private wells with no history of recent flooding and focused on quantification of DNA markers for Legionella spp. and L. pneumophila, as well as culturable L. pneumophila. The second survey was conducted in flood-impacted private wells in vi Texas, Florida, and Louisiana following Hurricanes Harvey and Irma in 2017 and the Louisiana Floods of 2016, quantifying five DNA markers for OPs (Legionella spp., L. pneumophila, Mycobacterium spp., M. avium, and N. fowleri). All water samples in both studies were also analyzed for total bacterial numbers (i.e., total number of copies of a gene present in all bacteria), certain bacteria that indicate environmental and fecal contamination (e.g. total coliform bacteria and Escherichia coli), and inorganics. Information about well system characteristics was obtained through questionnaires sent to participating residents. While it was not possible to sample before the flooding events and determine the background detection rates, this survey found that detection of the target OPs in the flood-impacted wells was widespread, but highly variable. Both culture-based and DNAbased testing methods were used to verify the results because each approach has strengths and weaknesses. Detection of DNA markers indicates the genetic material of the organism being tested for is present and detects DNA of organisms that are both dead and alive. Thus, DNA detection may overestimate the presence live (and infectious) pathogens. Detection of culturable pathogens indicates the organism is alive, but only detects pathogens that readily grow on culture media. There may be other pathogens in the water that do not readily grow on the media. Thus culture may underestimate the occurrence of pathogens. DNA markers for Legionella spp. (detectable in 50-100% of well waters) and Mycobacterium spp. (detectable in 13.2-45.0% of well waters) were the most commonly vii detected among the targets in this study. The detection of DNA markers for L. pneumophila (7.9-65.5%) and M. avium (7.9-32.5%) was less common. There were no private well characteristics, such as well depth, well type, or treatment that emerged as significant predictors of these OP detection or levels. Similarly, these OPs were not elevated in recently flooded homes reporting submerged wellheads or system damage. Thus, detection of these OPs was found to be widespread and sporadic. Detection rates of N. fowleri, which causes rare incidences of primary amebic meningoencephalitis (PAM), DNA was lower (5.0-12.7%) than other OPs, and was also not related to private well characteristics. However, N. fowleri DNA was more frequently detected in wells with wellheads that were submerged due to flooding than in wells with unsubmerged wellheads, as were total coliform bacteria (an indicator of environmental contamination) and total bacterial numbers. This demonstrates concern that submergence of wellheads during flooding is a concern for the introduction of microbial contamination in private wells. This work also explored two trends characteristic of municipal systems that were not observed in these surveys of private wells. First, positive associations between overall bacterial numbers and DNA markers for both Legionella spp. and Mycobacterium spp. were observed in private wells in all surveyed areas. This is contrary to what has been reported for overall bacterial numbers in municipal drinking water systems with a secondary disinfectant residual. Second, Legionella has been known to have an optimal growth range of 32 to 42 °C (90 to 108 °F) in municipal systems and recent surveys of municipal systems with both a free chlorine and chloramine secondary disinfectants viii showed a strong association between Legionella spp. and temperature. These associations were not observed in private wells in this work. Continuous disinfection treatment in municipal drinking water systems may eliminate certain microbes, lowering overall levels of bacteria, while OPs may persist by resisting disinfection, resulting in no correlation between total bacteria and OPs. Private wells do not generally use continuous disinfection and represent low-nutrient environments where naturally occurring OPs can grow alongside other bacteria. The results of this study are likely an overestimation of OPs infection risk associated with private wells, as infection can only be caused by live OPs, which cannot be directly determined by measuring DNA markers. In addition, sample sizes were limited by laboratory throughput and budget. Identifying key variables that impact the occurrence of OPs in private wells is necessary to minimize the risk of associated infections linked to private wells. This work provides strong preliminary evidence that OP occurrence in private wells is relatively commonplace. Science-based options for at-risk (e.g., immunocompromised) individuals to decrease their exposure to OPs in private well water need to be developed.

Magister Scientiae Dentium - MSc(Dent) / The initiation of highly active antiretroviral therapy has shown to result in successful suppression of viral replications followed by an increase in CD4 lymphocytes, a partial recovery of T-cell specific immune responses and decrease susceptibility to opportunistic pathogens. Aim: The aim of the present study was to determine the prevalence of oral lesions in patients before and after undergoing HAART. Methods: The study design was longitudinal and descriptive, investigating the prevalence of oral lesions presenting in HIV/AIDS patients at baseline, 3 and 6 months after taking HAART. A convenience sample size of 200 participants was targeted. Results: 210 HIV positive patients participated at baseline. At 3 months, 96 (46%) and at 6 months, 52 (25%) were available for review respectively. At baseline 210 HIV positive patients were recruited into the study from three hospitals. Two infectious disease hospitals belonged to the City of Harare and the other is a government hospital. Just over two thirds were female (64.3%) and the age ranged as follows: 21-30 (17%); 31-40 (44%); 41-50 (26% and 51-60 (9%).Discussion: HAART appears to be effective in reducing the prevalence of oral lesions in persons with AIDS likely due to the immunological reconstitution. Oral candidiasis remains the most prevalent oral opportunistic infection in immuno-suppressed individuals and hence its important predictive value for immuno-suppression defined as CD4-cell count level <200/mL of blood. All oral lesions strongly associated with HIV infection with the exception of non-Hodgkin’s lymphoma were diagnosed at baseline. CD4 cell count level increased after initiation of HAART. T-lymphocytes that are formed after the introduction of HAART may not provide sufficient protection against some lesions like parotid gland disease and HPV conditions (planar warts). HAART failure was detected in some patients who had negative CD4-cell count at 6 months compared to the baseline parameters. Conclusions: HIV-positive patients experience oral pain during the course of their disease, eating, drinking and swallowing. Further longitudinal studies are required in order to ascertain the prevalence of these lesions at three and six months and the effect of HAART.

In an era of pervasive water stress caused by population growth, urbanization, drought, and climate change, limiting the dissemination of microbial contaminants of emerging concern (MCECs) is of the utmost importance for the protection of public health. In this dissertation, two important subsets of MCECs, opportunistic pathogens (OP) and antibiotic resistant genes (ARG), are studied across several compartments of the urban water cycle, including surface water, stormwater, wastewater, recycled water, and potable water. Collectively, this dissertation advances knowledge about the occurrence of OPs and ARGs across these water systems and highlights trends that may be of value in developing management strategies for limiting their regrowth and transmission. Field studies of two surface water catchments impacted by stormwater runoff demonstrated the prevalence of ARGs in urban stormwater compared to pristine, unimpacted sites, or to days when no precipitation was recorded. The role of wastewater reuse in transmitting OPs and ARGs was also investigated. Traditional tertiary wastewater treatment plants producing water for non-potable use were found to be largely ineffective at removing ARGs, but plants using advanced oxidation processes or ozonation paired with biofiltration to produce direct potable reuse water were highly effective at removing ARGs. Non-potable reclaimed water consistently had greater quantities of sul1, a sulfonamide ARG, and Legionella and Mycobacterium, two OPs of significant public health concern, present than corresponding potable systems. Limited regrowth of OPs and ARGs did occur in simulated premise (i.e., building) plumbing systems operated with direct potable reuse waters, but regrowth was comparable to that observed in systems fed with potable water derived from surface or groundwater. Advancements were also made in understanding the role of several hypothesized driving forces shaping the antibiotic resistome in natural and engineered water systems: selection by antimicrobials and other compounds, horizontal gene transfer, and microbial community composition. Finally, whole-genome and metagenomic characterization were applied together towards profiling L. pneumophila in clinical and water samples collected from Flint, Michigan, where an economically-motivated switch to an alternative water source created conditions favorable for growth of this organism and likely triggered one of the largest Legionnaires' Disease outbreaks in U.S. history. / PHD

Opportunistic pathogens (OPs) are of special concern for immunocompromised populations and are known to grow in both drinking water and reclaimed water (i.e., non-potable recycled water) distribution systems, with aerosol inhalation and other non-ingestion exposures that are not addressed by existing regulatory frameworks. Factors enabling the growth of OPs in water distribution and premise (i.e., building) plumbing systems distributing reclaimed and other water sources systems are poorly understood especially for the emerging OP, Naegleria fowleri (i.e. brain-eating amoeba). Three phases of investigation were carried out to identify factors that facilitate the growth of OPs in main distribution and premise plumbing systems, with particular attention on reclaimed water systems, aging water mains, and private well systems. Phase one examined the role of biological treatment to remove organic carbon and disinfectant type on the occurrence of OPs during distribution of reclaimed water. Laboratory-scale simulated reclaimed water distribution systems were employed to systematically examine the effects of prior granular activated carbon (GAC) biofiltration of the water; chlorine, chloramines, or no disinfectant, and water ages ranging up to 5 days. The second and third phases of research explored the role of nitrification, iron corrosion, and disinfectant on the growth of N. fowleri both in municipal drinking water from a city grappling with aging water infrastructure and untreated private well water. Results from the simulated reclaimed water distribution systems suggested that biologically-active GAC filtration may unintentionally select for specific OPs, contrary to expectations and experiences with oligotrophic conditions in potable water systems. While GAC biofiltration was associated with lower total bacteria and Legionella spp. gene markers, there were no apparent benefits in terms of other OPs analyzed. Similarly, disinfectant treatments successful for controlling OPs in potable water were either ineffective or associated with increased levels of OPs, such as Mycobacterium spp. and Acanthamoeba spp., in the reclaimed water examined. In the potable water study, it was possible to recreate conditions associated with growth of N. fowleri in the aged main distribution system from where the water for the experiment was collected; including corroding iron mains, nitrification, and disinfectant decay. While the effects of nitrification could not be confirmed, there was a clear association of iron corrosion with N. fowleri proliferation. The role of iron was explored further in what, to the author's knowledge, was the first study of N. fowleri in private wells. Analysis of 40 wells found correlations between N. fowleri and stagnant iron levels, further supporting the hypothesis that iron corrosion or iron encourages the growth of N. fowleri, and, because wells are not routinely disinfected, not necessarily as a result of promoting disinfectant decay. As this study took place following a major flooding event, it provided insight not only into how surface water contamination may influence private well water microbial communities, but also added to the understanding that current recommendations for disinfecting private wells are inadequate and standards should be implemented to aid homeowners in the event of flooding. This exploratory research illuminated several factors influencing the OP growth in a range of water systems. Identifying key variables that control growth is crucial to improving the safety of these systems. / MS

Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Contamination of groundwater that is suitable for drinking is of growing concern as the water supply of South Africa is becomingincreasingly limited. This is especially the case in the North West province, with its semi – arid climate and variable rainfall patterns. The aim of the study was to evaluate the microbiological and physico – chemical qualities of groundwater obtained from selected DWA (Department of Water Affairs) monitoring boreholes in the Mooi River and Harts River catchment areas. Physico -chemical parameters included temperature, pH, electrical conductivity (EC), salinity, total dissolved solids (TDS), sulphate and nitrate concentrations. Physical parameters were measured using a calibrated submerge-able multimeter and chemical parameters using specialised kits and a spectrophotometer. Microbiological parameters included heterotrophic plate counts and total and faecal coliform enumeration. Membrane filtration and culture based methods were followed for enumeration of bacteria. During the identification procedures multiplex PCR for E. coli identification and 16S rRNA gene sequencing for identification of heterotrophic plate count bacteria and amoeba resistant bacteria were used. For antibiotic resistance, the Kirby- Bauer (1996) disk diffusion method was used. During the warm and wet season high electrical conductivity and salinity were observed in the Trimpark (65.3 mS/m; 325 ppm), School (125.1 mS/m; 644 ppm), Warrenton (166.9 mS/m; 867 ppm) and Ganspan (83.3 mS/m; 421 ppm) boreholes. Warrenton borehole had a high sulphate level (450 mg/l) as well. High chemical oxygen demand was observed in the Blaauwbank (62 mg/l) and Warrenton (98.5 mg/l) boreholes. In the dry and cold season similar observations were made for the various boreholes. Electrical conductivity and salinity levels remained high for the Trimpark (70.1 mS/m; 427.5 ppm), School (127 mS/m; 645 ppm), Warrenton (173.3 mS/m; 896.5 ppm) and Ganspan (88.1 mS/m; 444.5 ppm) boreholes. Nitrate levels for the Trimpark (14.1 mg/l) and School (137 mg/l), as well as sulphate levels for the Warrenton (325 mg/l) borehole were also high. Total coliforms, faecal streptococci and HPC bacteria were enumerated from water samples from all boreholes, except Blaauwbank where no faecal streptococci were enumerated. Faecal coliforms were enumerated from 5 of the possible 7 boreholes during a warm and wet season (Trimpark – 42 cfu/100ml; School – 2 cfu/100ml; Cemetery – 175 cfu/100ml; Warrenton – 3.84 x 10³ cfu/100ml; Ganspan – 1.9 x 10³ cfu/100ml). Indicator bacteria (FC, TC, HPC) exceeded target water quality ranges (TWQR) for drinking water in each case. During the cold and dry sampling season, faecal coliforms were enumerated mainly from the Trimpark (11 cfu/100ml) borehole. Total coliforms, faecal streptococci and HPC bacteria were enumerated from all the boreholes, except for Blaauwbank that contained no faecal streptococci or total coliforms. Enumerated indicator bacteria levels again exceeded TWQR for domestic use. Total coliform counts for the Pad dam borehole, however, complied with TWQR for domestic use. Identified E. coli were resistant to Erythromycin, Cephalothin and Amoxicillin and susceptible to Ciprofloxacin. Escherichia coli isolated from the Mooi River catchment shared the same antibiotic resistance phenotype. The most abundant HPC bacterial genus identified was Pseudomonas spp. (7 isolates). Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying degrees of antibiotic resistance were observed. Generally, the same pattern between the same genera were observed. All HPC isolates were resistant to Cephalothin and Amoxicillin and a lower degree Erythromycin and Streptomycin. The most abundant amoeba resistant bacteria was identified as Pseudomonas spp. Other isolates included Alcaligenes faecalis and Ochrobactrum sp. and Achromobacter sp. All of these are opportunistic pathogens, except for Achromobacter. Resistance to more antibiotics (Streptomycin, Chloramphenicol, Cephalothin, and Amoxicillin) was observed in ARBs compared to HPC (Cephalothin, Amoxicillin) from bulk water from the same borehole. The water of all the aquifers sampled is of very poor physico - chemical or microbiological quality or both. Water may be used for irrigation or livestock watering only in the case where these boreholes comply with TWQR for said purposes. Results obtained indicate that the groundwater is faecally contaminated. Amongst the bacteria, opportunistic pathogens displaying various degrees of antibiotic resistance were frequently isolated. These results indicate health risks if untreated groundwater is consumed. Therefore groundwater needs to be treated before distribution especially if the water is for human consumption. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.