Legionella pneumophila and other opportunistic pathogens of drinking water pose important problems at the interface of biology, environmental engineering, public health, and governance. In this thesis, I explore the molecular mechanisms permitting survival of L. pneumophila in built water systems is the nature of its physiology under different conditions and different life-phases. In the first chapter, I discuss how various physiological states of L. pneumophila affect the propensity for survival and virulence in relation to drinking water environments. This literature review should provide a perspective important for designing controlled laboratory experiments rooted in a robust understanding of how phenotype dictates experimental results. In the second chapter, I describe sequence and phylogenetic analyses performed to investigate the presence of a type 1 secretion system and virulence factor throughout the Legionella genus. While this system was previously believed to be conserved to L. pneumophila, our analysis indicates that this system is well-distributed throughout the Legionella genus, blurring the lines between "pathogenic" and "non-pathogenic" species. In the third chapter, I report the secretome of endemic Flint, Michigan L. pneumophila in corrosive water, simulating the environmental impact of the Flint Water Crisis on local L. pneumophila populations. Our results from this study have expanded the secretome of L. pneumophila, provided insight on mechanisms it may employ to resist stress in water, and created several novel lines of inquiry at the merging frontier of biochemistry and environmental engineering. / Master of Science in Life Sciences

Legionella pneumophila is a pathogen that can proliferate in premise (i.e., building) plumbing and, when aerosolized during water use, infect the lungs of exposed individuals and cause a deadly form of pneumonia known as Legionnaires' disease. Given that it is one of the primary sources of tap-water associated disease throughout much of the world, this organism has been the subject of intense research, ranging from aiming to understand key aspects of its physiology that allow it to proliferate in premise plumbing, to the specific virulence factors that make it so infectious to humans. The work presented here starts with a comprehensive review of published studies related to the L. pneumophila proteome, i.e., the set of expressed proteins associated with a given strain under a given set of environmental conditions, showing how the field has progressed in parallel to improvements in mass spectrometry technologies and how proteomics can be used as a tool to understand this unique and important organism. Copper is a natural antimicrobial that can be present in drinking water due to passive release from copper pipes or intentionally dosed (e.g., copper-silver ionization systems) for microbial control. However, some L. pneumophila strains have recently been found to exhibit copper resistance, an adaptive process that is not fully understood at the physiological level. Chapter Two describes the copper survivability of three outbreak-associated strains of L. pneumophila and examines the copper-induced proteome of QC1, a strain found to display high resistance to copper. Pairwise comparisons of the proteomes of copper-resistant and copper sensitive strains indicated that L. pneumophila QC1 adapts to copper exposure via the induction of redox and metal homeostasis proteins, while concomitantly inducing motility and pathogenesis related proteins, suggestive that copper induces a search for a host protozoan strain for protection. In 2014 and 2015, Flint, Michigan experienced the largest per capita community-wide Legionnaires' Disease outbreak in US history. The outbreak was associated with a change in the source of the municipal drinking water supply from Detroit water, which was sourced from the Great Lakes and subject to appropriate corrosion control, to the Flint River, which was not appropriately controlled for corrosivity. The underlying drivers of this outbreak have been debated and include: elevated iron in the water serving as a nutrient for L. pneumophila, diminished chlorine in the water due to reactions with iron, reduced copper in the water due to shifts in pH influencing release from copper pipes, and shifts in potentially key components of the microbial community. In Chapter Three of this dissertation, we employ controlled microcosm studies to establish a fundamental understanding of interactive effects of pipe material and water of varying iron bioavailability (ferric chloride, ferrous chloride and ferric pyrophosphate) on the microbial community and its relationship with L. pneumophila numbers. The combination of copper pipes and Flint River water decreased the diversity of the microbial community to a larger degree than copper pipes with Detroit water, implying greater copper bioavailability in the former condition. Several Order were found to be significantly associated with high or low numbers of culturable L. pneumophila recovered from the microcosms. Most notably, the Order Pseudomonadales was significantly associated to the reactors with low culturable L. pneumophila. This order contains Pseudomonas species known to inhibit the growth of L. pneumophila. The findings reported in this dissertation can be used to develop more informed management practices for drinking water systems to reduce the risk of Legionnaires' Disease outbreaks associated with premise plumbing. Specifically, 1) copper might be inducing a more pathogenic form of copper resistant L. pneumophila, 2) the use of corrosive control in municipal water systems goes beyond the influence on lead and copper pipes, but also on the microbial community, which in part influences L. pneumophila, and 3) there are organisms, such as Pseudomonadales species, associated with environments with low culturable L. pneumophila which might be introduced to the drinking water systems as probiotics. / Doctor of Philosophy / Legionella pneumophila is a microbe found in drinking water plumbing systems. This organism causes Legionnaires' Disease, a severe form of pneumonia that particularly affects immunocompromised individuals. Due to its health and economic impact, there are worldwide efforts to understand the biology of this organism, from the conditions that allows it to grow in the drinking water plumbing, to the specific components that allows it to infect humans. In this dissertation, we first review the published studies related to the L. pneumophila proteome, a powerful tool used to functionally describe biological organisms. This first chapter showed how proteomics can be used to understand this unique and important organism. In the next chapter we studied how copper metals may influence the proteome of L. pneumophila. Copper pipes have been extensively used to control the growth of microorganisms in drinking water systems, however some studies have reported that copper may promote the growth of L. pneumophila. In this chapter, we showed that a copper resistant strain of L. pneumophila adapts to copper exposure by inducing motility and pathogenesis related proteins, suggesting that it might be more infectious. In the last chapter of this dissertation, we investigated the combined effect of pipe material and water chemistry, on the microbial community and its relationship with L. pneumophila. The combination of copper pipes and a more corrosive water decreased the diversity to a larger degree, in comparison to the other evaluated conditions. Several organisms were also identified to be significantly associated with the high or low culturable L. pneumophila. This is of particular interest because they might be used as potential probiotics to control the growth of L. pneumophila. The findings reported in this dissertation can help to better understand the significance of water chemistry and pipe material, particularly copper pipes, for the purpose of reducing risk of Legionnaires' Disease outbreaks associated with drinking water systems.

abstract: Sustainability and environmental justice, two fields that developed parallel to each other, are both insufficient to deal with the challenges posed by institutional environmental violence (IEV). This thesis examines the discursive history of sustainability and critiques its focus on science-based technical solutions to large-scale global problems. It further analyzes the gaps in sustainability discourse that can be filled by environmental justice, such as the challenges posed by environmental racism. Despite this, neither field is able to contend with IEV in a meaningful way, which this thesis argues using the case study of the Flint Water Crisis (FWC). The FWC has been addressed as both an issue of sustainability and of environmental justice, yet IEV persists in the community. This is due in part to the narrative of crisis reflected by the FWC and the role that knowledge production plays in that narrative. To fill the gap left by both sustainability and environmental justice, this thesis emphasizes the need for a transformational methodology incorporating knowledge produced by communities and individuals directly impacted by sustainability problems. / Dissertation/Thesis / Masters Thesis Sustainability 2019

As waterborne disease originating in potable water plumbing systems (such as Legionnaires' Disease and Nontuberculous Mycobacterial (NTM) infections) continue to increase, it is important to better understand the cause(s), responsible parties and interventions to prevent disease. This dissertation begins with a literature review characterizing the propensity of building (premise) plumbing to enhance or diminish opportunistic pathogen growth, including Legionella. It then holistically examines the problem at the field, bench and pilot scale by first discovering problems with lead and Legionella in Flint, MI, during an event popularly referred to as the Flint Water Crisis in 2014-2016. Four years were then spent simulating critical factors hypothesized to have triggered the Legionella outbreak in residences and in a large hospital in Flint. In parallel with that work, pilot scale rigs were operated for several years, to examine the important role of water heater system design and operation on energy efficiency, hot water delivery, and Legionella. The first chapter literature review is entitled "Critical Review of the Propensity of Premise Plumbing Pipe Materials to Enhance or Diminish the Growth of Legionella and Other Opportunistic Pathogens." It examines the complex environments found in premise systems, focusing primarily on the role of pipe materials. The effects of metallic (copper, iron) and plastic pipe materials on opportunistic pathogens and Legionella include their effect on nutrient availability, disinfectant levels, and the composition of the broader microbiome. Design, configuration, and operation are also examined in terms of their potential for influencing opportunistic pathogens. This chapter demonstrates that pipe materials have the potential to stimulate or inhibit pathogen growth, dependent on circumstance and water chemistry. This chapter will be submitted to the journal Pathogens. The field study in this work first predicted, discovered and then exposed problems with lead and Legionella in Flint, Michigan. A citizen science project that sampled Flint water in August 2015, demonstrated a city-wide problem with water lead exceeding the EPA limit of 15 µg/L after corrosion control was interrupted. Follow-up sampling events between August 2015 and August 2017 demonstrated that the switch back to the original water source and addition of enhanced corrosion control in October 2015, dramatically reduced lead, copper and iron levels flowing into consumer homes. Entitled "Evaluating Water Lead Levels During the Flint Water Crisis," this work was published in Environmental Science and Technology in 2018. After our Virginia Tech team's work helped expose a Legionnaires' disease outbreak that killed twelve people and sickened nearly one hundred individuals, the started to explore possible links between corrosion control, plumbing materials and disinfection that could help explain the trajectory of disease in Flint and elsewhere. Three separate experiments were performed using bench-scale simulated glass water heaters. Two of the studies attempted to simulate what occurred in Flint homes before, during and after the water crisis in relation to factors that either encouraged or discouraged Legionella growth, while the third examined the more benign Blacksburg tap water and a broader range of influential plumbing conditions. The first study entitled "Copper Pipe, Lack of Corrosion Control, and Uncontrolled pH Influenced the Trajectory of the Flint Legionnaires' Disease Outbreak," determined that the very low pH levels in summer 2015 and interruption of phosphate corrosion control, could cause explosive growth of Legionella in PEX plumbing held at warm temperature, without disinfectant and with constant mixing. Under the same conditions copper pipe had antimicrobial properties that markedly reduced Legionella in our experiments. This work has been submitted for review to Environmental Science and Technology. The second companion study conducted at a higher pH, without mixing and with trace chlorine, found 2.5 log10 lower levels of Legionella compared to the worst-case conditions in the aforementioned study, demonstrating the importance of mixing and traces of chlorine. Higher levels of disinfectant and the presence of copper pipe also enhanced control of Legionella. This manuscript is titled "Interactive Effects of Copper Pipe, Stagnation, Corrosion Control, and Disinfectant Residual Influenced Reduction of Legionella pneumophila during Simulations of the Flint Water Crisis," and it has been published in Pathogens. The third simulated glass water heater study examined the disinfection of opportunistic pathogens in the presence of six different premise plumbing materials or conditions in Blacksburg tap water. Generally speaking, all of the premise plumbing materials reduced disinfection of opportunistic pathogens compared to a control condition with glass surfaces. Chlorine decay was catalyzed by iron pipe, warmer temperature and the presence of organic matter, increasing the persistence of Legionella. Magnesium anodes in particular, encouraged much higher Legionella growth compared to all other materials. This work titled "Chlorine and Chloramine Disinfection of Legionella spp., L. pneumophila, and Acanthamoeba Under Warm Water Premise Plumbing Conditions," has been submitted to Microorganisms. Results of a six-year pilot study titled "Elucidating the Role of Water Heater System Configuration in Energy Efficiency, Consumer Comfort and Legionella Proliferation," examined different types of residential-sized water heater systems with plastic pipes including: a standard tank system with water stagnant between uses, a recirculating tank system with flowing water between uses, and an on-demand system which only heated water and had flow during use. Considering the volume of water in each tank between 38 and 47 ° C as a measure of Legionella growth risk, with a heater setpoint at 48 °C (118 °F) the recirculating system had 90% of its volume at risk daily compared to only 24% of the standard system volume. The on-demand system used a minimum of 10% less energy than the standard tank, and 50% less energy than the recirculating tank, and had one tenth of the volume at risk of growing Legionella than either tank system. In fact, it was only by contriving a system to keep distal lines artificially warmed to above room temperature, that Legionella growth could occur in the on-demand system, whereas it rose to 107 L. pneumophila MPN per liter in a normally operating recirculating system. On the other hand, the on-demand heaters were repeatedly subject to mechanical malfunction during the study, and had difficulty delivering water at the desired temperature and flow rates versus traditional tank systems. This manuscript will be submitted to Water Research. / Doctor of Philosophy / Recent water crises in Flint, Michigan and Legionnaires' Disease outbreaks in Flint, New York City, and Quincy, Illinois have demonstrated the need to better understand the cause(s), responsible parties, and interventions required to prevent waterborne diseases. As waterborne disease originating in building plumbing systems (premise plumbing), such as Legionnaires' Disease and Nontuberculous Mycobacterial infections, continue to increase each year, the burden on healthcare systems and impact on public health also grows. In this dissertation, a literature review, a field study of water in Flint, small-scale laboratory studies, and residential-sized water heater systems were examined to study interactions between water chemistry, premise plumbing, and disease-causing opportunistic pathogens (OPs) with a focus on Legionella, the OP which causes Legionnaires' Disease. The first chapter literature review is entitled "Critical Review of the Propensity of Premise Plumbing Pipe Materials to Enhance or Diminish the Growth of Legionella and Other Opportunistic Pathogens." It examines the complex environments found in premise systems, and the important role of pipe material selection. The effects of metallic (copper, iron) and plastic pipe materials on opportunistic pathogens and Legionella include their impact on nutrient availability, disinfectant levels, and the composition of the broader microbiome. Design, configuration, and operation of plumbing systems are also examined in terms of their potential for influencing growth of opportunistic pathogens. This chapter demonstrates that pipe materials have the potential to stimulate or inhibit pathogen growth, dependent on circumstance and water chemistry. The field study in this work exposed problems with lead and Legionella in Flint, Michigan, during an event known in the popular press as the Flint Water Crisis 2014-2016. In August 2015, a citizen science sampling campaign demonstrated a citywide lead in water problem. After a federal emergency was declared, follow-up sampling events between August 2015 and August 2017 demonstrated that the switch back to the original water source, enhanced disinfection, and corrosion control under federal direction had reduced lead levels to half of the EPA limit. Additionally, the pipe material installed between each home and the water main (service lines) affected levels of lead and copper in water, with the lowest lead concentrations measured in homes with copper service lines. After our teams' work in Flint helped expose a Legionnaires' disease outbreak that killed twelve people and sickened nearly one hundred other individuals, we started to explore possible links between corrosion control, plumbing materials, and disinfection that could affect the trajectory of disease in Flint and elsewhere. Three follow-up studies, using small glass bottles to simulate water heaters, provided more specific comparisons between water conditions in premise plumbing and OP occurrence. Two studies expanded on phosphate corrosion control, chlorine (disinfectant) decay, and pH-related research questions, which arose during the Flint water crisis field study. The first determined that properly treated drinking water and some mixing could inhibit Legionella growth in copper pipes. The second study found that without mixing, copper could be antimicrobial and reduce Legionella growth even if the water chemistry was slightly altered with higher pH. The third simulated water heater study examined the reduction of OPs using a chlorine or chlorine plus ammonia disinfectant to reduce Legionella in the presence of six different plumbing conditions. The reduction of Legionella with chlorine was inhibited when carbon was increased and in the presence of a magnesium anode rod, a necessary water heater component. A six-year study using a residential-sized water heater system holistically examined three different types of water heater systems with plastic pipes: one using a standard water heater tank with water stagnant between uses, one using a water heater tank connected to a recirculating pump to provide constantly flowing water, and one tankless (on-demand) heater which only heated water and provided flow during use. Using temperature as an indicator of risk for Legionella growth, the recirculating system at a temperature setpoint of 48 °C (118 °F) would be at high risk for Legionella growth (water volume at 38-47 °C or 100-117 °F) in 90% of the tank volume each day, whereas the standard system would only be at high risk in 24% of the tank each day. The on-demand system provided the safest alternative for hot water distribution with virtually undetectable levels of Legionella risk when the pipes were kept at room temperature as per normal operation. The on-demand system also used at least 50% less energy than the recirculating system and 10% less energy than the standard system; however, we were not successful in finding a reliable on-demand system that could also provide hot water at the desired temperature and flow rate.