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 / Population growth, urbanization, drought, and climate change have all driven many U.S. municipalities to utilize alternative water sources, such as recycled wastewater, to offset demand on traditional potable water sources. Many water providers have moved towards a modern paradigm of utilizing multiple available water sources, recognizing the interconnectedness of various components of the urban water cycle, leading to opportunities to improve sustainability, optimize infrastructure use, stimulate economic growth, increase coordination among water agencies, and identify new water resources from which to meet consumer needs. Though advancements in treatment technologies throughout the twentieth century have largely succeeded in eliminating waterborne disease outbreaks associated with contamination of municipal water supplies by fecal pathogens in developed countries, several microbial contaminants of emerging concern (MCECs) have garnered attention. Two major groups of MCECs are considered in this dissertation: antibiotic resistance, including antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG), and opportunistic pathogens (OP), such as Legionella pneumophila, the causative agent of Legionnaires’ Disease. ARB are a rising cause of disease around the world and are a major challenge to modern medicine because they make antibiotics used for treatment ineffective. OPs, the leading cause of waterborne disease in the U.S. and other developed countries, have become prevalent in engineered water systems where low nutrient concentrations, warm water temperatures, and long stagnation times can facilitate their growth. Immunocompromised people, including smokers and the elderly, are especially vulnerable to infection with OPs. The role of the urban water cycle in facilitating the spread of these MCECs is not well understood. Here they were studied across several compartments of the urban water cycle, including surface water, stormwater, wastewater, recycled water (spanning a variety of intended uses, from non-potable to direct potable reuse), and potable water. Field studies were conducted of two watersheds impacted by stormwater runoff, one in the arid Colorado Front Range under conditions of a rare, 1-in-1,000 year rainfall event, and one in the humid climate of southwest Virginia, during three summer storms. Both studies 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. Wastewater treatment plants producing water for non-potable use (i.e. applications such as irrigation, but not for human consumption) were found to be largely inefficient at removing ARGs, and this reclaimed water consistently had greater quantities of the sul1 ARG present than in corresponding potable systems. In these systems, genes associated with the OPs Legionella and Mycobacterium as well as total bacteria were more abundant in reclaimed water than in corresponding potable systems. In more advanced treatment plants utilizing advanced oxidation processes or ozonation paired with biofiltration to produce direct potable reuse water (i.e. water fit for human consumption), ARGs were very effectively removed by treatment, with abundances often found to be higher in corresponding potable waters derived from surface or groundwater. Limited regrowth of ARGs as well as OPs did occur in simulated home plumbing systems operated with these waters, but regrowth was comparable to that observed in systems fed with potable water derived from surface or groundwater. Finally, a study of L. pneumophila in the Flint, Michigan drinking water system during use of an alternative water source that has been identified as a likely cause of two Legionnaires’ Disease outbreaks revealed presence of multiple strains of the OP in the system. Genomic comparisons revealed that strains isolated from hospital and residential water samples were highly similar to clinical strains associated with the outbreaks. Advancements were also made in understanding the role of several hypothesized driving forces in shaping the antibiotic resistome in natural and engineered water systems: selection by antimicrobials and other compounds, horizontal gene transfer, and microbial community composition. Together, these chapters describe an advancement in knowledge regarding the occurrence of OPs and ARGs in a variety of water systems, and highlight trends that may be of value in developing management strategies for limiting regrowth or transmission of these bacteria in various compartments of the urban water cycle.

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