Bacterial resistance is a formidable 21st-century global public health threat. If left unaddressed, we risk moving toward a “post-antibiotic era.” While resistance is a natural consequence of antibiotic use, the rate at which pathogenic bacteria have evaded multiple classes of drugs has markedly outpaced the introduction of new ones. New antibiotics are desperately needed to fill this void. Macrolides are one of the safest and most effective drug classes in medicine; however, resistance has compromised efficacy. To date, three generations have been developed with only the lattermost targeting bacterial resistance. Single next-generation macrolides will not keep pace with the inevitable onset of resistance; thus, there is a critical need to greatly accelerate the procurement of multiple future-generation antibiotics to tackle both current and future resistance mechanisms. My research is to meet this need by designing, synthesizing, and evaluating a novel, future-generation macrolide antibiotics that will serve as an armamentarium to be individually deployed on demand. In the previous research in Andrade group, we synthesized and evaluated various desmethyl ketolide analogs. The fact that 4-desmethyl telithromycin was fourfold less potent than telithromycin against A2058G mutants indicated replacing the 4-Me with hydrogen (i.e., desmethylation) to avoid a steric clash with the 2-amino group of G2058 was insufficient in rescuing bioactivity. Guided by MD simulation, we concluded a logical, superior alternative strategy was the replacement of the 4-Me group with one possessing a smaller vdW radius and capable of establishing favorable interactions with both wild-type and A2058G mutant ribosomes. Specifically, we reasoned that 4-fluoro solithromycin would be ideal candidate. The hypothesis was that the 4-fluoro moiety would engage in dipole-dipole interactions (C-F---H) with the exocyclic 2-amino group of guanine, which is based on accumulated evidence that strategic placement of organofluorine can strongly impact potency, selectivity, and physicochemical properties. In addition, the axially disposed of 4-fluorine would provide conformational stabilization from a gauche effect with the vicinal O5 group. The novel synthetic routes to unexplored desosamine analogs at the C3’-amino substituent to the macrolide antibiotic would play a role in bioactivity and resistance. Hofmann reaction was employed to execute the same 2,3-epoxide ring opening method without removing desosamine and re-glycosylating. This markedly reduces the steps, time, and cost involved in preparing novel desosamine-modified analogs. Significantly, this route enables the first synthesis of N,N’-disubstituted desosamine analogs from an epoxide, which was utilized to prepare novel analogs of clarithromycin. The application of in situ click chemistry toward the discovery of novel macrolide antibiotics first required the synthesis of suitable azide and aryl alkyne reactants. Alkyne partners were procured by commercial vendors or chemical synthesis. We targeted two logical, validated positions to tether the side chains, specifically N11 on the macrolactone and N3’ of desosamine. The first (N11) has been the most utilized. Moreover, extensive structure-activity relationships have revealed a four-carbon tether is ideal. Based on the solithromycin−E.coli X-ray structure, I designed, synthesized, and evaluated dehydro solithromycin, which possesses an (E)-alkene in the side-chain. The use of an unsaturated side chain would conformationally preorganize the bi-aryl side chain in order to pay the entropic penalty and thus favorably contribute to the overall binding. An insightful observation made from MD simulationed ribosomes bound with to solithromycin revealed that the interaction of the side-chain includes H-binding as well as π-stacking. The hypothesis was that employing tethered side-chains bearing motifs that maximize H-bonding and π-stacking would be superior antibiotics for treating resistant bacterial strains bearing erm¬-mediated N6 methyl and dimethylated ribosomes. To test this hypothesis, we developed various analogs with different alkynes by introducing different functional groups at the 3 and 5 positions on the aromatic ring. Another desosamine sugar modification is bis-azide. To date, the use of a two side chain strategy has not been reported. To access the requisite bis-azides, we employed a tactic the oxidative demethylation and alkylation of desosamine to afford bis-click solithromycin analogs. / Chemistry

Enterococcus faecalis is a commensal bacterium, which upon acquisition of virulence factors on mobile genetic elements can cause sepsis, urinary tract infections and endocarditis. E. faecalis isolates can be multi-drug resistant and have been implicated in the dissemination of antibiotic resistance genes to other genera. Although the host range of pheromone inducible conjugative plasmids is restricted to Enterococci, they often carry transposons, which are capable of transposing into the chromosome of other genera. The plasmid pCF10 contains the antibiotic resistance gene tetM on a conjugative transposon Tn925. Tn925 is a Tn916-like plasmid and is capable of pCF10-independent conjugative transfer to multiple bacterial species at low levels. Biofilms are communities of bacteria growing within a matrix. In biofilms, bacteria are more difficult to kill because of their lower susceptibility to antibiotics. In hospital settings, biofilms can grow on medically implanted devices, catheters or even human tissue. In mixed species biofilms, antibiotic resistances are able to be transferred through horizontal gene transfer from E. faecalis to other bacterial species. In mixed species biofilms, it has been show that Tn925 can transpose into S. aureus at rates of 10-8 by Ella Massie Schuh. Using static mixed species biofilms, the transfer of tetM from E. faecalis to S. aureus was studied, hoping to better understand the underlying mechanisms. The goal of these studies was to determine if residence on pCF10 increased the transfer frequency of Tn925 in mixed species biofilms. Mixed species biofilms containing E. faecalis (pCF10) and S. aureus (pALC2073aPSM) were established and pCF10 conjugation was induced with pheromone cCF10. Transfer of Tn925 / Biomedical Sciences

<p> Glycopeptide Antibiotics (GPAs) such as vancomycin are often used clinically as antibiotics of last resort against infections due to Gram-positive bacteria that are resistant to more commonly used antibiotics such as methicillin. The clinical emergence of vancomycin resistant enterococci (VRE) and vancomycin resistantS. aureus (VRSA) necessitates methods to evade this resistance. </p> <p> GP As consist ofa heptapeptide backbone that is cross-linked to create a pocket that binds the D-Alanyl-D-Alanine terminus of peptidoglycan intermediates, inhibiting strengthening ofthe cell wall and resulting in susceptibility to osmotic stress. Resistance to GPAs occurs when D-Ala-D-Lactate replaces D-Ala-D-Ala and the GPA pocket can no longer bind effectively. In order to create novel binding pockets, we must understand the specificity ofthe P450 monooxygenase enzymes that have been shown to catalyze the cross-links. The 4 P450-encoding genes ofthe GPA A47934 biosynthetic cluster of Streptomyces toyocaensis as well as genes encoding electron transport proteins necessary for P450 function from Streptomyces coelicolor were cloned in Escherichia coli for heterologous expression and characterization. One P450, StaJ was purified and shown to bind CO as expected using spectrophotometric tests. </p> <p> The genes responsible for GP A resistance are regulated by a two component regulatory system consisting ofa sensor kinase (VanS) and a response regulator (V an.R). In order to probe the events leading to VanS autophosphorylation and ultimately resistance activation we utilize a series of GP A derivatives harbouring the photo labile group benzophenone as well as the fluorescent and affinity moieties BODIPY and biotin. Benzophenone permits light controlled covalent binding of the GP A to proteins that bind them while BODIPY allows fluorescence detection and biotin allows enrichment and detection by Western analysis. We report that this system was insufficient to clearly identify vancomycin binding proteins due to background signals despite multiple rounds of troubleshooting. It must be our conclusion that under the conditions tested, there are no proteins that bind the GP A derivative used in this study. </p> / Thesis / Master of Science (MSc)

As the problem of resistance increases in the current health care system, new solutions to this problem are not emerging at a similar rate. The ability to discover novel antibiotics, and modify existing antibiotics, is competing with highly evolving resistance profiles. An alternate solution to this problem may be to search for inhibitors of these resistance mechanisms and pairing them with current antibiotics. Proof of this hypothesis lies in the great success of P-lactamase inhibitors already in the clinic. Inhibitors may be created using synthetic methods, however searching for inhibitors found in the natural environment may lead to a greater success. For example, bacteria in their natural setting must cope with constant exposure to antibiotics secreted by both themselves and by other species. As well, bacteria must be able to handle encounters with other species that are resistant to their own defense mechanisms. With this in consideration, it is possible that these bacteria have already established an ability to challenge resistance encountered in their own environment, such as through the secretion of compounds that inhibit these mechanisms. Screening of such inhibitors can be done against purified resistance elements or via cell-based screens with resistant bacteria. The focus of this research was to develop expression systems which contain inducible antibiotic resistance genes to be used for whole-cell screening for inhibitors of antibiotic resistance. The expression systems studied were pSWEET, for use in the Gram positive bacterium Bacillus subtilis, and pETcoco, for use in the Gram negative bacterium Escherichia coli. It was found that the pSWEET expression system integrated into the B. subtilis chromosome at unspecified locations and was not an ideal system for the proposed screen. pET coco holds promise as a suitable expression system but at this point in time it requires further examination to ensure plasmid stability and reproducibility of results. Therefore further examination of these two systems is needed if they are to be used in a screen for inhibitors, and a search for substitute systems must be undertaken. / Thesis / Master of Science (MSc)

The growing problem of antibiotic resistance has prompted the need for new and novel antimicrobial therapies. The bacterial cell division pathway holds great promise for the development of novel broad-spectrum antibiotics as the majority of the proteins are essential for viability. The wealth of information regarding bacterial cell division has come from studies of the model organisms Escherichia coli and Bacillus subtilis. Although much has been elucidated regarding this pathway, the functions of many individual proteins remain unsolved. An important model organism for the investigation of cell division is Streptomyces coelicolor. The mycelial Streptomyces are sporulating, Gram-positive bacteria that grow in long branching networks of filamentous cells much like filamentous fungi. The normally essential process of cell division is dispensable for growth and viability of S. coelicolor. More interestingly, there are two different modes of cell division in this organism, one for vegetative growth and one is utilized for synchronous septation during sporulation. It is still unclear how developmental regulators control this switch, but advancements in fluorescence microscopy have shed some insight into the cell division process by allowing direct visualization of many cellular components and their dynamics. To better understand bacterial cell division and its regulation in S. coelicolor, three additional fluorescent proteins (FPs), including m.RFP, CyPet and YPet, have been established in this work. An m.RFP shuttle vector was constructed and the utility of m.RFP was tested by translationally fusing it to a tip-localizing protein, DiviVA. This work demonstrated that m.RFP is functional and an efficient marker for localized proteins. Also, established in this work is a two-colour fluorescence reporter system, which includes the fluorescent proteins CyPet and YPet that can be used to study co-localization and protein-protein interactions within cells. Future plans are to use co-localization of FP fusions and fluorescence resonance energy transfer (FRET) between CyPet and YPet to investigate the assembly of protein complexes within the cells, such as those involved in cell division. These studies will reveal critical information that is needed for the development of drugs that have novel mechanisms of action. / Thesis / Master of Science (MSc)

The Streptomyces are invaluable as a natural source of antibiotics and other bioactive compounds used in medicine and agriculture. S. coelicolor is the model streptomycete, and is studied for its complex secondary metabolism and multicellular life cycle. The subject of this work is bldB, a gene essential for development and antibiotic production in S. coelicolor, and one of its many homologues, located in the abaA antibiotic regulatory locus. The aim was to study the transcriptional regulation of bldB using a luminescent reporter, and investigate the role of each of the genes in the abaA cluster in regulation of antibiotic production, in order to understand the function and mechanism of action of bldB and its homologues. Individual deletion of each of the four genes in the abaA cluster resulted in varying effects on production of the antibiotic CDA. The bldB homologue, SCO0703, was shown to be a positive regulator of CDA, as the null mutant was severely defective in CDA production. It was found that bldB is expressed in most other bld developmental mutants, with the exception of bldD. There was no direct interaction observed between BldD and the bldB promoter, and possible mechanisms of indirect regulation are proposed. / Thesis / Master of Science (MSc)

Genetic and biochemical studies were conducted on S. toyocaensis NRRL 15009, a gram-positive spomlating filamentous bacterium, and producer of the glycopeptide antibiotic A47934. This compound is structurally similar to the clinically important antibiotic vancomycin, and the recent spread of vancomycin-resistant enterococci (VRE) in North American hospitals has driven the need for new glycopeptides with enhanced activities. Studies were aimed at developing an understanding of the mechanism of A47934 biosynthesis inS. toyocaensis NRRL 15009, as well as the mechanism of resistance employed by this organism. Two cosmid clones, containing a partial A47934 biosynthesis gene cluster on a total of65 kilobases of S. toyocaensis NRRL 15009 chromosomal DNA, were isolated for study. Preliminary sequencing indicates the presence of several genes predicted in glycopeptide assembly, such as peptide synthetases and glycosyltransfe~ases. Furthermore, using a oligonucleotide probe designed to identify D-alanine-D-alanine ligases, an 8.1 kilobase chromosomal fragment was isolated from S. toyocaensis NRRL 15009 and found to contain genes very similar to VRE vanH, vanA and vanX. Phylogenetic analysis of the predicted products of these genes showed them to be more similar to the VRE enzymes than any other in each enzyme class. These genes were also found in the vancomycin producer A. orienta/is C329.2 and several other glycopeptide antibiotic producing organisms. Not only does this imply that these organisms employ a mechanism of resistance similar to clinical VRE, it also suggests that these organisms may have been the source of the VRE genes. The enzymes VanHst and DdlN were studied in some detail and found to have biochemical properties similar to their corresponding VRE enzymes VanH and VanA, respectively. Given that the latter group of enzymes has physical properties that have impeded detailed analysis of enzyme mechanism, these new enzymes could find use as model systems in drug development programs. / Thesis / Doctor of Philosophy (PhD)

Introduction and Justification: Diarrheal disease is a leading cause of death in children in low- and moderate-income countries. Food, food waste, and water are all vehicles that can promote the spread of diarrheal disease-causing bacteria like Campylobacter, Salmonella enterica, and E. coli. Resistance to commonly used antibiotics is on the rise, making them difficult to manage. This study aimed to determine prevalence and antibiotic resistance profiles of Campylobacter, S. enterica, and E. coli isolated from food, food waste, and water samples obtained from the Chobe Region of Botswana. In addition, the survival of two common pathogens, E. coli and C. jejuni, on kale, a type of leafy green commonly consumed raw, was determined. Methods: Samples were collected from the Chobe region of Botswana in 2022 including water from the local river, food (produce, beef, pork, and poultry) from local vendors, and food scraps from the landfill. Food samples were enriched in the appropriate selective media: Brilliant Green Bile Broth for E. coli, Bolton Broth for Campylobacter, and Rappaport Vassiliadis Broth for S. enterica. Water samples were collected using modified USEPA methods1103.1 and 1604, E.coli isolation was performed by plating on RAPID E.coli2 agar and incubation at 37°C for 2h and 44°C for 16-22h. Campylobacter, S. enterica, and E. coli were isolated from meat, poultry, and water samples before being sent to Virginia Tech, while enriched bacterial pellets from the produce were shipped for screening and isolation at Virginia Tech. E.coli were confirmed by PCR detecting the phoA gene (all E. coli), and classified as pathogenic through screening for the eae (present in enterohemorrhagic and enteropathogenic E.coli), stx1 and stx2 (present in enterohemorrhagic E. coli) and est1b ( present in Enterotoxigenic E.coli) genes. Campylobacter isolates were confirmed using a genera-specific PCR while S. enterica isolates were confirmed using invA primers. These enrichment and primer sets were tested as part of a study to determine the survival of E. coli O157:H7 and C. jejuni on kale during a 21-day shelf life. E. coli and S. enterica isolates were subjected to antibiotic resistance testing using the Kirby-Bauer Disk Diffusion method. Results: Methods for detection of inoculated E. coli O157:H7 on kale indicated survival for the majority of the shelf-life (up to 19 d), in comparison, C. jejuni was undetectable by day 13 using enrichment and PCR or plating. From the Botswanan samples, E. coli was isolated from 20% of produce, 49% of meat, and 84.7% of water. Salmonella was only isolated from produce samples (2.4%, 7/294). Resistance was uncommon among the Salmonella isolates with only one isolate being resistant to chloramphenicol. No Campylobacter were isolated from the screened produce, meat, or food waste. E. coli resistant to 3 or more classes of antibiotics (MCR) were identified in 15.5% of produce, and 22.2% of meat isolates. Isolation of E. coli or Salmonella from meat was not associated with a particular food type. In contrast, isolation of E. coli was more common from certain types of vegetables and fruits. Antibiotic-resistant E. coli were isolated more commonly from beef, poultry, and pork than from produce. Multi-class resistant E. coli were isolated from fruits, greens, soil associated, and above ground associated vegetables, beef, and poultry. Water samples were collected from the same time period as the food samples. E. coli isolation, especially pathogens (based on eae presence) was more frequent from environmental water samples collected during the wet season compared to the dry season. Water samples collected during periods of increased rainfall were more likely to contain E. coli isolates, especially pathogens. S. enterica and Diarrheagenic E. coli isolates, especially MCR isolates, pose a significant risk of illness to consumers. Strategies to reduce the circulation of these pathogens in foods and water sources are needed. / Master of Science in Life Sciences / People can get sick with diarrheal diseases after consuming contaminated food and water. These illnesses are difficult to treat and control when the bacteria causing them are resistant to antibiotics. Campylobacter, Salmonella, and diarrheagenic E. coli are three types of bacteria that can cause illness from food and water. These illnesses disproportionately affect people, especially children, in low-and moderate-income countries like Botswana. Little is known about the prevalence of Campylobacter, Salmonella, and diarrheagenic Escherichia coli in Botswana. This study aimed to determine the prevalence of these microorganisms as well as how resistant they are to different types of antibiotics. Samples from produce, beef, poultry, pork, and recreational water sources were collected in the Chobe region of Botswana over the course of 2022. Food samples were collected from different vendors and food scraps were obtained from the landfill. E. coli and Salmonella were isolated out of meat and water samples in Botswana while produce samples were shipped as mixed cultures to Virginia Tech where E. coli, Salmonella, and Campylobacter isolation or confirmation was done. Once the target bacteria were isolated, their resistance to certain antibiotics was tested. Salmonella was only found in produce from samples collected during October-December. No Campylobacter was found from produce, meat, or food waste. More E. coli was isolated from fruit or vegetable food waste collected from the landfill than from produce bought at local vendors. E. coli was obtained from meat purchased from local vendors more often than samples from the landfill. E. coli was found more often in water when there was more rainfall. E. coli that causes illness was also more likely to be obtained during the wet seasons. Resistant E. coli that could not be killed by the screened antibiotics, were classified as multi-drug resistant when it was resistant to more than three antibiotics. Rainfall, season, and the food source influenced if E. coli isolates were likely to be multi-drug resistant. While more research is needed to determine how these bacteria are moving in the environment and gaining resistance to antibiotics, the findings of this study show they are present in the environment and require further research.

Since penicillin was discovered in 1928, antibiotic usage in human and veterinary medicine and prevalence of antibiotic resistant bacteria (ARB), has been increasing. While antibiotics and antibiotic resistance genes (ARGs) naturally occur in soils, increasing abundances of ARGs correlate with increased antibiotic usage in agricultural settings. When livestock are treated with antibiotics, the antibiotic compounds, ARB, and ARGs can enter soil via manure excreted onto pastures or applied to other fields as fertilizer, thereby spreading antimicrobial resistance (AMR) in the environment. In addition to human health implications, increased AMR has negative impacts on ecosystem services such as carbon and nitrogen cycling. While many studies have researched antibiotic persistence in agricultural systems and their impacts on soil microbial communities, there are still significant knowledge gaps around the long-term effects of antibiotic exposure in soils, how those impacts differ among soils, and how elements of AMR may differentially transport through soil. To address these knowledge gaps, our objectives were to 1) examine the impact of multi-year repeated additions of manure from cattle administered antibiotics on soil microbial communities, 2) determine the interactive effects of soil moisture and type on soil microbial communities exposed to antibiotics and manure, and 3) differentiate between vertical transport of AMR in the form of viable ARB or ARGs in extracellular plasmids. Our results demonstrate that soil bacterial community structures were consistently altered by 3-year additions of manure from cattle administered antibiotics compared to soil amended with antibiotic-free manure. Furthermore, ARG abundances were higher in soils with manure additions compared to soil without manure, although this was true regardless of whether the cattle were administered antibiotics, suggesting that manure and antibiotic impacts on soil microbial communities can persist over multi-year of repeated manure applications. Additionally, in microcosms, effects of manure from cattle administered antibiotics on ARG abundances, microbial community structures, respiration, and nitrogen pools in soil were seen across multiple soil types and moisture contents, suggesting environmental conditions can alter how manure and antibiotics impact microbial community structure and nutrient cycling. Finally, ARB flowed readily through saturated soil, but were also detectable in the top 5 cm of soil columns. However, ARGs on extracellular plasmids did not flow through soil columns and were not detected in soil, indicating that extracellular DNA does not persist or transport through the soil to any meaningful degree. Overall, these results indicate a nuanced approach is required to mitigate the environmental spread of AMR. Soil management strategies for addressing the AMR crisis should consider the broader context of manure management, as high ARG abundances can come from application of manure from antibiotic-free cattle, and soil microbial communities in individual environments may have varied responses to manure antibiotic exposure. Furthermore, the transport of AMR through soil is complex and dynamic, as elements of AMR may transport differently through soil and require separate consideration in modeling and management. Future AMR management practices that consider diverse factors that affect persistence and spread of AMR in the environment can help protect livestock productivity and maintain the efficacy of antibiotics to protect human and animal health. / Doctor of Philosophy / Antibiotics are an important tool used to fight infections in humans, pets, and livestock. As antibiotics are used more frequently, the bacteria they target are more likely to develop resistance to the antibiotics, leading to increasing cases of infections that are harder to treat and higher risk. Antibiotic resistance can persist and spread in multiple forms, including the antibiotic compounds themselves, as antibiotic resistant bacteria (ARB), or as the genetic material that encodes for antibiotic resistance genes (ARGs). In agricultural systems, when livestock are treated with antibiotics they can excrete the antibiotics, along with ARB and ARGs, in the manure, which is then applied to land as fertilizer. In addition to the associated health risks, the spread of antibiotic resistance impacts microscopic bacteria and fungi in the soil, which are important for recycling nutrients for plants and maintaining ecosystem health. The overall goal of this dissertation was to gain a better understanding of how manure from cattle given antibiotics impacts these bacteria and fungi when manure is applied to the soil. The specific objectives were to 1) look impacts after long-term (multiple years) of manure addition, 2) examine how bacteria and fungi might respond differently to antibiotics in soils of different type or with different amounts of water, and 3) determine if ARGs that exist as free genetic material outside of living bacteria can be moved through the soil with flowing water in the same way as living bacteria. Results showed that while the composition of bacterial and fungal communities in the soil vary from year to year, adding manure with and without antibiotics had both caused different and consistent changes on the composition of bacterial communities. There were also higher concentrations of ARGs in soil that had manure added, however antibiotics in the manure did not cause ARGs to increase further, suggesting that even antibiotic-free manure can impact the spread of antibiotic resistance. Experimental work also demonstrated that the soil type and water content of soil can alter how bacteria and fungi respond to antibiotics in manure. The composition of bacterial and fungal communities, their activity rates, and the amount of nitrogen – an important plant nutrient with availability that is strongly affected by microbial activity – all differed with soil type and water content. Thus, while antibiotic resistance antibiotic resistance can cause measurable changes in soil across a range of environmental conditions, it is also likely to persist and spread in different ways in different environments. Finally, when water containing elements of AMR was added to soil, ARB were shown to both move through the soil easily and remain near the top of soil. In contrast, ARGs contained on genetic material outside of living cells did not move through the soil and were broken down within a few days, suggesting that antibiotic resistance likely spreads through living bacteria more than genes outside of cells. Overall, this work highlights the complexity of understanding the role of environmental transmission in the antibiotic resistance crisis and demonstrates the need for nuanced management approaches that take specific environments and conditions into account.

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.