Taxon composition and biodiversity analyses are known powerful parameters for environmental site status and environment diagnosis. Many ecological studies assess taxon composition through traditional species identification and use bioindicator species to evaluate environmental conditions. The recent breakthrough in bulk sample sequencing combined with DNA barcoding has created a new era for environmental monitoring. Metabarcoding approaches are more robust in studying alpha, and beta diversity compare to the DNA barcoding and the conventional method of species identification, particularly for rare and cryptic species. Here we built upon ecological studies of bioindicator species and transferred the traditionally named taxa to DNA-based approaches. We developed a small customized DNA database for biodiversity assessment and taxonomic identification of environmental DNA samples using high-throughput amplicon sequences. It contains macroinvertebrate species that are known as indicators of specific environmental conditions. By implementing this small database into the KRAKEN algorithm for the first time, we were able to assess environmental biodiversity compared to other popular methods of taxonomic classification, especially in polluted environments where the taxonomic composition globally change by the presence of anthropogenic drivers. Our method is incredibly faster, and it requires significantly less computational power in contrast to common homology-based techniques. To evaluate our approach, we have also studied the importance of database’s size and the depth of sequencing in taxonomic classification of high-throughput DNA sequences. / Thesis / Master of Science (MSc) / We developed a small customized DNA database for biodiversity assessment and taxonomic identification of environmental DNA samples using high-throughput amplicon sequences. It contains macroinvertebrate species that are known as indicators of specific environmental conditions. By implementing this small database into the KRAKEN algorithm for the first time, we were able to assess environmental biodiversity compared to other popular methods of taxonomic classification, especially in polluted environments where the taxonomic composition globally change by the presence of anthropogenic drivers. Our method is incredibly faster, and it requires significantly less computational power in contrast to common homology-based techniques.

Part I: Low complexity regions (LCRs) are common motifs in eukaryotic proteins, despite the fact that they are also mutationally unstable. For LCRs to be widely used and tolerated there must be regulatory mechanisms which compensate for their presence. I have endeavored to characterize the relationships and co-evolution of LCRs with the abundance of the proteins that host them as well as the transcripts which encode them. As the abundance of a gene product is ultimately responsible for its associated phenotype, any relationships have implications for the many neurodegenerative diseases associated with LCR expansion. I found that there are indeed relationships. LCRs are more associated with low abundance proteins, but the opposite is true at the RNA level: LCRs encoding transcripts have higher abundance. Investigating the co-evolution of LCRs and transcript abundance revealed that on short evolutionary timescales indels in LCRs influence the selective pressures on TAb. Viewing LCRs through the previously unexplored lens of abundance has generated new results. Results which, together with explorations of information flow and low-complexity in untranslated regions, expand our knowledge of the functional impacts of LCRs evolution. Part II: A commonly encountered problem in DNA sequencing is a situation where the DNA of interest makes up a small proportion of the DNA in a sample. This challenge can be compounded when the DNA of interest may come from many different organisms. Targeted metagenomics is a set of techniques which aim to bias sequencing results towards the DNA of interest. Many of these techniques rely on carefully designed probes which are specific to targets of interest. I have developed a bioinformatic tool, HUBDesign, to design oligonucleotide probes to capture identifying sequences from a given set of targets of interest. Using HUBDesign, and other methods, I have contributed to projects ranging in context from clinical to ancient DNA. / Thesis / Doctor of Science (PhD) / This thesis describes research in two fields: repetitive protein sequences and methods for sequencing the portions of a sample in which one is most interested. In the first part I describe the general properties of repetitive proteins, establish a connection between the presence of repeats in a protein and the amount of that protein which a cell maintains, and show that these two quantities evolve together. This informs our understanding of evolution and regulation with implications for repeat related diseases and further evolutionary research. In the second part I describe a method for selecting short nucleotide sequences which can be used to capture specifically the DNA of organisms of interest, as well as applications of this and other methods. These contributions are widely applicable as targeted sequencing is useful in fields as far apart as clinical sepsis diagnosis and determining the colour of ancient animals.

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.

Necrotic enteritis (NE), a devastating enteric disease caused by Clostridium perfringens type A, contributes to the losses of 6 billion dollars worldwide per year and is currently being considered as a major global threat to the poultry industry. In past decades, it has been well-controlled by ineed antimicrobial growth promoters (AGPs). The withdrawal of AGPs due to antibiotic-resistance concerns resulted in a spike in NE incidence and led to the re-emergence of NE in the modern broiler production system. To unveil the association of toxin genes of C. perfringens, particularly for netB, with clinical NE, a self-designed qPCR primer set targeting netB was developed to qualify and quantify netB in NE-producing and non-NE-producing isolates. The netB was demonstrated to exist in the majority of C. perfringens type A isolates. The presence and the amount of netB were not significantly different between two types of isolate, indicating that those indicators are insufficient to predict an association with the pathogenicity of NE. The virulence of netB is suggested to be expressed or triggered under certain conditions, further promoting NE. A side by side trial was implemented with different combinations of netB-positive C. perfringens (CP1) and two predisposing factors to assess their role in NE development. Both CP1 and predisposing factor(s) are required for consistent NE reproduction, and particularly, Eimeria exerts significant effects on NE induction. The use of CP1 without a predisposing factor failed to induce NE. The severity and incidence of NE were positively correlated with the number of predisposing factors given in the NE induction. Analyzing gut microbiota in chickens challenged with CP1 and/or Eimeria by metagenomic sequencing, significant overgrowth of Clostridium sensu stricto 1, the genus contains C. perfringens, was associated with NE. Eimeria infection precedent to CP1 challenge had a synergistic effect on the overrepresentation. In addition to C. perfringens, the other member under Clostridium sensu stricto 1 was found to participate in NE development. Given supplementary dose of 0.4 kg/ton in feed, lauric acid neither exerted the inhibitory effect against proliferation of Clostridium sensu stricto 1 and C. perfringens nor reduced the incidence and severity of NE.

No description available.

Plant-parasitic nematodes represent a substantial constraint on global food security by reducing the yield potential of all major crops, including soybean (Glycine max L.). The soybean cyst nematode (SCN) (Heterodera glycines I.) is widely distributed across all soybean production areas of the US, and is the major yield-limiting factor, especially in the Midwestern US. Double cropping (DC) is defined as producing more than one crop on the same parcel of land in a single growing season. Compared to conventional single annual crops, DC provides many advantages, including improving soil health, enhanced nutrient provisioning to plants, improvement of soil physical properties, control of erosion, decrease in tillage requirements, and enhanced profitability. In some double-cropping systems, soybean is planted following winter wheat (Triticum aestivum L.), and several reports suggest the potential of wheat to suppress SCN populations. Field trials were conducted from 2017 to 2018 to investigate the effect of wheat on SCN populations in double-cropping soybean. Nine fields with three levels of initial SCN populations (low, moderate, and high) were selected in Illinois. Wheat was planted in strips alternating with strips-maintained weed-free and under fallow over winter and early spring. Soybean was planted in all strips after the wheat harvest. Soybean cyst nematode egg densities were acquired at four time points: wheat establishment, post-wheat/pre-soybean, mid-soybean (R1 growth stage or beginning of flowering), and post-soybean harvest. Wheat strips reduced SCN egg densities compared with fallow strips at the R1 stage (−31.8%) and after soybean harvest (−32.7%). Field locations with noted SCN suppression were selected for a metagenomics study. The structure of fungal communities differed significantly between DC and fallow plots at soybean planting and after harvest (P<0.001). Fungal populations were affected by location at all sampling times and by treatments before planting and after soybean harvest. Several enriched fungal and bacterial taxa in wheat plots, including Mortierella, Exophiala, Conocybe, Rhizobacter spp., and others, were previously reported to parasitize SCN and other plant-parasitic nematodes. Knowing that phytocompounds with potential nematicidal activity are released via the root system of plants, we implemented a gas chromatography–mass spectrometry (GC-MS) pipeline to investigate the profile of phytochemicals in soil samples collected from these fields and reviewed the potential nematicidal activity of compounds with higher concentration in double cropping fields. A total of 51 compounds were detected with the GSMS analysis, 8 with unknown identification. Several compounds, including multiple fatty acids, had greater relative peak areas in double-cropping samples compared to fallow. This multi-approach study provides a better understanding of the mechanisms governing wheat's effect on SCN populations. Rather than a single mechanism, the suppression of SCN in soybean fields double-cropped with winter wheat is potentially linked to enriched microbial communities, increased populations of beneficial organisms, and higher concentrations of phytochemicals with nematicidal activity. This is the first study implementing metagenomics and GCMS to characterize soil microbial and chemical profiles in soybean fields double-cropped with winter wheat. Finally, a set of studies were conducted to evaluate the effect of two seed-applied succinate dehydrogenase inhibitors (SDHI) compounds, fluopyram and pydiflumetofen, on SCN population densities, plant injury, and plant growth. Next-generation sequencing was later employed to identify transcriptomic shifts in gene expression profiles of soybean seedlings treated with fluopyram and pydiflumetofen. Fluopyram reduced both SCN egg and cyst counts in comparison to pydiflumetofen and the untreated control. RNA expression patterns of seed treatments clustered by sampling time (5 DAP vs 10 DAP). Multiple DEGs identified in soybean seedlings treated with fluopyram are reported to be linked to systemic resistance, suggesting a role of systemic resistance on the suppression of SCN by fluopyram. The non-target inhibition of soybean succinate dehydrogenase genes by fluopyram may be the origin of the phytotoxicity symptoms observed and potentially the cause of the systemic resistance activation reported in the current study. To our knowledge, this is the first report of systemic resistance being activated by fluopyram in addition to the suppression of soybean succinate dehydrogenase (SDH) and ubiquinone oxidoreductase genes. This work helps to elucidate the mechanisms of suppression of SCN by fluopyram

<p>Surface mining of Alberta’s oil sands has led to significant land disturbance, making reclamation and sustainable development of this resource one of the largest challenges facing the industry today. Syncrude Canada Ltd. has developed an innovative technique to reclaim composite tailings (CT) through constructed wetland landscapes and is currently investigating the viability of a pilot-scale freshwater fen built over sandcapped CT. Unpredicted by abiotic geochemical modelling of CT behaviour, a minor episode of hydrogen sulfide (H₂S) gas release was encountered during the initial stages of fen construction indicating microbial activity was likely involved in H₂S generation within CT. This thesis investigates the S geochemistry of CT with depth and employed 454 pyrosequencing and functional enrichments to characterize the associated microbial communities in the first S biogeochemical study of oil sands CT. Porewater H₂S was detected extensively throughout the deposit with background levels ranging from 14 – 23 µM and a maximum of 301.5 µM detected at 22-24 m of depth. Reduced Fe (Fe²⁺) was also detected, but confined within surficial depths sampled, ranging from 1.2 – 38.5 µM. Mass balance calculations identify that the Fe²⁺ generated within the surficial zone of the CT deposit is sufficient to effectively sequester ambient concentrations H₂S generated in this deposit through FeS precipitates. Results identifying (1) distinct zones of porewater Fe²⁺ and H₂S, (2) co-occurrence of the highest [H₂S] and lowest dissolved organic C (DOC) at 22-24 m consistent with heterotrophic sulfate reducing bacteria (SRB) activity, and (3) the presence of mixed valence Fe biomineral, magnetite, throughout the deposit, are all consistent with microbially-mediated Fe and S cycling occurring within this CT deposit. The cultivation independent identification of several known iron reducing bacteria (IRB) and SRB within CT microbial communities, in conjunction with observed positive growth of IRB and SRB functional metabolic enrichments, demonstrates widespread capacity for microbial Fe and S activity throughout the CT deposit. Metagenomic characterization of CT microbial communities revealed high diversity (over 20 phyla) over the 5 depths examined. Multivariate statistical analyses (Unifrac) revealed that bacterial community composition and structure was driven by changed in DOC, ORP and salinity and that structuring corresponded with a surficial zone of Fe³⁺ reduction and an underlying zone of SO₄^2- reduction. Despite the high organic carbon (OC) content of oil sands tailings, much of that C is not considered to be labile and accessible to microbes. Based on the results of this thesis, CT SRB appear to have a greater ability than IRB to utilize recalcitrant OC (e.g. bitumen, naphthenic acids) given the widespread occurrence of porewater [H₂S] and surficially restricted [Fe²⁺] despite accessible pools of Fe³⁺ and OC with depth. This enhanced understanding of biogeochemical S cycling within CT newly establishes the importance of microbial activity in these processes, identifying the need to incorporate microbially based understanding into on-going development of reclamation strategies in order to manage these waste materials effectively.</p> / Master of Science (MSc)

Anaerobic digester design and operation influences the biomass degradation efficiency performed by complex and diverse microbial communities. Optimum anaerobic digester design and operational parameters in residential on-site wastewater treatment sites (OWTS) establishes physiochemical environments suitable for the growth and stability of the microbial communities responsible for organic waste degradation. A comparative study of the microbial communities and their functional profiles between different OWTS designs and operational parameters have not been done despite their functional importance in residential organic waste removal. Using whole-metagenome shotgun sequencing, microbial community compositions and functions were compared between two digester designs: conventional box septic tanks and septic tanks equipped with a novel closed-conduit tube called the InnerTubeTM. Wastewater was sampled along the length of each digester to explore the microbial community stratification during the anaerobic digestion treatment process. Additionally, the effect of effluent, aerobic recirculating-lines on the digester microbiome was also explored. Physiochemical characteristics in the form of oxygen demand, nitrogen and solids content was used as endpoints and correlated with microbial community and functional gene abundances to explore the microbes driving anaerobic digestion. Conventional digesters were characterized by syntrophic proprionate-oxidizing microbes and acetoclastic methanogens, while InnerTube™ digesters were characterized by syntrophic sulfate-reducing microbes and hydrogenotrophic methanogens. Recirculating digesters were enriched with denitrifying microbial consortia in syntrophy with hydrogenotrophic methanogens. Microbial communities were organized according to hydrolytic, acidogenic, acetogenic, and methanogenic groups along the digester treatment process. Insight into the core microbiome of OWTS can inform bioaugmentation and digester design and operation optimization strategies to improve the treatment of decentralized residential sewage sources. / Thesis / Master of Science (MSc) / Anaerobic digesters are used throughout North America to treat residential sewage. Despite their prevalence, the composition and function of the microbial communities driving sewage degradation in residential digesters has not been studied. We used DNA sequencing to compare the microbial communities and functional genes in different anaerobic digester designs across Southern Ontario. Our findings suggest there are successive microbial groups along the length of septic tanks and that different septic tank designs harbor characteristic sulfidogenic and methanogenic microbes. Characterization of these microbes could inform septic tank bioaugmentation, design and operational optimization strategies to improve sewage treatment performance.

Us as humans are colonised by many microbial communities (the human microbiome) that interact with and regulate the host's physiology, and have been linked with several diseases. The high number of interactions that intercurre between the microbiome and the host requires rigorous statistical approaches to link any condition of interest to microbiome data. Many publicly available microbiome datasets are available that allow to study such interactions. However, strong inconsistencies are found among the reported associations when looking at the same condition in different studies. On the road to consistent statistical microbiome analyses that rely on public data, lack of standardisation and availability are barriers to define reproducible and generalisable associations. The main aim of my PhD was the development of meta-analytical approaches to identify microbial signatures as general hallmarks of health versus disease, integrating diverse cohorts and conditions. During my PhD training, I first explored the associations between the oral microbiome and peri-implantitis, an oral disease of dental implants, in which I defined a microbial signature discriminating diseased from control samples. I further developed and applied discriminative models to multiple colorectal cancer (CRC) cohorts, showing that the microbial signature defined on CRC samples is shared across different populations. To be able to further generalised microbial signature with host's conditions through a meta-analysis approach, I collected and analysed 20,533 public metagenomes from 90 cohorts, that are available through the curatedMetagenomicData (cMD) version 3, an R package providing standardised taxonomic and functional profiles and manually curated metadata. The cMD3 resource was used to derive an easy-to-compute oral-to-gut introgression score that I found systematically associated in a large meta-analysis of twelve diseases and with ageing. Finally, I applied the meta-analysis approach to study diet interventions in mice, exploiting a novel approach able to profile the unexplored fraction of microbiomes, and showing associations driven by previously uncharacterised species. Overall, this thesis contributes to strengthening the links between human and animal microbiomes in normal and altered host conditions.

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.