Streptococcus gordonii, a commensal oral bacterium, is considered a good candidate to function as a live oral vaccine vector. The introduction of vaccine antigen genes into S. gordonii relies on the use of antibiotic resistance genes as selectable markers, which is undesirable. In this study, we used auxotrophic complementation (deletion of an essential gene from the chromosome and insertion into a plasmid) as a means to create an antibiotic marker-free gene delivery system in S. gordonii. S. gordonii ?thyA was created and complemented by an antibiotic marker-free expression plasmid containing the intact thyA gene, pDL276/thyAdelkan. Transformation of pDL276/thyAdelkan into the mutant gave an unexpected 100-fold increase in transformation efficiency as compared to pDL276. The transformants arose from both single and double crossing over. The increase in transformation efficiency suggests that a highly efficient antibiotic marker-free system to deliver genes to the chromosome has been created using thyA complementation.

Environmental bacteria and especially soil bacteria are active producers of antibiotic molecules and most drugs used nowadays are isolated from saprophytic soil bacteria and these microorganisms have also evolved numerous resistance pathways leading to an arsenal of Antibiotic Resistance Genes Determinants (ARGD) known as the environmental resistome. A survey of ARGD prevalence is required in order to characterize this natural phenomenon with critical implications in our current infectious diseases management. In order to perform such analysis we compiled a set of 71 metagenomic datasets from various environmental origins: soils, oceans, lakes, human feces, indoor air, etc., and compared their sequences with a database of known antibiotic resistance gene determinants (ARGD). ARGD-annotated reads are found in every environment analyzed confirming their ubiquity. Soil is found to be the richest and shares a large part of ARGD with the human gut microbiome, indicating ARGD transfers between these environments. Experiments using qPCR and metagenomic DNA sequencing on soil samples from two sites with known and distinct antibiotic pollution history were conducted to understand how ARGD abundance and diversity in soil are affected when impacted by antibiotic molecules. The first site is a reference soil from a long-term experiment without history of antibiotic pollution (Rothamsted Park Grass, UK). Soil microcosms are setup with addition of either antibiotic-containg animal manure or pure molecules and incubated for 6 months to monitor changes in ARGD concentration following these perturbations. Our second study-site is a very remote settlement in French Guiana where antibiotics are available since recently and may have impacted the local soil microbial community. Soil samples are taken following a line-transect going from the village (antibiotic source) to 3km deep in the forest in a gradient of human-impact. Our results all confirm prevalence of ARGD in soil at significant abundance but also that ARGD distribution is more correlated to environmental factors such as soil type, microbial taxonomy composition or microcosms incubation conditions than antibiotic molecules exposure in both sites. Pathogens ARGD diversity is far lower than ARGD diversity found in the environment and not all the soil resistome is readily accessible for transfer. In order to characterize the soil mobile gene pool, a strategy is proposed to isolate specifically mobile DNA directly from the environment for sequencing purposes. Better knowledge on the microbial ecology factors limiting ARGD transfers to pathogens may greatly help us reduce the current threat on our limited medical antibiotic molecules resource.

<div>Antibiotics have been widely used to treat bacterial diseases since the 1940s. However, the benefits offered by antibiotics have gradually faded due to the increased occurrence and frequency of antibiotic resistance. The widespread use of antibiotics has driven selection for resistance in bacteria and is becoming a global problem for human health and the environment. Antibiotic resistance is exacerbated by the ability of bacteria to share their antibiotic resistance genes (ARGs) with other bacteria via horizontal gene transfer (HGT). Many existing studies on HGT of ARGs focused on antibiotic concentrations at or above the minimal inhibitory concentration (MIC), which is the lowest concentration of an antibiotic that prevents visible growth of a bacteria culture. However, knowledge on the development of antibiotic resistance under different stressors at sub-MIC levels is still limited. In addition, carbon nanotubes (CNTs) have been widely studied in environmental, agricultural and biomedical areas due to their unique physical and chemical characteristics, but limited studies have been done to evaluate the effects of CNTs on the spread of ARGs. Electrochemical filtration has been shown to be a cost-effective technique to remove recalcitrant compounds and reduce antibiotic resistance, but limited studies have been done to evaluate the effectiveness of removal of ARGs with electrochemical filtration. Therefore, there is a critical need to evaluate the effects of trace levels of antibiotics and CNTs on the development of antibiotic resistance and electrochemical removal of ARGs. </div><div><br></div><div>The specific research objectives of this study were to evaluate: (1) selective pressure of sub-inhibitory concentrations of antibiotics on the development of antibiotic resistance and HGT, (2) development of antibiotic resistance and HGT under exposure to CNTs and antibiotics, and (3) effectiveness of using an electrochemical MWCNT filter to remove ARGs. </div><div><br></div><div>To evaluate the development of antibiotic resistance exposed to sub-MIC of erythromycin, HGT between environmental donor (<i>E. coli)</i> and pathogenic bacterial recipient (<i>B. cereus</i>) was quantified. The results indicated that extremely low concentration (0.4 ng/L to 4 µg/L) of erythromycin promoted HGT of <i>erm</i>80 gene, which is an erythromycin resistance gene. In addition to traditional culture-based method and quantitative real-time PCR (qPCR), a fluorescence <i>in situ</i> hybridization (FISH) approach was used to detect the occurrence and development of ARGs even the bacteria were in the viable but nonculturable (VBNC) state after treatment of sub-lethal level of erythromycin. Multi-walled carbon nanotubes (MWCNT) was selected as a representative stressor to evaluate the effects on HGT. The results showed that MWCNT enhanced HGT above 1 × MIC, which is the lethal level of erythromycin to recipients, and transfer frequencies of erm80 genes increased up to 101-fold under exposure to 1 × MIC erythromycin and MWCNT as compared to no MWCNT control. However, transfer efficiency of <i>erm</i>80 gene under exposure to sub-MIC of erythromycin was inhibited by MWCNTs. Moreover, transfer of antibiotic resistance plasmids was affected by antibiotics and MWCNTs. Although the concentration of individual stressor was not enough to confer antibiotic selection, effects of both antibiotics above 1 × MIC and MWCNTs could add up and select for antibiotic resistance. The results suggested that CNTs might create additional selective pressure for the spread of ARGs and their effects on HGT should be further investigated. Finally, an electrochemical MWCNT filtration was evaluated to remove genomic DNA and ARGs under the effects of operating conditions, such as pH, phosphate, and NOM. The results showed that the electrochemical MWCNT filtration reactor achieved 79% removal efficiency for genomic DNA and 91% removal efficiency for <i>erm</i>80 genes. The study suggested that electrochemical MWCNT filtration could be a promising technology for the removal of DNA and ARGs.</div><div><br></div><div>Overall, the results improved our understanding of the development of antibiotic resistance and ARGs under various selective pressures. Trace levels of antibiotics promoted the development and spread of ARGs. Conjugative transfer of resistance genes exposed to sub-MIC levels of erythromycin and MWCNTs also contributed to the spread and propagation of ARGs. As antibiotic concentrations detected in natural environment are often in trace levels, the results of this study may improve the understanding of health risks of trace levels of antibiotics and help develop effective mitigation strategies to control the spread of antibiotic resistance. Effective removal of ARGs with electrochemical MWCNT filtration may help the development of cost-effective treatment systems to remove ARGs to protect human health and the environment.</div><div><br></div>

The overuse and misuse of antibiotics to treat bacterial infections, the release of unmetabolized residuals into the sewer system, and the incomplete removal antibiotic residues by wastewater treatment plants (WWTPs) pose a severe threat to human health. The accumulation of antibiotic residue induces selective pressure on the bacterial population, resulting in the spread of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) in water. This study investigated the degradation of different types of ARGs in water matrices using a wide variety of treatment technologies. Real wastewater samples were collected from a WWTP in urban Philadelphia and the presence of single and multidrug-resistant bacteria and resistance genes were investigated using molecular-based techniques. Subsequently, an analytical method was developed and validated for the detection and quantification of the ARGs against a range of antibiotics, such as tetracycline (TCN), ciprofloxacin (CIP), and levofloxacin (LVX). Finally, to remove the ARGs from water matrices, different conventional and advanced oxidation processes were applied. At the very onset, conventional treatment processes such as chlorine treatment was used to inactivate the E.coli resistant strains. It was observed that chlorination can potentially deactivate the ARBs by applying a lower dose and contact time. However, the effectiveness of chlorine treatment in removing all types of ARGs from water matrices was limited. For instance, no significant degradation of extracellular ARGs (e-ARGs) was observed in DI water during chlorine treatment. Subsequently, a peracetic acid (PAA) based treatment process was used to degrade the genomic and plasmid-encoded ARGs from the water matrices. Similar to chlorine treatment, no significant changes were observed in the degradation of extracellular ARGs (e-ARGs) in DI and WW. Then, the degradation kinetics of ARGs across different types (gyrAR, tetAR, qnrSR) and forms (chromosomal, plasmids) were evaluated using the Ultraviolet (UV) disinfection process. Compared to chlorination and PAA, UV treatment showed better removal efficiencies for the degradation of different types of e-ARGs in DI water. The degradation profile of e-ARGs showed 1-4 log reductions at a UV fluence of 900 mj/cm2. The i-ARGs showed similar degradation rates as compared to e-ARGs in phosphate buffer saline (PBS) at the same UV dosage. On the other hand, the regrowth potential of ARBs at low UV dosage (60–180 mJ/cm2) showed the evidence of damage repairment after several hours of exposure to light (photoreactivation) and dark conditions, making it susceptible again to the resistance spread. To resolve this issue, process parameters were optimized, and no regrowth of the ARBs were found from the higher fluence from 300 to 600 mJ/ cm2. Later, UV/ H2O2 based AOP was applied to evaluate the degradation and deactivation of the same resistant genes. The addition of H2O2 during the UV treatment produces strongly reactive •OH radicals during the treatment and showed considerable improvements in e-ARGs degradation (1.2-5 logs) compared to UV treatment alone. However, this AOP showed minimal contribution to i-ARG degradation (1-2.4 logs), possibly due to the scavenging of •OH radicals by the cellular components in PBS. In contrast to PBS, the wastewater matrix moderately enhanced the gene degradation during the treatment. In terms of plasmid degradation, the conformational differences of the supercoiled structures showed 1.2-2.8 times slower degradation rates than chromosomal ARGs. In addition, the degradation kinetics of the free residual ARGs (f-ARGs) were assessed during the treatment to reduce the AMR dissemination risk from the treated sample. This study also examined the potential of ozone (O3) based oxidation process to degrade and deactivate the extracellular and intracellular ARGs, and MGE (plasmid, intl-1) from E.coli ARBs. The degradation kinetics of the ARGs across different sizes (118-454 bps) and types were evaluated in different water matrices (DI water, PBS, and WW), and showed a significantly higher removal for chromosomal, and plasmid encoded ARGs than other treatment technologies. For the e-ARGs in DI water, 3.8-5.2 logs removal was observed at ozone dosage of 2.0 × 10-2 M.s. i-ARGs in PBS and wastewater showed nearly similar degradation (3.8-5 logs) during O3, indicating the elimination of i-ARGs was not dependent on the cellular components and effluent organic matter. Moreover, an analysis of environmental DNA (eDNA) from wastewater was conducted to examine the degradation of DNA and ARGs for different storage periods and temperatures (-20°C, 0°C, 4°C, 22±0.87°C). Result indicated that water samples kept at -20°C and 0°C showed the best performance in preventing the DNA concentration and gene degradation over time. Additionally, the effectiveness of different preservatives (Longmire buffers: LB1 and LB2, benzalkonium chloride at 0.1%, 0.01%) were investigated in preserving the DNA integrity and the gene degradation at an ambient temperature. It was found that the Longmire buffer (LB1) exhibited lowest gene degradation during the three-week storage period. In summary, this research provided a comprehensive assessment on the degradation of e-ARGs, i-ARGs, and free ARGs from water using different treatment technologies (i.e., UV, UV/H2O2, O3, PAA, chlorine). Additionally, this study suggested valuable information on optimizing the process parameters of the selected methods and developed a comparative assessment of removing the ARGs from the water matrix (DI/PBS, WW). The estimation of Electrical Energy per Order (EEO, kWh/m3) during UV and ozone treatments provided a comparison of the energy consumption for ARGs degradation in the water. Overall, the findings of this study can be useful for evaluating different types and forms (chromosomal, plasmid) of ARG degradation from water matrices and can help to reduce the risk of AMR dissemination in the environment. / Civil Engineering

Anthropogenically generated antibiotic resistance genes (ARGs) are considered emerging contaminants, as they are associated with a critical human health challenge, are persist independent of a bacterial host, are subject to transfer between bacteria, and are present at amplified levels in human-impacted environments. Given the gravity of the problem, there is growing interest in advancing water treatment processes capable of limiting ARG dissemination. This study examined the potential for membrane treatment of microconstituent ARGs, and the effect of wastewater colloids on their removal. Native and spiked extracellular vanA (vancomycin resistance) and blaTEM (β-lactam resistance) ARGs were tracked by quantitative polymerase chain reaction through a cascade of membrane filtration steps. To gain insight into potential associations occurring between ARGs and colloidal material, the wastewater colloids were characterized by scanning electron microscopy, as well as in their protein, polysaccharide, and total organic carbon content. The results suggest that extracellular DNA (eDNA) containing ARGs interacts with wastewater colloids, and can both be protected against degradation and be removed more efficiently in the presence of wastewater colloidal material. Thus, ARG removal may be achievable in sustainable water reuse scenarios using lower cost membranes than would have been selected based on molecular size alone. As membranes are likely to play a vital role in water sustainability, the results of this study enable consideration of ARG removal as part of a comprehensive strategy to manage emerging contaminants and to minimize overall public health risks. / Master of Science

The purpose of this dissertation was to broadly investigate the fate of antibiotic resistance genes (ARGs) and engineered nanomaterials (ENMs) as representative contaminants of emerging concern in wastewater treatment plants (WWTPs). WWTPs may have their performance impacted by ENMs and may also serve as a reservoir and point of release for both ENMs and ARGs into the environment. Of interest were potential adverse effects of ENMs, such as stimulation of antibiotic resistance in the WWTP, toxicity to microbial communities critical for WWTP performance, and toxicity to humans who may be exposed to effluents or aerosols containing ENMs and their transformation products. Response of nine representative ARGs encoding resistance to sulfonamide, erythromycin and tetracycline to various lab-scale sludge digestion processes were examined, and factors that drove the response of ARGs were discussed. Mesophilic anaerobic digestion significantly reduced sulI, sulII, tet(C), tet(G), and tet(X) with longer solids retention time (SRT) exhibiting a greater extent of removal. Thermophilic anaerobic digesters performed similarly to each other and provided more effective reduction of erm(B), erm(F), tet(O), and tet(W) compared to mesophilic digestion. Thermal hydrolysis pretreatment drastically reduced all ARGs, but they generally rebounded during subsequent anaerobic and aerobic digestion treatments. Bacterial community composition of the sludge digestion process, as controlled by the physical operating characteristics, was indicated to drive the distribution of ARGs present in the produced biosolids, more so than the influent ARG composition. Effects of silver (nanoAg), zero-valent iron (NZVI), titanium dioxide (nanoTiO2) and cerium dioxide (nanoCeO2) nanomaterials on nitrification function and microbial communities were examined in duplicate lab-scale nitrifying sequencing batch reactors (SBRs), relative to control SBRs received no materials or ionic/bulk analogs. Nitrification function was only inhibited by high load of 20 mg/L Ag+, but not by other nanomaterials or analogs. However, decrease of nitrifier gene abundances and distinct microbial communities were observed in SBRs receiving nanoAg, Ag+, nanoCeO2, and bulkCeO2. There was no apparent effect of nanoTiO2 or NZVI on nitrification, nitrifier gene abundances, or microbial community structure. A large portion of nanoAg remained dispersed in activated sludge and formed Ag-S complexes, while NZVI, nanoTiO2 and nanoCeO2 were mostly aggregated and chemically unmodified. Thus, the nanomaterials appeared to be generally stable in the activated sludge, which may limit their effect on nitrification function or microbial community structure. Considering an aerosol exposure scenario, cytotoxicity and genotoxicity of aqueous effluent and biosolids from SBRs dosed with nanoAg, NZVI, nanoTiO2 and nanoCeO2 to A549 human lung epithelial cells were examined, and the effects were compared relative to outputs from SBRs dosed with ionic/bulk analogs and undosed SBRs, as well as pristine ENMs. Although the pristine nanomaterials showed varying extents of cytotoxicity to A549 cells, and gentoxicity was observed for nanoAg, no significant cytotoxic or genotoxic effects of the SBR effluents or biosolids containing nanomaterials were observed. Studies presented in this dissertation provided new insights in the fate of ARGs in various sludge digestion processes and ENMs in nitrifying activated sludge system in lab-scale reactors. The study also yielded toxicity data of ENMs to biological wastewater treatment microbial communities and human lung cells indicated by a variety of toxicity markers. The results will aid in identifying appropriate management technologies for sludge containing ARGs and will inform microbial and human toxicity assessments of ENMs entering WWTPs. / Ph. D.

The treatment of water and wastewater is energy intensive, and there is an urgent need to develop new approaches to address the water-energy challenges. Bioelectrochemical systems (BES) are energy-efficient technologies that can treat wastewater and simultaneously achieve multiple functions such as energy generation, hydrogen production and/or desalination. The objectives of this dissertation are to understand the fundamental microbiology of BES, develop cost-effective cathode catalysts, optimize the process engineering and identify the application niches. It has been shown in Chapter 2 that electrochemically active bacteria can take advantage of shuttle-mediated EET and create optimal anode salinities for their dominance. A novel statistical model has been developed based on the taxonomic data to understand and predict functional dynamics and current production. In Chapter 3, 4 and 5, three cathode catalyst (i.e., N- and S- co-doped porous carbon nanosheets, N-doped bamboo-like CNTs and MoS2 coated on CNTs) have been synthesized and showed effective catalysis of oxygen reduction reaction or hydrogen evolution reaction in BES. Chapter 6, 7 and 8 have demonstrated how BES can be combined with forward osmosis to enhance desalination or achieve self-powered hydrogen production. Mathematical models have been developed to predict the performance of the integrated systems. In Chapter 9, BES have been used as a research platform to understand the fate and removal of antibiotic resistant genes under anaerobic conditions. The studies in this dissertation have collectively demonstrated that BES may hold great promise for energy-efficient water and wastewater treatment. / Ph. D. / Water and energy are prerequisites to life. Every day, a lot of energy and money are spent on treating wastewater and producing fresh water. Bioelectrochemical systems (BES) are new technologies that can treat water and wastewater with low energy consumption. BES typically consist of an anode (where microorganisms break down organic matter) and a cathode, and work like a battery. Currently, BES are only studied in laboratories and not applied in real-world situations, because the performance needs to be improved and fundamentals remain to be better understood. The studies in this dissertation aim to address these problems and make BES toward practice. It has been shown in Chapter 2 that, under high salinity, some bacteria grow faster in the anode and the BES can produce higher electricity. It is difficult to understand the roles of every bacterium with current molecular techniques, and thus statistical methods are applied to estimate their possible functions. In Chapter 3, 4 and 5, three materials have been fabricated and functioned as the catalysts for electricity generation. Chapter 6, 7 and 8 have demonstrated how BES can be combined with forward osmosis, a spontaneous water diffusion process, to enhance desalination or achieve self-powered hydrogen production. Mathematical equations have been combined to simulate the process of biological metabolisms, water diffusion and ion migration. In Chapter 9, BES have been shown to remove antibiotic resistant gene, an emerging contaminant caused by the excessive use of antibiotics. The studies in this dissertation have collectively demonstrated that BES may be the answer to future water and wastewater treatment.

Many antibiotic resistance genes (ARGs) conferring resistance to a broad range of antibiotics have often been detected in aquatic environments such as untreated and treated wastewater, river and surface water. ARG proliferation in the aquatic environment could depend upon various factors such as geospatial variations, the type of aquatic body, and the type of wastewater (untreated or treated) discharged into these aquatic environments. Likewise, the strong interconnectivity of aquatic systems may accelerate the spread of ARGs through them. Hence a comparative and a holistic study of different aquatic environments is required to appropriately comprehend the problem of antibiotic resistance. Many studies approach this issue using molecular techniques such as metagenomic sequencing and metagenomic data analysis. Such analyses compare the broad spectrum of ARGs in water and wastewater samples, but these studies use comparisons which are limited to similarity/dissimilarity analyses. However, in such analyses, the discriminatory ARGs (associated ARGs driving such similarity/ dissimilarity measures) may not be identified. Consequentially, the reason which drives the dissimilarities among the samples would not be identified and the reason for antibiotic resistance proliferation may not be clearly understood. In this study, an effective methodology, using Extremely Randomized Trees (ET) Algorithm, was formulated and demonstrated to capture such ARG variations and identify discriminatory ARGs among environmentally derived metagenomes. In this study, data were grouped by: geographic location (to understand the spread of ARGs globally), untreated vs. treated wastewater (to see the effectiveness of WWTPs in removing ARGs), and different aquatic habitats (to understand the impact and spread within aquatic habitats). It was observed that there were certain ARGs which were specific to wastewater samples from certain locations suggesting that site-specific factors can have a certain effect in shaping ARG profiles. Comparing untreated and treated wastewater samples from different WWTPs revealed that biological treatments have a definite impact on shaping the ARG profile. While there were several ARGs which got removed after the treatment, there were some ARGs which showed an increase in relative abundance irrespective of location and treatment plant specific variables. On comparing different aquatic environments, the algorithm identified ARGs which were specific to certain environments. The algorithm captured certain ARGs which were specific to hospital discharges when compared with other aquatic environments. It was determined that the proposed method was efficient in identifying the discriminatory ARGs which could classify the samples according to their groups. Further, it was also effective in capturing low-level variations which generally get over-shadowed in the analysis due to highly abundant genes. The results of this study suggest that the proposed method is an effective method for comprehensive analyses and can provide valuable information to better understand antibiotic resistance. / MS / Antibiotic resistance is a natural and primordial process that predates the use of antibiotics in humans for disease treatment and occurs when a bacterium evolves to render the drugs, chemicals, or other agents meant to cure or prevent infections ineffective. Antibiotic resistance genes (ARGs) conferring resistance to a wide range of antibiotics have been widely found in rivers, surface waters, and hospital and farm wastewater discharges. Even treated wastewater from treatment plants is a concern as ARGs have frequently been detected in effluent discharges which poses questions on the effectiveness of treatment plants in removing ARGs. Since, these systems are interconnected there’s a possibility of dissemination and proliferation of ARGs which may pose serious threat to human health. Hence, it is desirable to perform comparative studies among these aquatic habitats. In previous studies, researchers compared different habitats which tells how similar and dissimilar the environments are in terms of ARGs present in these samples. While these analyses are important, it doesn’t tell which ARGs are unique or which ARGs are responsible to create those similarities or dissimilarities. This information is crucial in order to understand the water environments in terms of occurrence and presence of ARGs, the risk posed by them, and in identifying factors responsible for resistance gene proliferation. In this research, a methodology was developed which could capture such ARG variations in the environmental samples, using data analysis algorithms. Further the developed methodology was demonstrated using environmental samples such as wastewater samples from different geographical locations (to understand the spread of ARGs globally), untreated vs treated wastewater (to understand the effectiveness of treatment plants in removing ARGs), and different aquatic habitats (to understand the impact and spread of ARGs within these habitats). It was determined that the proposed method was efficient in differentiating samples and identifying discriminatory ARGs. The comparison between environmental samples showed that the samples from different locations have specific ARGs which were unique to wastewater samples from certain locations suggesting that site-specific factors can have certain effect in shaping the ARG profiles. Comparing untreated and treated samples revealed that treatment plants were able to remove certain ARGs but it was also observed v that some ARGs proliferated after the treatment irrespective of location and treatment plant specific variables. Analyzing different environments, the approach was able to identify certain ARGs which were specific to certain environments. The results of this study suggest that the proposed method is an effective method for comprehensive analyses and can provide valuable information to better understand antibiotic resistance. In essence, it is a valuable addition for improved surveillance of antibiotic resistance pollution and for the framing of best management practices.

Cattle are commonly treated with antibiotics that may survive digestion and promote antibiotic resistance when manure or composted manure is used as a soil amendment for crop production. This study was conducted to determine the effects of antibiotic administration and soil amendment practices on microbial diversity and antibiotic resistance of bacteria recovered from the surfaces of lettuce and radishes grown using recommended application rates. Vegetables were planted in field plots amended with raw manure from antibiotic-treated dairy cows, composted-manure from cows with different histories of antibiotic administration, or a chemical fertilizer control (12 plots, n=3). Culture-based methods, 16SrDNA amplicon sequencing, qPCR and shot-gun metagenomics were utilized to profile bacteria and characterize the different gene markers for antibiotic resistance. Culture-based methodologies revealed that lettuce grown in soils amended with BSAs had significantly larger clindamycin resistant populations compared to control conditions. Growth in BSAs was associated with significant changes to the bacterial community composition of radish and lettuce. Total sul1 copies were 160X more abundant on lettuce grown in manure and total tet(W) copies were 30X more abundant on radishes grown in manure. Analysis of shotgun metagenomic data revealed that lettuce grown in manure-amended soils possessed resistance genes for three additional antibiotic classes compared to other treatments. This study demonstrates that raw, antibiotic-exposed manure may alter microbiota and the antibiotic resistance genes present on vegetables. Proper composting of BSAs as recommended by the U.S. Department of Agriculture and Environmental Protection Agency is recommended to mitigate the spread of resistance to vegetable surfaces. / MSLFS / Antibiotics are drugs responsible for killing infectious diseases in both humans and animals. In cows, antibiotics are frequently used when they get infections in their udders. These drugs can be excreted through manure and urine and end up in the environment. Manure or composted manure is often applied as a soil amendment for crop production. The presence of antibiotics in soil may promote antibiotic resistance, meaning bacteria that carry antibiotic resistance genes (ARGs) are capable of surviving exposure to drugs that would normally kill them. Such bacteria may eventually pass their ARGs to pathogens, which then could no longer be treated effectively by antibiotics when there is an infection. Thus, there is concern that overuse of antibiotics in agriculture can contribute to reduced effectiveness of antibiotics and the growing global antibiotic resistance health crisis. This study sought to determine if prior antibiotic administration affected the antibiotic resistance of bacteria found on the surfaces of vegetables grown in soil amended with manure or compost from dairy cows. Lettuce and radishes were grown in the field in plots amended with raw manure from antibiotic-treated dairy cows, compost from cows with different histories of antibiotic administration, or a chemical fertilizer control. Mature vegetables were harvested and used to enumerate antibiotic-resistant bacterial colonies. Additionally, the 16S rRNA gene, which is a ubiquitous gene found in all bacteria, was sequenced to identify the kinds of microbes that colonized the radish and lettuce surfaces when grown under the different conditions. DNA was extracted from the bacteria collected from the vegetable surfaces to and different methods were used to identify the kinds of ARGs present and to which kinds of antibiotics they encode resistance. The results of the study indicated that raw, antibiotic-exposed manure may increase the bacteria found on vegetables in addition to their ARGs. Proper composting of manure, as recommended by the U.S. Department of Agriculture (USDA) and the Environmental Protection Agency (EPA), is recommended to mitigate resistance and control microbial populations on fresh vegetables.

Outputs of human-derived chemicals and constituents to the environment, and shifts in these outputs, can result in unintended consequences to human and ecological health. One such shift is the advent of the modern antibiotic era, in which mass production and outputs of antibiotics, which are mostly naturally-derived microbial defense compounds and include a few synthetic antimicrobials, has profound implications for contributing to the spread of antibiotic resistance. Antibiotic resistance arises from mutations and/or sharing of antibiotic resistance genes (ARGs) among bacteria via horizontal gene transfer, with carriage of ARGs by pathogenic bacteria of particular concern to human health. While most attention to stopping the spread of antibiotic resistance has been devoted to the clinic, it is critical to consider the environmental origin, ecology and pathways by which antibiotic resistance spreads in order to develop comprehensive strategies to combat antibiotic resistance. In particular, wastewater treatment plants (WWTPs) represent a potentially key critical control point given that they receive antibiotic resistant bacteria (ARB) and ARGs from the population, which are then routed to activated sludge biological treatment, consisting of high density, highly active microbial populations. The research projects described in this dissertation aimed to explore the occurrence of ARGs in WWTPs, particularly WWTPs in developing countries representing the extremes of what is expected to be encountered in terms of potential to spread antibiotic resistance, and to improve and apply novel technologies for monitoring key markers of antibiotic resistance in WWTPs and affected environments. The pathogen Staphylococcus aureus and a corresponding ARG (methicillin resistance mecA gene) were chosen as model biocontaminants of concern due to their environmental and public health relevance. The results reported in Chapters 3-5 advance the knowledge of bio(nano)sensing techniques and highlight areas of promise and challenge. The results reported in Chapter 2 provided insight into the baseline levels of ARGs expected in a highly impacted WWTP in India, thereby highlighting the magnitude and global scale of the problem of antibiotic resistance as well as the need for innovative solutions. / Ph. D. / Release of human-derived pollutants into the environment can result in unintended consequences to human and environmental health. The rise of antibiotic resistance in disease-causing bacteria serves as a notorious example. Antibiotic resistance arises from mutations and/or sharing of antibiotic resistance genes (ARGs), which are the genetic elements that enable the resistance to occur. While most attention to stopping the spread of antibiotic resistance has been devoted to the clinic, it is critical to consider the environmental factors by which antibiotic resistance spreads in order to develop wellinformed strategies to combat it. In particular, wastewater treatment plants (WWTPs) represent a potentially key critical control point given that they receive antibiotic resistant bacteria (ARB) and ARGs from the population, which are then routed to a highly active biological treatment process. The research projects described in this dissertation aimed to explore the occurrence of ARGs in WWTPs, particularly WWTPs in developing countries representing the extremes of what is expected to be encountered in terms of potential to spread antibiotic resistance, and to improve and apply novel technologies for monitoring key markers of antibiotic resistance in WWTPs and affected environments. The disease-causing bacterium <i>Staphylococcus aureus</i> and a corresponding ARG (methicillin antibiotic resistance mecA gene) were chosen as model biological contaminants of concern due to their environmental and public health relevance. The results reported in Chapters 3-5 advance the knowledge of integrated microbiology and nanotechnology techniques, and also highlight some associated limitations. The results reported in Chapter 2 provide insight into the baseline levels of ARGs expected in a highly impacted WWTP in India, thereby highlighting the magnitude and global scale of the problem of antibiotic resistance as well as the need for innovative solutions.