<p dir="ltr">Mycobacterium abscessus (Mab) is a type of mycobacterium that is known for its remarkable resistance to a variety of antibiotics. This pathogen poses a significant risk for individuals with weakened immune systems as it can cause skin and soft tissue infections, pulmonary disease and disseminated infections. Mab's ability to expel antibiotics through efflux pumps and form strong biofilms makes it even more challenging to treat infections. Lipids form a major part of the extracellular matrix of Mab biofilms. Efflux pumps have been shown to export lipids to the cell surface. Despite ongoing research into Mab's antibiotic tolerance, there is still much to learn about the impact of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis during biofilm development in Mab. In this study, we investigated the impact of the EPIs; CCCP (carbonyl cyanide m-chlorophenyl hydrazone), piperine (PIP), reserpine (RES), berberine (BER), and verapamil (VER) on efflux activity, biofilm formation, antibiotic resistance, and lipid biosynthesis in Mab during planktonic and biofilm growth conditions. We found that Mab cells had a higher tolerance to EPIs in biofilm-stimulating medium and that the presence of EPIs led to a decrease in minimum inhibitory concentrations of frontline antibiotics, reduced efflux activity within Mab cells, and significantly inhibited biofilm formation. We examined the effects of EPIs that inhibited biofilm formation on lipid metabolism in Mab using radiolabeling with 14C?palmitic acid and 14C-acetic acid which are precursors of lipid biosynthesis. We observed that the EPI berberine inhibited the incorporation of 14C-palmitic acid into glycopeptidolipids in the surface lipids of planktonic cells and increased cellular glycopeptidolipid (GPL) in biofilm cells. Verapamil-treated cells showed a 55 % increase in cellular trehalose monomycolate (TMM) compared to controls. Piperine-treated cells exhibited a 50 % increase in cardiolipin. The incorporation of 14C-acetate into biofilm cells showed that piperine-treated biofilm cells showed a 146 % increase in surface glycopeptidolipids. Overall, our study enhances our understanding of lipid biosynthesis in Mab, the effects of EPIs on Mab biofilms, efflux mechanisms, and antibiotic resistance and offers insights for combating Mab-related infections.</p>

The discovery of antibiotics has been considered as one of the most remarkable scientific accomplishments of the last century. However, the extensive usage of antibiotics has led to the rapid emergence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB), which have been recognized as one of the biggest threats to human and environmental health. While ARGs and ARB are ubiquitous in diverse environments, aquatic environments play a particularly crucial role in their prevalence and dissemination. Furthermore, the microbial complexity and various pollutants persisting in aquatic environments significantly contribute to the evolution and spread of ARGs and ARB. However, the knowledge regarding the distribution pattern of ARGs on a large scale, as well as the interaction between microbial community, specific pollutants, and ARGs and ARB, is currently limited. In this study, I conducted systematic work at multiple scales, to fill crucial knowledge gaps that could support the future management of the spread of antimicrobial resistance. In particular, on the ecosystems level I depict the biogeographical patterns of ARGs in freshwater reservoirs, on the community level I explored the selection patterns of combinations of antibiotics on multidrug resistant strains in complex community context, and finally, on the population level I investigated the impact of cigarette smoke, and waste products on the dissemination of ARGs. In my first study, samples were collected from 24 freshwater reservoirs across southeast China and the biogeographical patterns of bacterial communities and ARG profile were characterized using 16S rRNA gene high-throughput sequencing and high-throughput-quantitative PCR. A distance-decay pattern for both, microbial communities and ARG profiles, were observed. However, larger differences between reservoir ARG profiles than microbial community compositions were detected. Further, I found that the biogeographical patterns of bacterial communities were simultaneously driven by stochastic and deterministic processes, while ARG profiles were not explained by stochastic processes, and the relationship between bacterial communities and ARG profiles was weak. In summary, this study indicated a decoupling of bacterial community composition and ARG profiles in inland waters under relatively low-human-impact at a large scale. In a second study, the selection dynamics for multidrug resistance between isogenic pairs of E. coli strains under exposure to multiple selective agents in the absence and presence of the microbial community were investigated using microcosm experiments. The presence of the community significantly decreased the selection for multidrug resistant strain under exposure to a single antibiotic. While pressure through the second antibiotic significantly decreased the activity and diversity of the community, its ability to reduce selection was consistently maintained at levels comparable to those recorded in the absence of the second antibiotic. This indicates that the observed effects of community context on selection dynamics are rather based on competitive or protective effects between the focal strains and a small proportion of bacteria within the community, than on general competition for nutrients. Last but not least, the effect of cigarette-derived pollutants on the proliferation of ARGs was explored using multifaced approaches. Cigarette smoke condensate in an artificial lung sputum medium significantly elevated the transfer rates of a multi-drug-resistance encoding plasmid between Pseudomonas strains. The overproduction of reactive oxygen species (ROS) as part of the bacterial stress response was directly connected to the increasing transfer rates. Similarly, cigarette ash leachate in an environmental medium significantly increased the plasmid transfer rates, and overproduction of ROS was equally detected. Furthermore, used cigarette filters with entrapped toxicants were submerged in a wastewater stream and colonized by distinct microbial communities compared to those colonizing unused control filters. The microbial communities colonizing used cigarette filters were significantly enriched in AMR, potential pathogenic bacteria and mobile genetic elements. Overall, the insights gained within this thesis into the spread of AMR at multiple scales constitute a valuable contribution to support future management and monitoring of ARGs in diverse ecosystems.

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.

Bakgrund: Antibiotikaresistens är ett globalt folkhälsoproblem vars ökning kan leda till att antibiotikabehandlingens verkan uteblir. Att förebygga antibiotikaresistens kan göras på många olika sätt, där utgångspunkten är att minska dagens antibiotikaanvändning för att i sin tur minska förekomsten av resistenta bakterier så framtida generationer kan behandla svåra infektioner. Sjuksköterskan är central i detta arbete till följd av omvårdnadsarbetets täta patientkontakt.  Syfte: Syftet var att beskriva sjuksköterskans kunskaper om förebyggande arbete gällande antibiotikaresistens.  Metod: Metoden var en allmän litteraturöversikt baserad på tio vetenskapliga artiklar.  Resultat: Tre teman identifierades: Sjuksköterskans kunskap om antibiotika och antibiotikaresistens, där sjuksköterskans kunskap om antibiotika generellt visades god medan kunskapen om antibiotikaresistens var något mer varierande. Sjuksköterskans förmåga och kunskap att arbeta enligt förebyggande praxis, vilket visade att sjuksköterskan till stor del hade god följsamhet att förebygga infektioner och därmed antibiotikaresistens. Desto högre utbildning och erfarenhet, desto bättre kunskap och följsamhet, vilket visade olika orsaker som ledde till bättre kunskap och följsamhet.  Slutsats: Litteraturöversikten visade att kunskap om antibiotikaresistens är nödvändigt för sjuksköterskor att ha för att de ska kunna arbeta förebyggande. Att öka kunskapen hos sjuksköterskor gällande antibiotikaresistens och hur det förebyggs i kliniskt arbete kan leda till en framtid där vi fortfarande kan behandla patienter med antibiotika. / Background: Antibiotic resistance is a global public health problem whose increase can cause antibiotic treatment to fail. Preventing antibiotic resistance can be done in many ways, where the basis is to reduce today’s antibiotic use in order to reduce the occurrence of resistant bacteria so that future generations can treat severe infections. The nurse is central in this work because of the frequent patient contact in nursing care.  Aim: The aim was to describe nurse’s knowledge of preventative work regarding antibiotic resistance.  Method: The method used was a general literature review based on ten research articles.  Results: Three themes were identified: Nurse’s knowledge regarding antibiotics and antibiotic resistance, where the nurse’s knowledge of antibiotics was generally shown to be good, while the knowledge of antibiotic resistance was somewhat more variable. Nurse’s ability and knowledge to work preventative, which showed that the nurse largely had good compliance to prevent infections and thus antibiotic resistance. The higher the education and experience, the better the knowledge and compliance, which showed different reasons that led to better knowledge and compliance.   Conclusions: This literature review showed that knowledge about antibiotic resistance is necessary for nurses to possess in order to prevent it in their profession. Increased nursing knowledge regarding antibiotic resistance and how to prevent it in clinical work results in a future where we are still able to treat patients with antibiotics.

In an era of pervasive water stress caused by population growth, urbanization, drought, and climate change, limiting the dissemination of microbial contaminants of emerging concern (MCECs) is of the utmost importance for the protection of public health. In this dissertation, two important subsets of MCECs, opportunistic pathogens (OP) and antibiotic resistant genes (ARG), are studied across several compartments of the urban water cycle, including surface water, stormwater, wastewater, recycled water, and potable water. Collectively, this dissertation advances knowledge about the occurrence of OPs and ARGs across these water systems and highlights trends that may be of value in developing management strategies for limiting their regrowth and transmission. Field studies of two surface water catchments impacted by stormwater runoff demonstrated the prevalence of ARGs in urban stormwater compared to pristine, unimpacted sites, or to days when no precipitation was recorded. The role of wastewater reuse in transmitting OPs and ARGs was also investigated. Traditional tertiary wastewater treatment plants producing water for non-potable use were found to be largely ineffective at removing ARGs, but plants using advanced oxidation processes or ozonation paired with biofiltration to produce direct potable reuse water were highly effective at removing ARGs. Non-potable reclaimed water consistently had greater quantities of sul1, a sulfonamide ARG, and Legionella and Mycobacterium, two OPs of significant public health concern, present than corresponding potable systems. Limited regrowth of OPs and ARGs did occur in simulated premise (i.e., building) plumbing systems operated with direct potable reuse waters, but regrowth was comparable to that observed in systems fed with potable water derived from surface or groundwater. Advancements were also made in understanding the role of several hypothesized driving forces shaping the antibiotic resistome in natural and engineered water systems: selection by antimicrobials and other compounds, horizontal gene transfer, and microbial community composition. Finally, whole-genome and metagenomic characterization were applied together towards profiling L. pneumophila in clinical and water samples collected from Flint, Michigan, where an economically-motivated switch to an alternative water source created conditions favorable for growth of this organism and likely triggered one of the largest Legionnaires' Disease outbreaks in U.S. history. / PHD / Population growth, urbanization, drought, and climate change have all driven many U.S. municipalities to utilize alternative water sources, such as recycled wastewater, to offset demand on traditional potable water sources. Many water providers have moved towards a modern paradigm of utilizing multiple available water sources, recognizing the interconnectedness of various components of the urban water cycle, leading to opportunities to improve sustainability, optimize infrastructure use, stimulate economic growth, increase coordination among water agencies, and identify new water resources from which to meet consumer needs. Though advancements in treatment technologies throughout the twentieth century have largely succeeded in eliminating waterborne disease outbreaks associated with contamination of municipal water supplies by fecal pathogens in developed countries, several microbial contaminants of emerging concern (MCECs) have garnered attention. Two major groups of MCECs are considered in this dissertation: antibiotic resistance, including antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG), and opportunistic pathogens (OP), such as Legionella pneumophila, the causative agent of Legionnaires’ Disease. ARB are a rising cause of disease around the world and are a major challenge to modern medicine because they make antibiotics used for treatment ineffective. OPs, the leading cause of waterborne disease in the U.S. and other developed countries, have become prevalent in engineered water systems where low nutrient concentrations, warm water temperatures, and long stagnation times can facilitate their growth. Immunocompromised people, including smokers and the elderly, are especially vulnerable to infection with OPs. The role of the urban water cycle in facilitating the spread of these MCECs is not well understood. Here they were studied across several compartments of the urban water cycle, including surface water, stormwater, wastewater, recycled water (spanning a variety of intended uses, from non-potable to direct potable reuse), and potable water. Field studies were conducted of two watersheds impacted by stormwater runoff, one in the arid Colorado Front Range under conditions of a rare, 1-in-1,000 year rainfall event, and one in the humid climate of southwest Virginia, during three summer storms. Both studies demonstrated the prevalence of ARGs in urban stormwater compared to pristine, unimpacted sites, or to days when no precipitation was recorded. The role of wastewater reuse in transmitting OPs and ARGs was also investigated. Wastewater treatment plants producing water for non-potable use (i.e. applications such as irrigation, but not for human consumption) were found to be largely inefficient at removing ARGs, and this reclaimed water consistently had greater quantities of the sul1 ARG present than in corresponding potable systems. In these systems, genes associated with the OPs Legionella and Mycobacterium as well as total bacteria were more abundant in reclaimed water than in corresponding potable systems. In more advanced treatment plants utilizing advanced oxidation processes or ozonation paired with biofiltration to produce direct potable reuse water (i.e. water fit for human consumption), ARGs were very effectively removed by treatment, with abundances often found to be higher in corresponding potable waters derived from surface or groundwater. Limited regrowth of ARGs as well as OPs did occur in simulated home plumbing systems operated with these waters, but regrowth was comparable to that observed in systems fed with potable water derived from surface or groundwater. Finally, a study of L. pneumophila in the Flint, Michigan drinking water system during use of an alternative water source that has been identified as a likely cause of two Legionnaires’ Disease outbreaks revealed presence of multiple strains of the OP in the system. Genomic comparisons revealed that strains isolated from hospital and residential water samples were highly similar to clinical strains associated with the outbreaks. Advancements were also made in understanding the role of several hypothesized driving forces in shaping the antibiotic resistome in natural and engineered water systems: selection by antimicrobials and other compounds, horizontal gene transfer, and microbial community composition. Together, these chapters describe an advancement in knowledge regarding the occurrence of OPs and ARGs in a variety of water systems, and highlight trends that may be of value in developing management strategies for limiting regrowth or transmission of these bacteria in various compartments of the urban water cycle.

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.

Biofilms are communities of microorganisms that can form in the human microbiome and on medical implants among other locations. These communities provide greater protection for their member cells resulting in an increase in resistance to antibiotic treatment and persistent infections. There are several factors that may contribute to antibiotic resistance of biofilms. These studies were done concurrently with biological experiments to test the hypothesis that dense, rigid structures within the biofilm may be an additional mechanism for protection from antibiotics. A computational tool and workflow was developed to analyze bead movement for the characterization of biofilm biomaterial properties including rigidity. With this tool, the analysis revealed that the amyloid, curli, confers rigidity in biofilms, thereby restricting bead movement. Greater movement of the beads is seen in biofilms lacking curli and biofilms that produced complex heterogeneous rigid structures. A new model was also developed that uses microscopy imaging data to simulate diffusion-reaction of antibiotics within heterogeneous biofilms. This model was used to investigate the effect of the dense, rigid structures on antibiotic treatment through test simulations and simulations using biological imaging data. These studies reveal various properties about the dense, rigid structures that confer protection. / Mathematics

As the population in water stressed areas increases, it is critical that wastewater treatment plants (WWTPs) continue to replenish depleted water supplies, and serve as an alternative water source. WWTPs depend on microorganisms in activated sludge to remove pollutants from wastewater and therefore an understanding of how these microorganisms are affected by various conditions and pollutants is needed. Also, as consumer products and industrial processes evolve, so do the pollutants they discharge to wastewater. In order to keep pace with these changes, understanding the effects of emerging contaminants to WWTP processes is essential. The research herein assesses microbial community dynamics of the response of nitrifying microorganisms in activated sludge to variation in ammonia concentration and evaluates the impact of engineered nanoparticles on activated sludge microbial communities and other emerging pollutants, such as antibiotic resistance genes and disinfection by-products. In order to assess microbial community dynamics of the response of nitrifying microorganisms to removal of ammonia in the feed, nitrifying activated sludge reactors were operated at various relevant temperatures and the nitrifying microbial community was characterized using activity assays and bio-molecular techniques. We found that Nitrospira spp. were the dominant nitrifying microorganisms, exhibiting stable relative abundance across multiple trials and over a range of temperatures. These results indicate the possibility of comammox bacteria in the system and highlight the complexity of nitrifying microbial communities in activated sludge relative to past understanding. Both microbial and chemical impacts of engineered nanoparticles on WWTP processes were also investigated. Metagenomic analysis of DNA extracted from activated sludge sequencing batch reactors dosed with gold nanoparticles with varied surface coating and morphology indicated that nanoparticle morphology impacted the microbial community and antibiotic resistance gene content more than surface coating. However, nanoparticle fate was controlled by surface coating more than morphology. Disinfection by-product formation in the presence of nanoparticles during WWTP disinfection was assessed using silver, titanium dioxide, ceria, and zero valent iron nanoparticles. Silver nanoparticles were found to enhance trihalomethane formation, which was attributed to the citrate coating of the nanoparticles. These studies both raise concern over the relationship between engineered nanoparticles and other emerging concerns in WWTPs, and take a step towards informing nanoparticle design in a manner that limits their associated environmental impact. / Ph. D. / Wastewater treatment plants (WWTPs) are crucial to protect human and environmental health by removing pathogens and pollutants in sewage before they are released into aquatic environments used for recreation and drinking water. As populations living in water stressed areas continues to rise, the continued recovery of clean water from WWTPs is essential to both replenish water supplies and serve as an alternative water source. WWTPs depend on a complex mixture of microorganisms called activated sludge to remove pollutants from water. In order for WWTPs to continue discharging acceptable water in the future, a greater understanding of how these important microorganisms respond to environmental changes such as temperature and sewage content is needed. Sewage flowing into WWTPs is also evolving as advances in technology and chemicals used in consumer products and industrial settings discharge new pollutants into waste streams. Therefore, an understanding for how these new pollutants affect WWTP processes is also needed. In this dissertation, two challenges facing WWTPs were evaluated: 1) how bacteria responsible for nitrogen removal in WWTPs respond to the stress of starvation, and 2) how engineered nanoparticles in sewage impact the microorganisms in activated sludge and disinfection in WWTPs. Nitrogen removal is important because it can cause algal blooms when treated wastewater is discharged and because some forms, like ammonia, are toxic. The first step of nitrogen removal in WWTPs involves forming nitrate from ammonia, performed by nitrifying bacteria and archaea. This nitrate is then transformed into nitrogen gas by other microorganisms and therefore removed from the wastewater. How nitrifying microorganisms responded to decreased ammonia concentrations in the feed was determined using nucleic acid based techniques. Traditionally it is thought that in wastewater treatment, ammonia is oxidized to nitrite by one group of microorganisms, and nitrite is then oxidized to nitrate by separate microorganisms. However, in this study only microorganisms from the latter group were detected, which demonstrates the possibility of microorganisms capable of both ammonia and nitrite oxidation present in our system (as has been found in other environments). Also, the increased use of engineered nanoparticles in consumer products and industrial processes has led to their increased presence in wastewater. Nanoparticle are particles that are 1-100 nm in one dimension and have unique properties compared to larger forms of the material they are made of. These particles are sometimes utilized for their antimicrobial activity and therefore may impact the microorganisms used in WWTPs. Using activated sludge bioreactors dosed with gold nanoparticles with various morphologies and surface coatings, implications of these nanoparticle properties on activated sludge microorganisms was assessed. We found that nanoparticle morphology was more important than surface coating in affecting the activated sludge microbial communities. However, gold nanoparticle fate in the bioreactors was determined more by surface coating than morphology. These results and further research on how nanoparticle properties affect WWTPs and the environment may inform nanoparticle design that can be tailored to decrease environmental impact. The impact of nanoparticles on WWTP disinfection processes was also evaluated. WWTPs often use chlorine and/or ultraviolet (UV) disinfection in order to inactivate pathogens in wastewater. Chemical reactions between organics in the wastewater and chlorine produce disinfection by-products which can be toxic. Nanoparticles are used to enhance desired chemical reactions in industry, and therefore may enhance the undesired reactions of disinfection by-product formation in WWTPs. Here several types of nanoparticle (silver, titanium dioxide, ceria, and zero valent iron) were dosed to WWTP effluents and then subjected to chlorine and/or UV disinfection, then this was analyzed for trihalomethanes (a common type of disinfection by-product). It was found that the citrate coating on silver nanoparticles led to increased trihalomethane formation. More research is needed to determine the mechanisms involved with this phenomenon, and to determine other nanoparticle-coating combinations that may have similar effect.

The antibiotic resistance is a natural phenomenon that can be considered harmful for humans when antibiotic resistant bacteria transfers resistance genes to the human pathogens at uncontrollable rate. One of the potential environments for spreading of antibiotic resistance is found to be in biogas reactors due to their mesophilic conditions that are optimal for the bacterial reproduction. The sudden changes in the environment of biogas reactors could negatively affect both biogas production rate but also microbial reproduction ability. This experimental research was done to determine to what extend does changes in pH with the increased volume dosing of acetic acid affect the persistence of antibiotic resistant enterobacteria in biogas reactors. Additionally, this experimental research is aiming to determine the difference in the enterobacterial persistence between year 2023 and year 2024 for the two biogas reactors (Selena and Taylor) fed with the chemically based substrate. The results show that the general persistence of antibiotic resistant enterobacteria in both reactors increased year 2024 compared to year 2023. According to the blank samples, the average persistence of enterobacteria increased in Selena by 500% and in Taylor by 250%. However, overall increase of enterobacteria that are antibiotic resistant cannot be determined where it can only be observed that the resistance has increased against majority of tested antibiotics. The obtained results also show a trend that in most tested groups quantity of antibiotic resistant enterobacteria is gradually increasing with the higher volume of acetic acid. The pH level decreased with the increased volume of acetic acid prior the incubation process with an average of 6,4 for Taylor biogas reactor and 6,7 for Selena biogas reactor. This indicates that the composition of fatty acid affected the environment so that it became more acidic. However, after the incubation process, the increased pH was detected in all groups compared to the results prior the incubation with the pH average of 8,1 for Taylor biogas reactor and 7,9 for Selena biogas reactor. This indicates that the acetic acid was consumed by the present bacteria and a decrease of acetic acid quantity resulted in an increase of pH. With that being said, the acetic acid can be considered as the enrichment substrate for the enterobacterial colonies adequate for efficient exchange of antibiotic resistant genes hence reproduction. However, the statistical analysis (nonparametric Kruskal-Wallis) results show that only between minority of tested groups the significant difference was detected.