The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners in detergents) were used in the summers of 2004 and 2005 to determine the origins of fecal pollution at beaches with a past history of, or the potential for, high enterococci counts and posted advisories. At Hilton and Anderson beaches, ARA and fluorometry in the summer of 2004 detected substantial human-origin pollution in locations producing consistently high counts of Enterococcus spp. Investigations by municipal officials led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of these mitigation efforts was confirmed during the summer of 2005 using ARA and fluorometry, with the results cross-validated by pulsed-field gel electrophoresis (PFGE). Results at other beaches indicated that birds and/or wildlife were largely responsible for elevated enterococci levels during 2004 and 2005. The application of fluorometry proved difficult in opens waters due to high levels of dilution, but showed potential for use in storm drains. An additional study developed and tested a new library-based MST approach based on the pattern of DNA band lengths produced by the amplification of the 16S-23S rDNA intergenic spacer region, and subsequent digestion using the restriction endonuclease MboI. Initial results from small known-source libraries yielded high average rates of correct classification (ARCC). However, an increase in the library size was accompanied by a reduction in the ARCC of the library and the method was deemed unsuccessful, and unsuitable for field application. A final study focused on the potential for classification bias with disproportionate source category sizes using discriminant analysis (DA), logistic regression (LR), and k-nearest neighbor (K-NN) statistical classification algorithms. Findings indicated that DA was the most robust algorithm for use with source category imbalance when measuring both correct and incorrect classification rates. Conversely k-NN was identified as the most sensitive algorithm to imbalances with the greatest levels of distortion obtained from the highest k values. Conclusions of this project include: 1) application of a validation set, as well as a minimum detectable percentage to known-source libraries aids in accurately assessing the classification power of the library and reducing the false positive identification of contributing fecal sources; 2) the validation of MST results using multiple methods is recommended for field applications; 3) fluorometry displayed potential for detecting optical brighteners as indicators of sewage leaks in storm drains; 4) the digestion of the 16S-23S rDNA intergenic spacer region of Enterococcus spp. using MboI does not provided suitable discriminatory power for use as an MST method; and 5) DA was the least, and k-NN the most, sensitive algorithm to imbalances in the size of source categories in a known-source library. / 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.

Yes / N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. / University of Malaya High Impact Research (HIR) Grant (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027) given to K.G.C. and National Natural Science Foundation of China (no. 81260481) given to H.W.

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.

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.

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.

Tesis por compendio / [ES] Los residuos de antibióticos en la leche constituyen un aspecto de gran importancia para la salud pública y pueden tener un efecto negativo en la industria láctea. Para proteger al consumidor se han establecido los Límites Máximos de Residuos (LMR) para diferentes antibióticos en la leche cruda. Sin embargo, la transferencia de estas sustancias de la leche al queso ha sido poco estudiada y, por lo tanto, se desconoce el impacto de la presencia de antibióticos sobre el proceso de elaboración y la seguridad del queso. El objetivo de la tesis ha sido evaluar la transferencia de los antibióticos más empleados en el ganado caprino lechero, desde la leche a quesos curados y frescos, así como estudiar el efecto sobre el proceso de fabricación y la calidad de los quesos durante la maduración. Para alcanzar el objetivo se han realizado elaboraciones de quesos curados (60 días) a partir de leche en presencia de diversos antibióticos (amoxicilina, bencilpenicilina, cloxacilina, eritromicina, ciprofloxacina, enrofloxacina y oxitetraciclina) a niveles admisibles (LMR) y fabricaciones de leche libre de antibiótico como referencia. Los resultados obtenidos sugieren que la mayor parte de los antibióticos en la leche no afectaron al proceso de fabricación del queso. Solamente la eritromicina y oxitetraciclina incrementaron el tiempo requerido en el proceso de elaboración. Respecto a las características del queso evaluadas a diferentes tiempos de maduración (0, 30 y 60 días), las pocas diferencias encontradas se refirieron a la concentración de ácidos grasos libres, el color y las propiedades de textura, pero en su mayoría no fueron detectadas en la evaluación sensorial. Sin embargo, cantidades variables de antibióticos, se transfirieron de la leche al queso (entre 7,4 y 68%). Las quinolonas y oxitetraciclina presentaron las más elevadas tasas de retención, así como persistencia a lo largo de la maduración, con altas concentraciones de quinolonas (enrofloxacina: 148±12 µg/kg; ciprofloxacina: 253±24 µg/kg) y de oxitetraciclina (20±5,7 µg/kg) después de los 60 días de curado. En el estudio de quesos frescos, se evaluó la presencia de residuos de medicamentos en la leche pasteurizada y en los quesos frescos a partir de leche de cabra con antibióticos (amoxicilina, bencilpenicilina, cloxacilina, neomicina, eritromicina, ciprofloxacina, enrofloxacina y oxitetraciclina) a niveles de seguridad (LMR). Los resultados mostraron que altas cantidades de antibióticos, entre el 71 y el 100% de la concentración inicial en la leche cruda, permanecieron en la leche pasteurizada y que fueron transferidas al queso en gran medida, con porcentajes de retención que oscilaron entre 37,5 y 75%. Con respecto a los márgenes de seguridad de estos productos, se calcularon teniendo en cuenta diferentes grupos de edades (niños, adolescentes y adultos), y los efectos negativos de estos antibióticos sobre la salud. Los resultados indicaron que el mínimo margen de seguridad en la leche pasteurizada lo presentaron la ciprofloxacina, enrofloxacina, y eritromicina para el grupo de niños. En relación al queso fresco, se obtuvo un elevado margen de seguridad para todos los antibióticos y grupos de edad considerados, lo que sugiere que este producto probablemente no tenga efectos negativos en la salud del consumidor. Es importante enfatizar que concentraciones relativamente altas de antibióticos podrían permanecer en la leche de cabra pasteurizada y productos relacionados, como los quesos frescos y de corta maduración. La presencia de estos antibióticos podría contribuir al desarrollo y propagación de la resistencia a los antimicrobianos que actualmente constituye un grave problema a nivel mundial. Los resultados de este trabajo podrían servir a las autoridades de salud pública para evaluar si los sistemas actuales de control de antibióticos en la leche y los productos lácteos son adecuados o deberían ser revisados. / [CA] Els residus d'antibiòtics en la llet animal constitueixen un aspecte de gran importància per a la salut pública, i poden tindre un efecte negatiu en la indústria làctia. Per a protegir al consumidor s'han establit els Límits Màxims de Residus (LMR) per a diferents medicaments veterinaris en la llet crua. No obstant això, la transferència d'estes substàncies de la llet al formatge ha sigut poc estudiada i, per tant, es desconeix el impacte de la presència de antibiòtics sobre el procés d'elaboració i la seguretat del formatge. L'objectiu de la tesi ha sigut avaluar la transferència dels antibiòtics més empleats en el bestiar caprí lleter, des de la llet a formatges curats i frescos, així com l'efecte sobre el procés de fabricació i la qualitat dels formatges durant la maduració. Per a aconseguir l'objectiu s'han realitzat elaboracions de formatges curats (60 dies) a partir de llet en presència de diversos antibiòtics (amoxicilina, bencilpenicilina, cloxacilina, eritromicina, ciprofloxacina, enrofloxacina i oxitetraciclina) a nivells admissibles (LMR) i fabricacions de llet lliure d'antibiòtic (control) com a referència. Els resultats obtinguts suggerixen que la major part dels antibiòtics en la llet no van afectar el procés de fabricació del formatge. Només l'eritromicina i l'oxitetraciclina van incrementar el temps requerit en el procés d'elaboració del formatge. Respecte a les característiques del formatge avaluades a diferents temps de maduració (0, 30 i 60 dies), les poques diferències trobades es van referir a la concentració d'àcids greixos lliures, el color i les propietats de textura, però majoritàriament no van ser detectades en l'avaluació sensorial. No obstant això, quantitats variables d'antibiòtics, es van transferir de la llet al formatge (entre 7,4 i 68%). Les quinolones i l'oxitetraciclina van presentar les més elevades taxes de retenció, així com persistència al llarg de la maduració, amb altes concentracions de quinolones (enrofloxacina: 148±12 µg/kg; ciprofloxacina: 253±24 µg/kg) i d'oxitetraciclina (20±5,7 µg / kg) després dels 60 dies de curació. En l'estudi de formatges frescos, es va avaluar la presència de residus de medicaments en la llet pasteuritzada i en els formatges frescos a partir de llet de cabra amb antibiòtics (amoxicilina, bencilpenicilina, cloxacilina, neomicina, eritromicina, ciprofloxacina, enrofloxacina i oxitetraciclina) a nivells de seguretat (LMR). Els resultats van mostrar que altes quantitats d'antibiòtics, entre el 71 i el 100% de la concentració inicial en la llet crua, van romandre en la llet pasteuritzada i que van ser transferides al formatge en gran manera, amb percentatges de retenció que van oscil·lar entre 37,5 i 75%. Respecte als marges de seguretat d'estos productes, es van calcular tenint en compte diferent grups d'edats (xiquets, adolescents i adults), i els efectes negatius d'estos antibiòtics sobre la salut. Els resultats van indicar que el mínim marge en la llet pasteuritzada ho van presentar la ciprofloxacina, la enrofloxacina, i l'eritromicina per al grup de xiquets. En relació al formatge fresc, es va obtindre un elevat marge de seguretat per a tots els antibiòtics i grups d'edat considerats, la qual cosa suggereix que este producte probablement no tinga efectes negatius en la salut del consumidor. És important emfatitzar que concentracions relativament altes d'antibiòtics podrien romandre en la llet de cabra pasteuritzada i productes relacionats, com els formatges frescos i de curta maduració. La presència d'estos antibiòtics podria contribuir al desenrotllament i propagació de la resistència als antimicrobians que actualment constitueix un greu problema a nivell mundial. Els resultats d'este treball podrien servir les autoritats de salut pública per a avaluar si els sistemes actuals de control d'antibiòtics en la llet i els productes lactis són adequats o haurien de ser revisats. / [EN] Antibiotic residues in milk are a great concern for public health, and they have negative implications for the dairy industry. To guarantee the safety of milk and derivates, Maximum Residues Limits (MRL) for different veterinary drugs in raw milk have been established. However, the transfer of these substances from milk to cheese, has been poorly studied and, therefore, the impact of the use of raw milk containing antibiotics on the cheese-making and the cheese safety is thus far unknown. The aim of this thesis was to evaluate the transfer of the most widely used antibiotics used in dairy goats, from milk to matured and fresh cheese, as well as their effect on the cheese-making process and the quality characteristics of the cheeses during ripening. To this end, antibiotic-free milk was spiked individually with seven veterinary drugs (amoxicillin, benzylpenicillin, cloxacillin, erythromycin, ciprofloxacin, enrofloxacin and oxytetracycline) and used to make mature Tronchón cheeses, which were analyzed at different ripening times (0, 30, and 60 days) to determine antibiotic residues, pH, chemical composition, proteolytic and lipolytic activities, color, and textural properties. The cheese-making process was unaffected by the presence of most antibiotics in milk. Only erythromycin and oxytetracycline significantly increased the time required for cheese production. Regarding cheese characteristics, the few differences found were related to the free fatty acid concentration, color and textural properties, which remained mostly undetected by the sensory analysis panelists. However, variable amounts of antibiotics, ranging from 7.4 to 68 %, were transferred from milk spiked with antibiotics, to cheese. Oxytetracycline and quinolones presented the highest retention rates as well as persistence along maturation, with high concentrations of quinolones (enrofloxacin: 148±12 µg/kg, ciprofloxacin: 253±24 µg/kg) and oxytetracycline residues (20±5.7 µg/kg) in cheeses after 60 days of ripening. In the study of fresh cheeses was evaluated the presence of drug residues in pasteurized fluid milk and fresh cheeses obtained from goat milk containing antibiotics (amoxicillin, benzylpenicillin, cloxacillin, neomycin, erythromycin, ciprofloxacin, enrofloxacin and oxytetracycline) at safety levels (MRLs). The safety margin of these dairy products for consumers was also evaluated. Results showed that high amounts of antibiotics, between 71-100% of the initial concentration in raw milk, remained in pasteurized goat milk and were transferred to cheese to a high extent, with retention percentages ranging from 37.5 to 75%. Regarding the safety margin of goat milk products, calculated taking into account different age groups (children, teenagers and adults), and the published negative effects of such antibiotics on consumer health, results indicate that the minimum safety margin of pasteurized milk was obtained for ciprofloxacin, enrofloxacin and erythromycin in the group of children. Regarding fresh cheese, an elevated safety margin was obtained for all antibiotics and age groups considered. It is important to emphasize that relatively high concentrations of antibiotics could remain in pasteurized fluid goat milk and related products such as fresh cheese and cheeses of a short ripening period. The presence of these antibiotics in dairy products might contribute to the development and spread of antimicrobial resistance, which is considered an important public health concern worldwide. The result of this research could serve the public health authorities to assess if current control systems of antibiotics in milk and dairy products are adequate or have to be revised. / Por último, quiero dar las gracias a la educación pública que permite que la economía no sea una barrera para la formación y al “Programa de ayudas a la investigación” (FPI-2014) de la Universitat Politècnica de Valencia, que me concedió la oportunidad de realizar está investigación durante los 4 años de trabajo. / Quintanilla Vázquez, PG. (2019). Effect of antibiotics in goat milk on the manufacture and characteristics of cheese [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125701 / Compendio

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.