Tesis por compendio / [EN] Legionella pneumophila is a strictly environmental and opportunistic pathogen that can cause severe pneumonia after inhalation of aerosols with enough bacterial load. Outbreaks and sporadic cases are usually localized in temperate environments, and the reservoirs are often water-related sources where biofilms are created. The existence of non-cultivable forms of the bacteria increases the risk for public health, as culture-based methods may miss them, thus complicating the environmental investigations of the sources. Genetic classification through the Sequence-Based Typing (SBT) technique allowed an increased discrimination among L. pneumophila strains compared to previous methods. SBT data can also be used for genetic variability and population structure studies, but a more exhaustive analysis can be performed using high-throughput genome sequencing strategies. This thesis describes the use of both SBT and genomic sequencing to evaluate and provide solutions to different public health needs in L. pneumophila epidemiology. We have focused in the Comunidad Valenciana (CV), the second region in Spain with the highest incidence of Legionellosis, with special interest in the city of Alcoy, where recurrent outbreaks have occurred since 1998. Firstly, SBT data were used to gain a deeper insight into the genetic variability and distribution of the most abundant Sequence Types (ST) in the CV area. We have shown that the level of variability in this region is comparable to that from other countries, revealing the existence of both locally and broadly extended profiles. Approximately half of the observed genetic diversity was found to result from geographical and temporal structure. Secondly, L. pneumophila detection from environmental sources remains a challenge for public health. A comparison between water and biofilm samples using a sensitive touchdown PCR (TD-PCR) strategy revealed that the use of biofilms increased by ten-fold the detection rate. This method allowed evaluating the hidden uncultivable L. pneumophila diversity in the locality of Alcoy and the real-time investigation of a Legionellosis outbreak affecting a hotel in Calpe (Southeast of Spain) in 2012. Thirdly, genomic sequencing was applied to a set of 69 strains isolated during 13 outbreaks occurred in Alcoy in the period 1999-2010, mainly the recurrent ST578. Higher intra-outbreak variability than expected was observed, pointing to the potential existence of multiple sources in this endemic area or high environmental diversity. Interestingly, above 98% of the genomic variability in this ST was found as being incorporated through recombination processes rather than through point mutations. Finally, a metagenomic analysis of environmental biofilms from Alcoy revealed a microbial community dominated by Proteobacteria, Cyanobacteria, Actinobacteria and Bacteroidetes. Despite the known endemism of Legionella in this area, the genus was only found in a relative abundance ranging 0.01-0.07%, which explains the low recovery from environmental sources. In summary, the results from this thesis can benefit public health efforts to control this pathogen in the environment, as we provide new insight into its molecular epidemiology, with immediate applications to surveillance and outbreak investigations. / [ES] Legionella pneumophila es un patógeno oportunista estrictamente ambiental capaz de causar neumonía debido a la inhalación de aerosoles con suficiente carga bacteriana. Los brotes y casos esporádicos suelen producirse en ambientes templados y los reservorios encontrarse en zonas con agua donde pueden crearse biopelículas microbianas. La existencia de formas no cultivables de la bacteria aumenta el riesgo para la salud pública, ya que los métodos estándar basados en cultivo microbiológico no pueden detectarlas, complicando las investigaciones ambientales. La clasificación genética basada en el método Sequence-Based Typing (SBT) permite un mayor poder de discriminación entre cepas de L. pneumophila en comparación con métodos previos. Los datos derivados del SBT pueden utilizarse para estudios de variabilidad genética y estructura poblacional. Sin embargo, puede llevarse a cabo un análisis más exhaustivo mediante técnicas de secuenciación genómica de alto rendimiento. Esta tesis describe la utilización tanto de SBT como de secuenciación genómica para evaluar e incluso proponer soluciones a diferentes necesidades en salud pública relacionadas con la epidemiología de L. pneumophila. Nos centramos en la Comunidad Valenciana (CV), la segunda región en España con mayor incidencia de Legionelosis, con especial interés en la localidad de Alcoy, donde ocurren brotes de forma recurrente. En primer lugar, utilizamos datos derivados de SBT para conocer mejor la variabilidad y la distribución de los perfiles genéticos (Sequence Types, ST) en el área de la CV. Mostramos que el nivel de variabilidad en sólo esta región es comparable a la de otros países, con perfiles extendidos local y globalmente. Aproximadamente la mitad de la diversidad genética observada se estima que procede de estructuración geográfica y temporal. En segundo lugar, la detección de L. pneumophila a partir de fuentes ambientales sigue suponiendo un reto para la salud pública. En esta tesis realizamos una comparación entre la detección mediante touchdown PCR (TD-PCR) a partir de muestras de agua y biopelículas microbianas y mostramos que estas últimas proporcionan un aumento de 10 veces en la tasa de detección de la bacteria. Este método permitió evaluar la diversidad no cultivable de L. pneumophila en la localidad de Alcoy y la investigación a tiempo real de un brote en un hotel en Calpe (Sudeste de España) en 2012. A continuación, aplicamos la secuenciación genómica a 69 cepas aisladas durante 13 brotes ocurridos en Alcoy en el período 1999-2010, principalmente el recurrente ST578. Se observó mayor variabilidad entre cepas de un mismo brote que la esperada, lo cual apunta a la existencia potencial de múltiples fuentes en este área, o alta diversidad ambiental. Además, se observó que más del 98% de la variabilidad genómica fue introducida por procesos de recombinación y no de mutación puntual. Finalmente, se realizó un análisis metagenómico de biopelículas ambientales recogidas en Alcoy. Se encontró que la comunidad está dominada por Proteobacteria, Cyanobacteria, Actinobacteria y Bacteroidetes. A pesar del conocido endemismo de Legionella en el área, este género sólo se encontró en una abundancia relativa entre 0.01-0.07%, lo cual explica su baja tasa de recuperación a partir de muestras ambientales. En resumen, los resultados de esta tesis pueden ser de utilidad para los programas de control de este patógeno llevados a cabo por las autoridades de salud pública, ya que proporcionan una nueva percepción de su epidemiología molecular, con aplicación inmediata a la vigilancia e investigación de brotes. / [CA] Legionella pneumophila és un patogen oportunista estrictament ambiental capaç d'ocasionar pneumònia degut a la inhalació d'aerosols amb la suficient carga bacteriana. Els brots i casos esporàdics solen ocórrer en ambients temperats, i els reservoris solen trobar-se en zones amb aigua on poden crear-se biopel·lícules microbianes. La existència de formes no cultivables del bacteri augmenten el risc per a la salut pública, ja que els mètodes estàndard basats en el cultiu microbiològic no poden detectar-les, complicant les investigacions ambientals. La classificació genètica basada en el mètode Sequence-Based Typing (SBT) permet un major poder de discriminació entre soques de L. pneumophila en comparació amb previs mètodes. Les dades derivades del SBT poden utilitzar-se per a estudis de variabilitat genètica i estructura poblacional, però un anàlisis més exhaustiu pot dur-se a terme a través de tècniques de seqüenciació genòmica d'alt rendiment. Esta tesis descriu la utilització tant del SBT com de la seqüenciació genòmica per a avaluar i proposar solucions a diferents necessitats en salut pública relacionades amb l'epidemiologia de L. pneumophila. Ens centrem en la Comunitat Valenciana (CV), la segona regió d'Espanya amb la major incidència de Legionel·losi, amb especial interès en la localitat d'Alcoi, on els brots ocorren de forma recurrent des de 1998. Primer, hem utilitzat dades derivades del SBT per a conèixer millor la variabilitat i la distribució dels perfils genètics (Sequence Types, ST) en l'àrea de la CV. Mostrem que el nivell de variabilitat en només aquesta regió és comparable a la d'altres països, amb perfils estesos tant de forma local com més amplia. Aproximadament la meitat de la diversitat genètica observada s'estima que procedeix d'estructuració geogràfica i temporal. Segon, la detecció de L. pneumophila a partir de fonts ambientals continua suposant un repte per a la salut pública. En aquesta tesis realitzem una comparació entre la detecció mitjançant touchdown PCR (TD-PCR) a partir de mostres d'aigua i biopel·lícules microbianes i mostrem que aquestes últimes proporcionen un augment de deu vegades en la tassa de detecció. A més, aquest mètode ens va permetre avaluar la diversitat no cultivable de L. pneumophila a la localitat d'Alcoi i la investigació a temps real d'un brot de Legionelosis que va afectar a un hotel en Calp (Sud-est d'Espanya) a l'any 2012. Tercer, vam aplicar la seqüenciació genòmica a 69 soques aïllades durant 13 brots ocorreguts a Alcoi en el període 1999-2010, principalment el recurrent ST578. Es va observar una major variabilitat entre soques d'un mateix brot de l'esperada, apuntant a l'existència potencial de múltiples fonts en aquesta àrea, considerada endèmica, o alta diversitat ambiental. A més, es va observar que més del 98% de la variabilitat genòmica havia sigut introduïda a partir de processos de recombinació i no de mutació puntual. Finalment, es va realitzar una anàlisi metagenòmica de biopel·lícules ambientals recollides a Alcoi. Varem trobar que la comunitat està dominada per Proteobacteria, Cyanobacteria, Actinobacteria i Bacteroidetes. A pesar del conegut endemisme de Legionella en l'àrea, aquest gènere només es va trobar en una abundància relativa entre 0.01-0.07%, el qual explica la seua baixa tassa de recuperació a partir de mostres ambientals. En resum, els resultats d'aquesta tesi poden ser d'utilitat per als programes de control d'aquest patogen duts a terme per les autoritats de salut pública, ja que proporcionen una nova percepció de la seua epidemiologia molecular, amb aplicació immediata a la vigilància i la investigació de brots. / Sánchez Busó, L. (2015). Genetic and genomic variability of Legionella pneumophila: applications to molecular epidemiology and public health [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52854 / Premios Extraordinarios de tesis doctorales / Compendio

[EN] One of the main challenges of drinking water utilities is to ensure high quality supply, in particular, in chemical and microbiological terms. However, biofilms invariably develop in all drinking water distribution systems (DWDSs), despite the presence of residual disinfectant. As a result, water utilities are not able to ensure total bacteriological control. Currently biofilms represent a real paradigm in water quality management for all DWDSs. Biofilms are complex communities of microorganisms bound by an extracellular polymer that provides them with structure, protection from toxics and helps retain food. Besides the health risk that biofilms involve, due to their role as a pathogen shelter, a number of additional problems associated with biofilm development in DWDSs can be identified. Among others, aesthetic deterioration of water, biocorrosion and disinfectant decay are universally recognized. A large amount of research has been conducted on this field since the earliest 80's. However, due to the complex environment and the community studied most of the studies have been developed under certain simplifications. We resort to this already done work and acquired knowledge on biofilm growth in DWDSs to change the common approaches of these studies. Our proposal is based on arduous preprocessing and posterior analysis by Machine Learning approaches. A multi-disciplinary procedure is undertaken, helping as a practical approach to develop a decision-making tool to help DWDS management to maintain, as much as possible, biofilm at the lowest level, and mitigating its negative effects on the service. A methodology to detect the more susceptible areas to biofilm development in DWDSs is proposed. Knowing the location of these hot-spots of the network, mitigation actions could be focused more specifically, thus saving resources and money. Also, prevention programs could be developed, acting before the consequences of biofilm are noticed by the consumers. In this way, the economic cost would be reduced and the service quality would improve, eventually increasing consumers' satisfaction. / [ES] Uno de los principales objetivos de las empresas encargadas de la gestión de los sistemas de distribución de agua potable (DWDSs, del inglés Drinking Water Distribution Systems) es asegurar una alta calidad del agua en su abastecimiento, tanto química como microbiológica. Sin embargo, la existencia de biofilms en todos ellos, a pesar de la presencia de desinfectante residual, hace que no se pueda asegurar un control bacteriológico total, por lo que, hoy en día, los biofilms representan un paradigma en la gestión de la calidad del agua en los DWDSs. Los biofilms son comunidades complejas de microorganismos recubiertas de un polímero extracelular que les da estructura y les ayuda a retener el alimento y a protegerse de agentes tóxicos. Además del riesgo sanitario que suponen por su papel como refugio de patógenos, existen muchos otros problemas asociados al desarrollo de biofilms en los DWDSs, como deterioro estético del agua, biocorrosión y consumo de desinfectante, entre otros. Una gran cantidad de investigaciones se han realizado en este campo desde los primeros años 80. Sin embargo, debido a la complejidad del entorno y la comunidad estudiada la mayoría de estos estudios se han llevado a cabo bajo ciertas simplificaciones. En nuestro caso, recurrimos a estos trabajos ya realizados y al conocimiento adquirido sobre el desarrollo del biofilm en los DWDSs para cambiar el enfoque en el que normalmente se enmarcan estos estudios. Nuestra propuesta se basa en un intenso pre-proceso y posterior análisis con técnicas de aprendizaje automático. Se implementa un proceso multidisciplinar que ayuda a la realización de un enfoque práctico para el desarrollo de una herramienta de ayuda a la toma de decisiones que ayude a la gestión de los DWDSs, manteniendo, en lo posible, el biofilm en los niveles más bajos, y mitigando sus efectos negativos sobre el servicio de agua. Se propone una metodología para detectar las áreas más susceptibles al desarrollo del biofilm en los DWDSs. Conocer la ubicación de estos puntos calientes de biofilm en la red permitiría llevar a cabo acciones de mitigación de manera localizada, ahorrando recursos y dinero, y asimismo, podrían desarrollarse programas de prevención, actuando antes de que las consecuencias derivadas del desarrollo de biofilm sean percibidas por los consumidores. De esta manera, el coste económico se vería reducido y la calidad del servicio mejoraría, aumentando, finalmente, la satisfacción de los usuarios. / [CA] Un dels principals reptes dels serveis d'aigua potable és garantir el subministrament d'alta qualitat, en particular, en termes químics i microbiològics. No obstant això, els biofilms desenvolupen invariablement en tots els sistemes de distribució d'aigua potable (DWDSs, de l'anglès, Drinking Water Distribution Systems), tot i la presència de desinfectant residual. Com a resultat, les empreses d'aigua no són capaces de garantir un control bacteriològic total. Actualment el biofilms representen un veritable paradigma en la gestió de la qualitat de l'aigua per a tots les DWDSs. Els biofilms són comunitats complexes de microorganismes vinculats per un polímer extracel·lular que els proporciona estructura, protecció contra els tòxics i ajuda a retenir els aliments. A més del risc de salut que impliquen els biofilms, com a causa del seu paper com a refugi de patògens, una sèrie de problemes addicionals associats amb el desenvolupament del biofilm en els DWDSs pot ser identificat. Entre altres, deteriorament estètic d'aigua, biocorrosión i decadència de desinfectant són universalment reconeguts. Una gran quantitat d'investigació s'ha realitzat en aquest camp des dels primers anys de la dècada del 80. No obstant això, a causa de la complexitat de l'entorn i la comunitat estudiada, la major part dels estudis s'han desenvolupat sota certes simplificacions. Recorrem a aquest treball ja realitzat i a aquest coneixement adquirit en el creixement de biofilms en els DWDSs per canviar el punt de vista clàssic del biofilm en estudis en els DWDSs. La nostra proposta es basa en l'ardu processament previ i posterior anàlisi mitjançant enfocaments d'aprenentatge automàtic. Es va dur a terme un procediment multidisciplinari, ajudant com un enfocament pràctic per desenvolupar una eina de presa de decisions per ajudar a la gestió dels DWDS a mantenir, en la mesura possible, els biofilm en els nivells més baixos, i la mitigació dels seus efectes negatius sobre el servei. Es proposa una metodologia per detectar les àrees més susceptibles al desenvolupament de biofilms en els DWDSs. En conèixer la ubicació d'aquests punts calents de la xarxa, les accions de mitigació podrien centrar-se més específicament, estalviant recursos i diners. A més, els programes de prevenció es podrien desenvolupar, actuant abans que les conseqüències del biofilm es noten pels consumidors. D'aquesta manera, el cost econòmic seria reduït i la qualitat del servei podria millorar, finalment augmentant la satisfacció dels consumidors. / Ramos Martínez, E. (2016). Assessing biofilm development in drinking water distribution systems by Machine Learning methods [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/63257

<p dir="ltr"><i>Stenotrophomonas maltophilia</i> is an emerging multidrug-resistant, Gram-negative opportunistic pathogen. It causes many hospital-acquired infections such as sepsis, endocarditis, meningitis, and catheter-related urinary tract infections. It also affects individuals with cystic fibrosis, exacerbating their lung condition. <i>S. maltophilia</i> often causes pathogenesis through the formation of biofilms. However, the molecular mechanisms <i>S. maltophilia</i> uses to carry out these pathogenic steps are unclear. The SMF-1 chaperone/usher pilus has been thought to mediate <i>S. maltophilia</i> attachment. To confirm this role, we created an isogenic deletion of the <i>smf-1</i> pilin gene and observed a defect in biofilm compared to wild type. We also discovered 2 additional chaperone/usher pilus operons, mutation of which also caused attenuation in biofilm levels. Analysis of <i>S. maltophilia</i> clinical strains and <i>S. maltophili</i><i>a</i> complete genomes listed in NCBI showed that these three pili are prevalent and highly conserved, suggesting a vital role in infection. Intriguingly, through TEM studies, we found that the mutation of one pilus is not phenotypically compensated by another. Infection of <i>Galleria mellonella</i> larvae revealed increased virulence of the pilus mutants. Additionally, we also demonstrated a relationship between pilus and flagella contributing to the overall biofilm development of <i>S. maltophilia</i>. Understanding their activity may help identify therapeutic targets for this pathogen.</p>

Pseudomonas aeruginosa (PA) is a gram-negative bacillus well known for colonizing human respiratory airways and causing opportunistic infections. Children with neuromuscular disease (NMD) including cerebral palsy (CP) and severe upper airway obstruction who get infected with PA, their chances of experiencing a severe illness, being admitted to a pediatric intensive care unit, and extended or repeated hospital stays increase dramatically. These patients often need a surgical procedure called tracheostomy which act as a channel for microbes to enter lower respiratory tract and increase infections, despite its well documented impact as an opportunistic pathogen comprehensive investigation into the diversity of PA in such vulnerable populations is limited. To fill this gap in knowledge we perform whole genome sequencing (WGS) and phenotypic analysis of 40 PA isolates from the respiratory tract of this susceptible population with and without tracheotomies. Pangenome analysis showed highly variable genome content with 16,212 total genes of which 2326 are core genes. MLST revealed diverse sequence types (STs) among the studied population with 21 known and 10 new STs. Genotypic analysis revealed moderate variations in the antimicrobial resistance determinants and virulence factors among all isolates. In total 8 serogroups were identified, with serogroups O6 and O11 accounting for 70% of all the isolates. Genotypic diversity was observed in overall population however comparative analysis among tracheostomized and non-tracheostomized patient groups showed significant similarity which aligns with the phenotypic analysis revealing significant similarity with minor differences in biofilm formation, motility, hemolysis production, and pigment production. Last, we explored putative healthcare transmission and identified three potential transmission events. These findings provide insight into how WGS along with phenotypic analysis can help us better understand population dynamics, epidemiology, virulence profile and antibiotic resistance profile of PA contributing to respiratory infections which has valuable therapeutic implications for epidemiology and disease management.

Central Appalachian headwater streams in coalfield areas are prone to mining disturbances, and compensatory mitigation is required in cases of documented impacts. Stream construction on reclaimed mines is a common mitigation strategy. Streams constructed as compensatory mitigation are meant to restore structural and functional attributes of headwater streams and are often evaluated by measuring structural ecosystem characteristics. However, replacement of stream ecosystem functions is essential for mitigation of mining disturbances from an ecosystem perspective. This research compared selected structural and functional measures in eight constructed streams on mined areas to those of four forested reference streams across two years. Three organic matter functions were evaluated: riparian litterfall input, leaf breakdown, and periphyton accrual. Constructed streams were typically warmer than reference streams and also had elevated specific conductance, elevated oxidized nitrogen concentrations, depressed benthic macroinvertebrate richness, and lower levels of canopy cover. Functionally, litterfall input and total leaf breakdown means for constructed streams were approximately 25% and 60% of reference means, respectively. Leaf breakdown in constructed streams appeared to be inhibited as a result of reduced processing by benthic macroinvertebrates as well as inhibition of microbial and physicochemical pathways. Constructed streams with total breakdown rates most similar to reference-stream levels had the coldest stream temperatures. Areal periphyton biomass, benthic algal standing crop, and senescent autotrophic organic matter in constructed streams were roughly quadruple, double, and quintuple those of reference streams, respectively. Indicator ratios also suggested stream-type differences in periphyton structure. Mean algal accrual was greater in constructed streams than in reference streams during leaf-on seasons. My results suggest that light is likely the primary factor driving accrual rate differences during summer and fall, but that temperature may also be important during fall. Planting a diverse assemblage of native riparian trees and ensuring their successful development can inhibit benthic irradiance and thermal energy inputs while providing similar quantity and quality of OM to constructed streams, thereby fostering replacement of reference-like OM functions in some streams. / Ph. D.

<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>

<p dir="ltr"><i>Serratia marcescens </i>is a bacterium with a ubiquitous environmental distribution and the ability to cause opportunistic infections. This research explores three different group behaviors in <i>S. marcescen</i>s. These are biofilm formation, microbial hitchhiking, and responses to cannabinoid-induced stress. To study biofilm development, we used a crystal violet assay to measure biofilm and compared that to the bacterial growth of those cultures. We looked at the role of nutrients and temperature in biofilm produced by <i>S. marcescens</i> and tested four <i>S. marcescens</i> strains. We found that there were differences in the ratio of biofilm to growth when <i>S. marcescens</i> was grown in different media. The exact relationship between temperature and media composition requires more information than could be attained from these studies. Next, we wanted to investigate whether <i>S. marcescens</i> could also utilize movement of other, more highly motile species of bacteria through a process called microbial hitchhiking. <i>S. marcescens</i> was grown with a highly motile <i>Paenibacillus</i> sp. isolate. <i>S. marcescens</i> was tracked by the red pigment that it produces. It was observed that <i>S. marcescens</i> consistently spread farther across a surface when it was co-cultured with <i>Paenibacillus</i> sp. than when grown alone. This was repeated with three other <i>S. marcescens</i> strains and three different species of <i>Paenibacillus.</i><i> </i>Hitchhiking behavior was observed in all cases. To understand the mechanism by which <i>S. marcescens</i> hitchhikes on <i>Paenibacillus </i>spp., a <i>S. marcescens </i>flagellar mutant was used. Even in the absence of a flagellum, <i>S. marcescens</i> was still able to hitchhike implying that another mechanism must be involved. Finally, we evaluated the response of <i>S. marcescens </i>to cannabidiol (CBD) a phytocannabinoid with antimicrobial and antibiofilm properties, though it has limited potency against Gram-negative bacteria like <i>S. marcescens</i>. We found that CBD did not kill <i>S. marcescens </i>nor did it affect its biofilm development. Interestingly, <i>S. marcescens </i>cultures showed enhanced pigment production in response to high-dose CBD exposure compared to vehicle controls. This response was also observed with exposure to three additional phytocannabinoids. Results from these studies add to our understanding of how <i>S. marcescens</i> can access new areas and persist in a broad range of environments.</p>

H. somni is capable of forming a prominent biofilm, and luxS is known to play an important role in biofilm formation through quorum sensing, but has also been postulated to function in gene regulation. In order to further study the function of H. somni LuxS, mutants 2336::TnluxS and 2336::TnuspE were identified from a bank of mutants generated with EZ-Tn5 <KAN-2>Tnp transposome (EpiCentre). The 2336::TnluxS and 2336::TnuspE mutants were highly attenuated in mice, but only 2336::TnuspE was deficient in biofilm formation. However, the electrophoretic profiles of the LOS and serum sensitivity of both mutants were substantially altered compared to the parent strain, but exopolysaccharide production during biofilm formation also only decreased in 2336::TnuspE. The altered phenotypes were partially restored in complemented recombinant clones obtained using shuttle vector pHS649S. To clarify whether luxS regulates the expression of various virulence genes, mRNA from both the parent strain and 2336::TnluxS was sequenced. It was determined that the transcription level of 53 genes in 2336::TnluxS and 42 genes in 2336::TnuspE in planktonic form were changed. In biofilm, 320 genes in 2336::TnluxS and 230 genes in 2336::TnuspE were differentially regulated compared to biofilm formed by strain 2336. The immunogloblin binding protein A (IbpA) of H. somni is known to be cytotoxic to phagocytic cells. In this study, we found that strains with a mutation in ibpA were less capable of early replication in monocytes. The IbpA protein concentrated from the culture supernatant of strain 2336 facilitated the intracellular survival of strain 129Pt, which lacks IbpA. However, the ability of several ibpA mutants to resist intracellular killing was not significantly impaired by 48 h post-infection. Two transposon mutants 2336::TnluxS and 2336::TnuspE replicated in monocytes in a similar manner as the ibpA mutants. Confocal microscopy revealed that the intracellular-replicable strains (2336, 64Vc, 2336::TnluxS, 2336::TnuspE and the ibpA mutants) prevented the acidification of the bacterial-containing phagosome and the expression of lysosome marker LAMP-2, which may facilitate survival of H. somni in monocytes. An enzyme-linked immunosorbent assay was developed to detect bovine antibodies to the H. somni exopolysaccharide that is formed during biofilm formation. When an index value of 0.268 was used the sensitivity of the assay for experimentally- and naturally-infected calves was 90.5% at 3 weeks post-infection, and the specificity of the assay for healthy calves was 92.5%. The EPS ELISA may aid in identifying calves with diseases due to H. somni. / Ph. D.

Part I: Signaling Role of the Sensor Kinase RetS in Biofilm formation Regulation of Pseudomonas aeruginosa-<br />The opportunistic human pathogen Pseudomonas aeruginosa causes both acute and chronic infections in predisposed individuals. Acute infections require a functional Type Three Secretion System (TTSS), which mediates the translocation of select cytotoxins into host cells. Chronic infections, the leading cause of death among cystic fibrosis patients, are characterized by drug-resistant biofilms formation. To regulate gene expression, Pseudomonas aeruginosa utilizes two-component regulatory systems (TCS). Specifically, we focus on the TCS signaling kinase RetS, which is a critical repressor of biofilm formation. The signaling mechanism of RetS is unusual. According to recent findings and one hypothesis, RetS employs a novel signaling mechanism involving direct binding to the signaling kinase GacS, thereby repressing the GacS-induced biofilm formation. RetS is believed to be regulated by the interaction of its periplasmic sensory domain (RetSperi) with an unknown ligand. As such, RetSperi is a potential drug target. We hypothesized that ligand-binding shifts the equilibrium between the formation of a RetS homo-dimer and the RetS-GacS complex by tuning the homo-dimerization of the RetSperi. While the molecular signal that regulates RetS is unknown, our structural studies of the sensory domain suggest that this ligand is a carbohydrate-based moiety. Unchanged biofilm-EPS production phenotype of RetSperi ligand binding site mutants indicates that the natural ligand is not from Pseudomonas aeruginosa.<br />Additional experiments unambiguously determined that the sensory domain forms a stable homodimer. Adding to the complexity of the system, we have identified<br />two possible dimer interfaces in our in vitro assays. However, inconsistent with the current model, elimination of RetSperi results in a slightly increased biofilm EPS production phenotype. Therefore, with the previous demonstration that RetS is able to dephosphorylate GacS, we propose an alternative hypothesis: the RetS kinase domain serves as a phosphatase for phosphorylated GacS; this phosphatase activity is tuned by signaling sensing on RetSperi. Finally, to provide an important piece of information for understanding the molecular basis of RetS-GacS signaling, we have developed a crystallization-based structure determination strategy in order to reveal the precise RetS-GacS interaction pattern.<br /><br />PartII: The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology-<br />germination-specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic-"?lactam-containing cortex peptidoglycan. The protein also contains a metal-binding site that is positioned directly at the entrance of the substrate-binding cleft. / Ph. D.

As demand for alternative water sources intensifies, increased use of reclaimed water is important to help achieve water sustainability. In addition to treatment, the manner in which reclaimed water is distributed is a key consideration as it governs the water quality at the point of use. In this work, simulated reclaimed water distribution systems (SRWDSs) were operated for more than two years to examine the role of system design, biofiltration, residual disinfectant type (i.e., chlorine, chloramine, no residual) and temperature on important aspects of chemistry and microbial regrowth under laboratory-controlled conditions. Turbidity decreased to 0.78 NTU after biofiltration and chlorinated treatments from 10.0-12.6 NTU for conditions with chloramine and no residuals. SRWDSs were susceptible to sediment accumulation, which occupied 0.83-3.2% of the volume of the first pipe segment (1 day of hydraulic residence time), compared to 0.32-0.45% volume in the corresponding chlorinated SRWDSs. The mass of accumulated sediment positively correlated (R2 = 0.82) with influent turbidity. Contrary to experiences with potable water systems, chlorine was found to be more persistent and better at maintaining biological stability in the SRWDSs than chloramine, especially at the higher temperatures >22°C common to many water scarce regions. The severe nitrification at the warmer temperatures rapidly depleted chloramine residuals, decreased dissolved oxygen, and caused elevated levels of nitrifiers and heterotrophic cell counts. A metagenomic taxonomic survey revealed high levels of gene markers of nitrifiers in the biofilm samples at 22°C for the chloraminated system. Non-metric multidimensional scaling analysis confirmed distinct taxonomic and functional microbial profiles between the chlorine and chloramine SRWDSs. Reflecting on multiyear experiences operating two different SRWDSs reactor designs, including thin tubes (0.32-cm diameter) and pipe reactors (10.2-cm), illustrated strengths and weaknesses of both approaches in recreating key aspects of biochemical changes in reclaimed water distribution systems. It is clear that approaches deemed successful with drinking water distribution systems may not always directly transfer to simulating reclaimed distribution systems, or to proactively managing full-scale reclaimed systems that have long periods of stagnation and where minimally-treated wastewater with high levels of nutrients and turbidity are used. / Doctor of Philosophy / Increasing water scarcity is creating an impetus for creating more sustainable water supplies. Wastewater effluent is increasingly viewed as in important resource that can reduce both water and energy demand. Reclaiming moderately to minimally-treated secondary wastewater effluent for non-potable reuse (NPR) applications; such as agricultural irrigation, landscaping, and toilet flushing, helps reduce demand for higher quality potable water sources. NPR presently accounts for more than 50% of total reuse and is projected to become increasingly important. While NPR is attractive, important knowledge gaps remain in terms of managing water quality and safety as it is transported through distribution pipes to the point of use. A comprehensive literature review revealed that NPR distribution systems are distinct from conventional drinking water distribution systems (DWDSs) and that it is doubtful if our current understanding of DWDSs would directly transfer to NPR systems. Unlike drinking water systems, NPR systems are currently unregulated at the national level and corresponding state-to-state regulations vary widely. The levels of water treatment can vary from simply distributing untreated effluent from wastewater treatment plants to very high-level treatment with membranes that produces water of equal or even higher quality than many existing tap waters. A common treatment train for minimally-treated NPR involves biologically activated carbon (BAC) filtration and the use of disinfectants (e.g., chlorine or chloramine) to control microbial water quality to the point of use. Prior studies from DWDSs have demonstrated water quality degradation in terms of disinfectant loss, bacterial growth, and aesthetic problems, with the settling of trace particulate matter producing sediment within pipe distribution systems. In particular, accumulated sediment can become a hotspot for water quality deterioration. Considering that minimally-treated reclaimed water can have much higher levels of particulate matter and nutrients than drinking water, it was predicted that NPR distribution systems could suffer from faster water quality degradation than corresponding drinking water systems, especially at the warmer temperatures common in water-scarce regions. This work was the first multi-year attempt to examine the effects of disinfectant (i.e. free chlorine, chloramine, no residual), BAC filtration versus no filtration, water age (up to 5-d versus 28-min), and temperature (14°C, 22°C, 30°C) in different types of lab-scale reactors. Two simulated reclaimed water distribution systems (SRWDSs) including 4-in. diameter Pipe SRWDSs versus 1/8-in. diameter Tube SRWDSs, were designed to study key aspects of full-scale NPR systems and were operated for more than two years to study chemical and microbial changes as distributed water traveled through the two systems. The Pipe SRWDSs were designed to assess the impacts on final water quality after long-term operation that allowed sediment to slowly accumulate, whereas the complementary Tube SRWDS design did not allow sediment to accumulate and only held the water for 28 minutes. Water was sampled regularly to track the trends of key water quality parameters, including disinfectant residuals, dissolved oxygen, nitrogen compounds involved in nitrification reactions, and various types of bacteria of interest. Sequencing of the biological genetic materials on selected samples was conducted to understand the types of bacteria present and their functions under the different circumstances. High levels of sediment were found to accumulate near the beginning of the Pipe SRWDSs, which caused loss of oxygen and disinfectants at the bottom of the pipes. Chlorine was more persistent and better at preventing bacteria growth as water traveled through the distribution system. In contrast, a type of bacteria that used ammonia as a nutrient (i.e., nitrifying bacteria) were observed in the pipes with chloramine (i.e., ammonia plus chlorine) as the disinfectant. The nitrifying bacteria caused rapid depletion of chloramine residuals, especially at temperatures above 22°C. At 30°C both chlorine and chloramine were almost immediately consumed in the pipe reactors. Nitrification is known to trigger water quality problems in chloraminated DWDSs, and we expect that chloraminated RWDSs would be even more susceptible to nitrification and associated water quality degradation issues in Compare the Tube SRWDSs to the Pipe SRWDSs, aside from heavy accumulations of sediment in the pipes versus no sediment in the thin tubes, the tubes clogged repeatedly from formation of thick biofoulants in the systems treated with no disinfectant and chloramine, whereas they remained relatively free of biofoulants and clogging in the tubes with chlorine. Even in just 28 minutes, it took water to move from the start to the end of the tube, both chlorine and chloramine were almost completely consumed in the tubes, due to the unrealistically high pipe surface area to the small flow volume inherent to this reactor design. As NPR becomes increasingly common to help achieve water sustainability, it will be important to deploy laboratory simulations, that are capable of testing and revealing key chemical and microbial processes that affect the operation of these systems and water safety at the point of use. The insights from this first long-term effort of simulating RWDSs highlight some unique characteristics and challenges of RWDSs, and reveals key concepts to help guide future research.