Dissertations / Theses on the topic 'Bacterii'

Made available in DSpace on 2015-06-17T19:34:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-26. Added 1 bitstream(s) on 2015-06-18T12:49:23Z : No. of bitstreams: 1 000829428_20170301.pdf: 100482 bytes, checksum: 894af24aad4ca7d75f9898581c07f398 (MD5) Bitstreams deleted on 2017-03-03T11:01:31Z: 000829428_20170301.pdf,. Added 1 bitstream(s) on 2017-03-03T11:02:40Z : No. of bitstreams: 1 000829428.pdf: 275587 bytes, checksum: 79098a3390015f4449a4b64413d932a8 (MD5)<br>Estudos que visam a interação das proteínas Cry de Bacillus thuringiensis, a fim de encontrar combinações adequadas para o desenvolvimento de plantas Bt são ferramentais fundamentais no controle de lepidópteros-praga. A lagarta H. armigera causa danos severos nas culturas agrícolas e sua introdução no Brasil levou a busca de formas de controle eficientes e nesse contexto B. thuringiensis pode ser um bom agente de controle. Diante do exposto o presente trabalho objetivou avaliar a toxicidade das proteínas Cry1Aa, Cry1Ab, Cry1Ac e Cry1Ca de B. thuringiensis à H. armigera, assim como interação dessas proteínas aos receptores do mesêntero do inseto. A toxicidade foi estimada com bioensaios de dose resposta com as proteínas testadas e a interação das proteínas com os receptores foram verificadas em análise de união entre a proteína ativada e marcada com a vesícula da borda em escova da membrana apical das células do intestino (brush border mambrane vesicle- BBMV) do mesêntero larval de H. armigera, e ensaios de competição heteróloga. Dentre as proteínas testadas, a Cry1Ac destacou-se como a mais efetiva, seguida das proteínas Cry1Ab e Cry1Aa. A proteína Cry1Ca não apresentou toxicidade. As proteínas Cry1Aa, Cry1Ab e Cry1Ac se ligaram aos receptores da membrana do intestino médio das lagartas de H. armigera de forma especifica. Os ensaios de competição heteróloga revelaram que as proteínas Cry1Aa, Cry1Ac e Cry1Ab competem entre si pelo mesmo receptor<br>Studies attempting interaction of Bacillus thuringiensis Cry proteins in order to find combinations for developing Bt plants are fundamental in controlling lepidopteran pests. H. armigera causes severe damage to agricultural crops and their introduction in Brazil has led the search for efficient control and B. thuringiensis may be a good control agent. The aim of this research was to evaluate the toxicity of Cry1Aa, Cry1Ab, Cry1Ac and Cry1Ca proteins from B. thuringiensis to H. armigera, as well as interaction of these proteins with the receptors present in insect midgut. Toxicity was estimated from the lethal concentration LC50 of the tested proteins and protein interactions with the receptors were found in a binding analysis between activated and biotinylated protein with the midgut brush border vesicle membrane (BBMV) of H. armigera, and heterologous competitive binding assays. Among the tested proteins, Cry1Ac protein was the most toxic, followed by the Cry1Ab and Cry1Aa proteins. The Cry1Ca protein showed no toxicity. The Cry1Aa, Cry1Ab and Cry1Ac proteins showed specific binding to the midgut membrane receptors of H. armigera caterpillars. Heterologous competitive binding assays revealed that Cry1Aa, Cry1Ab, Cry1Ac compete for a common receptor in the midgut larvae

Orientador: Manoel Victor Franco Lemos<br>Coorientador: Ricardo Antonio Polanczyk<br>Banca: Janete Apparecida Desidério<br>Banca: Camila Chiaradia Davolos<br>Resumo: Estudos que visam a interação das proteínas Cry de Bacillus thuringiensis, a fim de encontrar combinações adequadas para o desenvolvimento de plantas Bt são ferramentais fundamentais no controle de lepidópteros-praga. A lagarta H. armigera causa danos severos nas culturas agrícolas e sua introdução no Brasil levou a busca de formas de controle eficientes e nesse contexto B. thuringiensis pode ser um bom agente de controle. Diante do exposto o presente trabalho objetivou avaliar a toxicidade das proteínas Cry1Aa, Cry1Ab, Cry1Ac e Cry1Ca de B. thuringiensis à H. armigera, assim como interação dessas proteínas aos receptores do mesêntero do inseto. A toxicidade foi estimada com bioensaios de dose resposta com as proteínas testadas e a interação das proteínas com os receptores foram verificadas em análise de união entre a proteína ativada e marcada com a vesícula da "borda em escova" da membrana apical das células do intestino ("brush border mambrane vesicle"- BBMV) do mesêntero larval de H. armigera, e ensaios de competição heteróloga. Dentre as proteínas testadas, a Cry1Ac destacou-se como a mais efetiva, seguida das proteínas Cry1Ab e Cry1Aa. A proteína Cry1Ca não apresentou toxicidade. As proteínas Cry1Aa, Cry1Ab e Cry1Ac se ligaram aos receptores da membrana do intestino médio das lagartas de H. armigera de forma especifica. Os ensaios de competição heteróloga revelaram que as proteínas Cry1Aa, Cry1Ac e Cry1Ab competem entre si pelo mesmo receptor<br>Abstract: Studies attempting interaction of Bacillus thuringiensis Cry proteins in order to find combinations for developing Bt plants are fundamental in controlling lepidopteran pests. H. armigera causes severe damage to agricultural crops and their introduction in Brazil has led the search for efficient control and B. thuringiensis may be a good control agent. The aim of this research was to evaluate the toxicity of Cry1Aa, Cry1Ab, Cry1Ac and Cry1Ca proteins from B. thuringiensis to H. armigera, as well as interaction of these proteins with the receptors present in insect midgut. Toxicity was estimated from the lethal concentration LC50 of the tested proteins and protein interactions with the receptors were found in a binding analysis between activated and biotinylated protein with the midgut brush border vesicle membrane (BBMV) of H. armigera, and heterologous competitive binding assays. Among the tested proteins, Cry1Ac protein was the most toxic, followed by the Cry1Ab and Cry1Aa proteins. The Cry1Ca protein showed no toxicity. The Cry1Aa, Cry1Ab and Cry1Ac proteins showed specific binding to the midgut membrane receptors of H. armigera caterpillars. Heterologous competitive binding assays revealed that Cry1Aa, Cry1Ab, Cry1Ac compete for a common receptor in the midgut larvae<br>Mestre

Blast of pear caused by Pseudomonas syringae pv. syringae is one of the bacterial disease that limit pear production throughout the world. Symptoms are characterized by blast of buds and blossoms wich causes significant loss of fruit production, and necrotic spots on leaves or fruits. Control of bacterial blast of pear with chemicals is difficult and is based on copper compounds and antibiontics. However, its use is limited by the low efficacy, phytotoxicity to the plant or emerging resistance of the pathogen. The activity of several phosphonates (fosetil-Al, potassium phosphonate, etephon and fosfomycin) for control of P. syringae pv. syringae infection on pear was determined in this work, and laboratory models for studying P. syringae pv. syringae-pear interaction were developed<br>Pseudomonas syringae pv. syringae és un bacteri que ha estat descrit com agent causant de diverses malalties en més de 200 especies vegetals. En perera causa la necrosi bacteriana, que afecta la majoria de zones productores de pera del món, provocant un debilitament dels arbres i una disminució de la productivitat. En el treball que es presenta s'ha determinat l’activitat de diversos fosfonats (fosetil-AI, fosfonat potàssic, etefon i fosfomicina) en el control de la infecció per P. syringae pv. syringae en perera. Per això s'han desenvolupat models d'estudi de la interacció P. syringae pv. Syringae-perera i s'han determinat els factors que afecten la interacció. Aquests models de laboratori, com que han permès conèixer aspectes concrets de la interacció hoste-patogen i definir de forma clara el tipus d'interacció, s'han aplicat a l'estudi de l'activitat dels fosfonats en la interacció P. syringae pv. syringae -perera i en el control de la malaltia

Blood stream infections are challenging infections to rapidly diagnose. The current clinical diagnostic methods for blood stream infections require culturing the blood sample prior to identifying the bacteria and any resistance the bacteria may contain. Removing the culturing step from the bacterial identification process of a blood stream infection provides a significant reduction in the processing time. However, eliminating the culturing step shifts the difficulty from processing time to concentration, since clinical concentration levels can be as low as 10 CFU/mL in blood. This dissertation developed and evaluated many aspects of the process required to identify bacteria from a blood stream infection without culturing the bacteria. Two new methods of separating the bacteria from the blood cells were developed: inducing clotting using a centrifugal-sedimentation on a hollow disk, and filtering whole blood. Inducing clotting achieved 69\% bacterial recovery from 7 mLs of whole blood in 117 s. Filtering whole blood achieved 100\% bacterial removal from 5 mLs of whole blood in $\approx 90$ s, but the bacteria were difficult to remove from the filter. Bacterial removal from the filter after blood filtration was also investigated. At a very low bacterial concentration of 200 CFU/mL, a blood lysis solution of 3\% Tween 80 followed by a 3\% Pluronic F108 backflush solution achieved 60\% removal of the bacteria from the filter. In addition to developing two new methods, a previously developed technique using centrifugal-sedimentation on a hollow disk underwent a stability analysis in order to decrease the occurrence of mixing. This analysis yielded the development of the analytical solution to the Navier-Stokes equations for a two-fluid flow with a moving wall boundary and a free surface. The analysis also experimentally identified a stability boundary that was found to be in good agreement with the Kelvin-Helmholtz instability model. After exploring the methods to recover bacteria from blood, experiments were performed to identify a bacterial lysing solution that could lyse \textit{E. coli}, \textit{E. cloacae} and \textit{K. pneumoniae} bacteria. The best bacterial lysing solution consisted of incubating the bacteria with 1 mg/mL lysozyme for 10 min followed by the addition of 6 M GHCl and 1\% SDS. This solution obtained a 46\% DNA recovery. The DNA were then fragmented by ultrasound to reduce the segment length for DNA labelling. In addition to lysing and fragmenting the DNA, a microfluidic device was prototyped and tested for incorporating the lysing, capturing, releasing, and fragmenting of the DNA all on a single device. Whole experiments were performed which extracted the bacteria from the blood, removed and collected the DNA from the bacteria, and fragmented the DNA. The best overall recovery from an experiment performing the whole process was 26.8\%. The 26.8\% recovery was achieved with a 68\% recovery of the bacteria from spinning and a 54.1\% removal of bacteria from off of the filter and a 72.9\% recovery of the DNA from the bacteria.

Probiotics are live microorganisms which when administered in adequate amounts confer one or more health benefits on the host. Different processing conditions, the acidic condition of the stomach and exposure to hydrolytic enzymes affect the viability and efficacy of probiotic organisms. This study investigated the protective effects of two biopolymers poly-gamma-glutamic acid (γ-PGA) and bacterial cellulose (BC) on probiotics during freeze drying and during exposure to simulated intestinal juices and bile salts. The antibacterial property of Bifidobacterium strains was also investigated against four pathogenic bacteria. γ-PGA, a naturally occurring biopolymer was produced by two bacteria (Bacillus subtilis ATCC 15245 and B. licheniformis ATCC 9945a) in GS and E media, γ-PGA yields of about 14.11g/l were achieved in shake flasks and molecular weight of up to 1620 k Da was recorded, γ-PGA production was scaled up in a fermenter with B. subtilis using GS medium. BC, an edible biopolymer was produced by Gluconacetobacter xylinus ATCC 23770 in HS medium and a modified HS (MHS) medium. A yield of about 1.37g/l was recorded and BC production with MHS medium was used for probiotic application. B. longum NCIMB 8809 B. breve NCIMB 8807 and B. animalis NCIMB 702716 showed the best antimicrobial properties against the investigated pathogens. Survival of Bifidobacterium strains was improved when protected with powdered BC (PBC) although γ-PGA offered better protection than PBC. Viability of B. longum NCIMB 8809, B. breve NCIMB 8807 and B. animalis NCIMB 702716 in simulated gastric juice (SGJ) and simulated intestinal juice with bile salts was improved when protected with 5% γ-PGA and 5% γ-PGA+PBC with a reduction of < 1 Log CFU/ml while a reduction of ≤2 Log CFU/ml was recorded in PBC protected cells. Protecting Bifidobacterium strains with γ-PGA, PBC or a novel γ-PGA + PBC combination is a promising method to deliver probiotic bacteria to the target site in order to confer their health benefits on the host.

A novel Gram negative, capsule-forming bacterium was previously isolated in Dr. G. Roland Vela's laboratory. The distribution of this bacterium in soils from various locations was investigated. Soil samples from 188 locations around the world were examined. Isolates of the bacterium were obtained from 50 of these soils, with 48 of the isolates found in soils from the southwestern United States and northern Mexico. This suggests that this region is the natural habitat of the bacterium. The other two isolates were obtained from Madrid, Spain and Taipei, Taiwan. None were found in soils from South America or Australia. A lack of variation in morphology and physiological properties in the isolates suggests that a homogeneous population exists, even from widespread geographical locations.

The regulation of the expression of virulence genes in Salmonella enterica serovar Typhimurium is an intensively studied feature of Salmonella lifestyle. How Salmonella integrates environmental signals to activate virulence-related genes within the host has been explored. The genes encoded in Salmonella pathogenicity island I (SPI-1) required for the invasion of epithelial cells are well characterized, and many regulators involved in the activation of SPI-1 genes have been described. However, little is known about mechanism involved in the repression of SPI-1 under conditions were Salmonella does not require the expression of virulence-related genes. The expression of SPI-1 encoded genes have been reported to be a burden for Salmonella physiology and therefore mechanism to control shut down of SPI-1 under non permissive conditions might play a crucial role in Salmonella physiology. Here we describe that at exponential phase, CRP-cAMP, which acts as an activator at stationary phase, represses the expression of SPI-1 genes. The overall objective of this thesis was to characterize how CRP-cAMP silences SPI-1 expression under non-permissive conditions and to describe the molecular mechanism behind this phenotypic observation. In this thesis we i) define the target gene for the CRP-mediated regulation of SPI-1 ii) elucidate at which level of regulation CRP-cAMP modulates the expression of the SPI-1 genes master regulator hilD iii) characterize the involvement of CRP-cAMP dependent sRNA in hilD regulation iv) characterize the interaction of the CRP-cAMP dependent sRNA Spot 42 with the hilD 3’UTR region to regulate SPI-1 and v) explore the role of CRP-cAMP in the modulation of the SPI-1 repressor CsrA through the regulation of the long non-coding RNA csrB and csrC. CRP-cAMP represses hilA expression at exponential phase (non-permissive conditions for SPI-1 expression) and acts as an activator at stationary phase (permissive conditions for SPI-1 expression). CRP-cAMP mediated repression of hilA causes a concomitant attenuation in the expression level of SPI-1 encoded effector proteins. The regulation of SPI-1 during logarithmic growth phase occurs upstream of HilA by repressing hilD, hilC and rtsA expression and is mediated by the regulation of hilD expression at the post transcriptional level through the hilD 3’UTR. CRP-cAMP mediated regulation of hilD requires, in addition to the hilD 3’UTR, the sRNA chaperone Hfq and the major endonuclease RNAse E. CRP-cAMP represses the expression of the sRNA Spot 42 at exponential phase. We show that Spot 42 positively regulates hilD expression at exponential growth phase and requires of the presence of the hilD 3’UTR, the sRNA chaperone Hfq and the major endonuclease RNAse E. Interestingly, Spot 42 and the hilD 3’UTR region physically bind to Hfq. Spot 42 physically interacts with the last 150 nt of the hilD 3’UTR and unstructured region III of Spot 42 is required for the regulation of hilD. CRP-cAMP represses csrC but not csrB expression at exponential phase to regulate the expression of hilD. Remarkably, the CRP-cAMP dependent sRNA Spot 42 positively regulates the expression of csrC.<br>La regulación de la expresión de genes de virulencia en Salmonella enterica serovar Typhimurium es una característica intensamente estudiada en Salmonella. La forma en que Salmonella integra señales ambientales para activar los genes relacionados con la virulencia dentro del huésped ha sido explorada. Los genes codificados en la isla de patogenicidad I de Salmonella (SPI-1) son necesarios para la invasión de células epiteliales, están bien caracterizados y se han descrito muchos reguladores implicados en su activación. Sin embargo, poco se sabe sobre el mecanismo implicado en la represión de SPI-1 en condiciones en las que Salmonella no requiere la expresión de genes relacionados con la virulencia. Es sabido que la expresión de genes codificados por SPI-1 son una carga para la fisiología de Salmonella y por lo tanto el mecanismo para controlar la represión de SPI-1 en condiciones no permisivas podría desempeñar un papel crucial en la fisiología de Salmonella. Aquí se describe que en fase exponencial, CRP-cAMP, que actúa como un activador en fase estacionaria, reprime la expresión de los genes SPI-1. El objetivo general de esta tesis fue caracterizar cómo el CRP-cAMP silencia la expresión de SPI-1 en condiciones no permisivas y describir el mecanismo molecular detrás de esta observación fenotípica. El CRP-cAMP reprime la expresión de hilA en la fase exponencial (condiciones no permisivas para la expresión de SPI-1) y actúa como un activador en fase estacionaria (condiciones permisivas para la expresión de SPI-1). La represión mediada por CRP-cAMP de hilA provoca una atenuación concomitante en el nivel de expresión de proteínas efectoras codificadas por SPI-1. La regulación de SPI-1 durante la fase de crecimiento logarítmico se produce aguas arriba de HilA mediante la represión hilD, hilC y rtsA expresión y está mediada por la regulación de hilD a nivel post transcripcional a través de la hilD 3'UTR. La regulación mediada por CRP-cAMP de hilD requiere, además de la hilD 3'UTR, la chaperona Hfq y la endonucleasa RNAsa E. CRP-cAMP reprime la expresión del sRNA Spot 42 en la fase exponencial. Mostramos que Spot 42 regula positivamente la expresión hilD en la fase de crecimiento exponencial, Spot 42 interacciona físicamente con los últimos 150 nt de la hilD 3'UTR.

Los bacteriófagos son componentes abundantes y omnipresentes en muchos ambientes naturales, y el cuerpo humano no es la excepción. Del mismo modo que pasa en la naturaleza, los fagos en el cuerpo humano pueden desempeñar un papel importante en el control y la evolución de las poblaciones bacterianas, promoviendo la selección de ciertos tipos de bacterias e influyendo notablemente en la evolución de los genomas bacterianos mediante transferencia genética horizontal. Además, teniendo en cuenta su gran abundancia, podría preveerse que las partículas fágicas incluso lleguen a afectar a las bacterias presentes en muestras clínicas humanas, interfiriendo de esta manera con el diagnóstico microbiológico de diversas patologías infecciosas. El objetivo principal de la presente tesis doctoral fue evaluar la presencia de los bacteriófagos en biomas humanos. Específicamente, se evaluó la interferencia que pueden tener en pruebas de diagnóstico clínico, y su papel como reservorios y vehículos en la diseminación de genes de resistencia a antibióticos (ARGs) en la microbiota del tracto digestivo y respiratorio. Este trabajo se presenta en cuatro capítulos: (1) Los bacteriófagos presentes en muestras clínicas pueden interferir con las herramientas de diagnóstico microbiológico; (2) Genes de resistencia a antibióticos en partículas fágicas aisladas de heces humanas e inducidas de aislamientos bacterianos clínicos; (3) El fagoma fecal de individuos sanos y las variaciones causadas por el tratamiento con ciprofloxacina; (4) Genes de resistencia a antibióticos en partículas fágicas aisladas de esputos de pacientes con fibrosis quística. Los resultados derivados de esta tesis sugieren que la presencia de fagos en muestras humanas puede influir y sesgar los resultados de pruebas diagnósticas microbiológicas y moleculares. Además, se destaca el papel de las partículas fágicas como vehículos para la movilización y diseminación de resistencias a antibióticos, tanto en el microbioma intestinal de individuos sanos, como en el microbioma respiratorio individuos sanos y de pacientes con fibrosis quística. Las partículas fágicas pueden, por tanto, contribuir a la generación de bacterias multirresistentes, consideradas como un problema de salud mundial de primera magnitud.<br>Bacteriophages are abundant and ubiquitous components in many natural environments and the human body is not an exception. As in natural environments, phages in the human body can play an important role in the potential control and evolution of the bacterial populations, by promoting the selection of certain types of bacteria and by significantly influencing the evolution of bacterial genomes through horizontal gene transfer. Furthermore, considering their great abundance, it could be envisaged that phage particles can even affect the bacteria present in human clinical samples, interfering with the microbiological diagnosis of several infectious diseases. The main objective of this PhD thesis was to evaluate the presence of bacteriophages in human biomes. Specifically, it was evaluated the interference of phages in clinical diagnostic tests, and their role as reservoirs and vehicles in the dissemination of antibiotic resistance genes (ARGs). This thesis is presented in four chapters: (1) Bacteriophages present in clinical samples can interfere with microbiological diagnostic tools; (2) Antibiotic resistance genes in phage particles isolated from human feces and induced from clinical bacterial isolates; (3) Fecal phageome of healthy individuals and variations caused by ciprofloxacin treatment; (4) Antibiotic resistance genes in phage particles isolated from sputum of cystic fibrosis patients. The results derived from this research suggest that the presence of phages in human samples can influence and bias the results of microbiological and molecular diagnostic tests. In addition, the role of phage particles as vehicles for the mobilization and dissemination of antibiotic resistance is highlighted, both in the intestinal microbiome of healthy individuals, and in the respiratory microbiome of healthy individuals and of cystic fibrosis patients. Therefore, phage particles can contribute to the generation of multiresistant bacteria, which are considered as a global health problem of the first magnitude.

L’aparició de farmacoresistència bacteriana als antibiòtics convencionals és una situació d’alarma mundial. Aquest escenari ha obligat a implementar mesures com la millora de les pràctiques d’higiene o l’administració controlada d’antibiòtics per reduir el seu ús en totes les àrees en les que s’utilitzen comunament, inclosa la medicina humana i animal i la indústria animal de producció d’aliments . Totes aquestes mesures estan destinades a disminuir l’aparició i propagació de la resistència als antibiòtics entre els bacteris, però quan es tracta de combatre als bacteris que ja presenten una o múltiples resistències, es necessiten amb urgència alternatives als antibiòtics. En aquest context, s’han proposat diferents estratègies, entre les quals s’inclouen l’ús de citoquines i pèptids antimicrobians (AMPs). Les citoquines són petites proteïnes reguladores intercel·lulars que tenen un paper central en l’inici, manteniment i regulació de la resposta immune innata. Concretament, l’IFN-γ té un paper fonamental en la promoció de la immunitat protectora contra les infeccions. D’altra banda, els AMPs, com GWH1, són generalment petits pèptids catiònics de naturalesa amfipàtica que tenen activitats antibacterianes, antifúngiques i antivirals d’ampli espectre, la capacitat de modular la resposta immune de l’hoste i una possibilitat reduïda d’induir resistència bacteriana. Malgrat el seu potencial com a agents antiinfecciosos, les citoquines i els AMPs estan subjectes a diversos desavantatges que s’han d’abordar abans de la seva possible aplicació biomèdica. En particular, la baixa estabilitat és un inconvenient comú associat a aquest tipus de compostos proteics. En aquest sentit, s’han aplicat diferents estratègies per superar aquesta limitació i millorar l’eficàcia d’aquests fàrmacs després de la seva administració. La majoria d’aquestes estratègies consisteixen en la vehicularización d’aquestes proteïnes o pèptids en estructures superiors, proporcionant un entorn protector i permetent la possibilitat d’alliberar-los en localitzacions concretes. La formació d’aquestes estructures pot ser modulada a través d’un disseny racional sobre el gen de la proteïna recombinant, com, per exemple, la incorporació de certs pèptids en l’estructura de la proteïna per generar components individuals amb capacitat autoensamblant (nanopartícules solubles), o per obtenir proteïnes propenses a l’agregació (cossos d’inclusió -IBs-). Encara que aparentment diferents, tots aquests complexos s’originen a partir d’interaccions proteiques impulsades per factors termodinàmics i cinètics similars que finalment condueixen a la formació de diferents formats de proteïnes; arranjaments estructurals superiors que involucren a través d’interaccions dirigides o no dirigides, la convergència de formes proteiques aïllades. En aquesta tesi s’ha caracteritzat i avaluat la nanoformulación d’aquestes molècules (citocines i AMPs) en diferents formats proteics incloent IBs i nanopartícules solubles autoensamblants amb l’objectiu de desenvolupar alternatives terapèutiques als antibiòtics. A més, s’ha llançat una mica de llum sobre les forces que governen el procés d’agregació i com es pot modular per promoure la formació d’aquests IBs.<br>La aparición de farmacorresistencia bacteriana a los antibióticos convencionales es una situación de alarma mundial. Este escenario ha obligado a implementar medidas como la mejora de las prácticas de higiene o la administración controlada de antibióticos para reducir su uso en todas las áreas en las que se utilizan comúnmente, incluida la medicina humana y animal y la industria animal de producción de alimentos. Todas estas medidas están destinadas a disminuir la aparición y propagación de la resistencia a los antibióticos entre las bacterias, pero cuando se trata de combatir a las bacterias que ya presentan una o múltiples resistencias, se necesitan con urgencia alternativas a los antibióticos. En este contexto, se han propuesto diferentes estrategias, entre las que se incluyen el uso de citoquinas y péptidos antimicrobianos (AMPs). Las citoquinas son pequeñas proteínas reguladoras intercelulares que desempeñan un papel central en el inicio, mantenimiento y regulación de la respuesta inmune innata. Concretamente, el IFN-γ tiene un papel fundamental en la promoción de la inmunidad protectora contra las infecciones. Por otro lado, los AMPs, como GWH1, son generalmente pequeños péptidos catiónicos de naturaleza anfipática que tienen actividades antibacterianas, antifúngicas y antivirales de amplio espectro, la capacidad de modular la respuesta inmune del huésped y una posibilidad reducida de inducir resistencia bacteriana. A pesar de su potencial como agentes antiinfecciosos, las citoquinas y los AMPs están sujetos a varias desventajas que deben abordarse antes de su posible aplicación biomédica. En particular, la baja estabilidad es un inconveniente común asociado a este tipo de compuestos proteicos. En este sentido, se han aplicado diferentes estrategias para superar esta limitación y mejorar la eficacia de estos fármacos tras su administración. La mayoría de estas estrategias consisten en la vehicularización de estas proteínas o péptidos en estructuras superiores, proporcionando un entorno protector y permitiendo la posibilidad de liberarlos en localizaciones concretas. La formación de estas estructuras puede ser modulada a través de un diseño racional sobre el gen de la proteína recombinante, como, por ejemplo, la incorporación de ciertos péptidos en la estructura de la proteína para generar componentes individuales con capacidad autoensamblante (nanopartículas solubles), o para obtener proteínas propensas a la agregación (cuerpos de inclusión -IBs-). Aunque aparentemente diferentes, todos estos complejos se originan a partir de interacciones proteicas impulsadas por factores termodinámicos y cinéticos similares que finalmente conducen a la formación de diferentes formatos de proteínas; arreglos estructurales superiores que involucran a través de interacciones dirigidas o no dirigidas, la convergencia de formas proteicas aisladas. En esta tesis se ha caracterizado y evaluado la nanoformulación de estas moléculas (citoquinas y AMPs) en diferentes formatos proteicos incluyendo IBs y nanopartículas solubles autoensamblantes con el objetivo de desarrollar alternativas terapéuticas a los antibióticos. Además, se ha arrojado algo de luz sobre las fuerzas que gobiernan el proceso de agregación y cómo se puede modular para promover la formación de dichos IBs.<br>The emergence of bacterial drug resistance to conventional antibiotics is a global alarming situation. This worrying scenario has forced the implementation of measures such us improved hygiene practices or antibiotic stewardship to reduce antimicrobial usage in all areas in which these therapeutics are commonly used, including human and animal medicine and food-producing animal industry. All these measures are intended to diminish the appearance and spread of drug resistance among bacteria, but when it comes to combat against drug or multidrug resistant bacteria, alternatives to traditional antibiotics are urgently needed. In this context, different strategies have been proposed as promising alternatives to antibiotics, including the use of cytokines and antimicrobial peptides (AMPs). Cytokines are small intercellular regulatory proteins that play a central role in initiating, maintaining, and regulating the innate immune response. Specifically, IFN-γ has a pivotal role in promoting protective immunity against infections. On the other hand, AMPs such as GWH1, are generally small cationic peptides with an amphipathic nature that have a broad-spectrum antibacterial, antifungal and antiviral activities, the ability to modulate the host immune response and a reduced possibility of inducing bacterial drug resistance. Despite their potential as anti-infective agents, cytokines and AMPs are subjected to several disadvantages that must be addressed prior to their possible biomedical application. In particular, low stability is a common drawback associated to these type of protein compounds. In this sense, different technologies and strategies have been applied in order to overpass this limitation and improve the efficiency of these drugs after administration. Most of these strategies consist on the vehicularization of these proteins or peptides into superior complexes, providing a protective environment and allowing the possibility of deliver them to the target site. The formation of these superior structures may be modulated by a direct rational design over the recombinant protein gene, such as que incorporation of certain peptides into the protein structure to generate building blocks for spontaneous self-assembling (soluble nanoparticles), or to obtain prone-to-aggregate proteins (inclusion bodies -IBs-). Although seemingly different, these small-scale complexes all originate from fundamental protein interactions and are driven by similar thermodynamic and kinetic factors finally leading to the formation of different proteins formats; superior structural arrangements that involve through directed or not directed interactions, the convergence of isolated protein forms. In this thesis, the nanoformulation of these molecules (cytokines and AMPs) into different protein formats including IBs and soluble self-assembling nanoparticles have been characterized and evaluated with the aim to develop therapeutic alternatives to antibiotics. In addition, some light has been shed on the forces that govern the aggregation process and how it can be modulated to promote the formation of IBs.<br>Universitat Autònoma de Barcelona. Programa de Doctorat en Biotecnologia

A unique, previously undescribed Gram-negative bacterium was isolated from several soils in Texas and extensively characterized in this study. The cells measured 1-2 by 4-6 μm. The distinguishing characteristic of the bacterium is the extraordinary capsular material which surrounds the cells. The new isolates are aerobic, mesophilic, non motile and have the ability to utilize a variety of organic compounds as the sole source of carbon and energy. The organism grows optimally at 30° C and the optimal pH lies between 7.0-8.0. The isolates produce catalase but oxidase is not produced. They do not produce indole or hydrogen sulfide. The organism can hydrolyze gelatin and Tween 80 but not starch, esculin and casein. The major cellular fatty acid is anteiso 15:0. The guanine and cytosine content is 58-62 mole%. The organism's taxonomic position was further established by specific gene probes, 16S rRNA homology, DNA homology and "ribotyping." These data showed that it was most closely related to members of the genus Paenibacillus, although somewhat divergent from other species classified in this genus. After careful evaluation of the results obtained during this study, it is proposed that this unique bacterium be named Paenibacillus velasolus sp. nov.

Aproximadament el 40% de les vaques lleteres desenvolupen una malaltia uterina durant el post-part que acaba provocant infertilitat. Alguns estudis indiquen que la infecció uterina, causada principalment per Escherichia coli durant la primera setmana post-part, està associada amb la metritis, caracteritzada per la inflamació de l’úter on la vaca és incapaç d’eliminar els bacteris patògens. El tractament antibacterià tradicional que s’utilitza per contrarestar la metritis pot no ser efectiu en tots els casos, sobretot quan hi ha una inflamació prolongada. El primer estudi està enfocat en avaluar l’efecte de 4 possibles probiòtics del grup de bacteris de l’àcid làctic (BAL): Lactobacillus rhamnosus, Pediococcus acidilactici, Lactobacillus sakei i Lactobacillus reuteri, en un cultiu primari d’endometri amb infecció bacteriana i/o inflamació. Els principals resultats obtinguts van ser que P. acidilactici disminuïa la infecció d’E. coli, que L. rhamnosus reduïa significativament la inflamació cel·lular i que L. reuteri era capaç de disminuir la infecció d’E. coli quan les cèl·lules epitelials estaven prèviament inflamades. Durant el segon estudi es van provar 4 combinacions diferents de BAL a partir dels resultats obtinguts en el primer estudi. Es va avaluar la capacitat d’aquestes combinacions de disminuir la infecció d’E. coli i reduir la inflamació en el mateix cultiu primari. La combinació composta per L. rhamnous/ P. acidilactici/ L. reuteri amb una ràtio de 12/12/1 va ser la seleccionada. Llavors es va decidir comprovar que aquesta combinació continués sent eficaç en un model ex vivo (explants d’endometri). Els resultats obtinguts confirmaven la capacitat de la combinació de BAL de reduir la inflamació tissular. Per altra banda els assajos amb microscòpia electrònica mostraven un efecte protector de BAL en les cèl·lules epitelials. Hi havia menys necrosis, menys dany mitocondrial i més moc en les cèl·lules tractades amb BAL que en les no tractades. En el tercer estudi es va aplicar la combinació de BAL intravaginalment a vaques i al cap de 3 setmanes es va recol·lectar els seus endometris per obtenir-ne explants i infectar-los amb E. coli ex vivo. No es van observar diferències en els marcadors d’inflamació entre les vaques tractades amb BAL o les vaques control, ni en el número de Lactobacillus que hi havia a l’endometri de les vaques. Per altra banda, les vaques tractades amb BAL tendien a tenir menys presència d’E. coli a la vagina que les vaques control, i a més, expressaven menys B-defensins i MUC1, considerats marcadors d’infecció. Finalment, en el quart estudi, es van analitzar els efectes in vivo de la combinació de BAL sobre la incidència de metritis i inflamació de l’endometri en vaques de llet quan s’administrava intravaginalment durant 3 setmanes pre-part i o a l’úter un dia post-part. Els principals resultats obtinguts van ser que el tractament vaginal reduïa un 58% la prevalença de metritis comparada amb el grup control mentre que el tractament endometrial no la variava. No es va observar cap diferència amb els marcadors d’inflamació però els dos tractaments disminuïen l’activitatneutrofílica.<br>Approximately 40% of dairy cows develop a uterine disease during the post-partum leading to infertility. Several studies indicate that uterine infection, mainly caused by Escherichia coli during the first week post-partum, is associated with metritis, characterized by inflammation in the endometrium where the cow is not able to clear pathogenic bacteria. The traditional antimicrobial treatment may lack efficacy, especially in cases of sustained inflammation. The first study is focused in the evaluation of 4 possible probiotics belonging to the lactic acid bacteria (LAB): Lactobacillus rhamnosus, Pediococcus acidilactici, Lactobacillus sakei, and Lactobacillus reuteri, in an endometrial primary culture against bacterial infection and inflammation. The main results were that P. acidilactici was able to reduce E. coli infection, L. rhamnosus diminished cellular inflammation, and L. reuteri reduced E. coli infection when the epithelial cells were inflammated. On the second study, 4 different LAB combinations based on the results of the first study, were tested using the same primary culture. The combination composed by L. rhamnosus/ P. acidilactici/ L. reuteri with a ratio of 12/12/1 was selected. Then, this combination was tested in an ex vivo model (endometrial explants). The obtained results confirmed the capacity of this LAB combination to reduce tissular inflammation. On the other hand, electron microscopy assays showed a protective effect of LAB in endometrial epithelial cells. There was less necrosis, mitochondrial damage, and more mucus in the surface of LAB-treated cells than not-treated cells. In the third study, LAB combination was applied in vivo in the vagina of several cows, and 3 weeks later, the endometrium of those animals were collected. Explants were made from the endometrium and then infected with E. coli. No differences were observed in the inflammation markers between LAB-treated and control cows, or in the final quantification of Lactobacillus in the endometrium. On the other hand, LAB-treated cows tended to have less presence of E. coli in the vagina than control cows and, moreover, they expressed less B-defensins and MUC1, considerate markers of infection. Finally, on the fourth study, the effects of LAB combination were analyzed in vivo quantifying metritis prevalence and endometrial inflammation in dairy cows when the LAB combination was applied intravaginally during 3 weeks pre-partum or intra-uterine, 1 day after calving. The main results were that the vaginal treatment reduced metritis prevalence up to 58% compared with the control cows while no differences were observed with the endometrial treatment. No differences were found in the inflammation markers whereas both treatments (vaginal and endometrial) were able to modulate neutrophilic activity

L’administració d’antibiòtics a dosis subterapèutiques va ser una estratègia extensament utilitzada a partir dels anys 50 en la producció animal per a la prevenció d’infeccions i com a promotors del creixement. Tanmateix, la creixent aparició de bacteris resistents als antibiòtics va comportar que l’any 2006, la Unió Europea (UE) prohibís l’ús dels antibiòtics com a promotors del creixement (AGP) en la nutrició animal (CE n. 1831/2003). Aquesta prohibició, però, va fer repuntar el nombre de casos d’infeccions en animals i toxiinfeccions alimentàries en humans que fins aleshores havien estat controlades. La salmonel·losi, segona causa d’infeccions alimentàries a la UE, és causada per diferents serotips del bacteri gramnegatiu Salmonella que contaminen ous i carn d’au de corral (Salmonella Enteritidis) i carn de porc i de boví (Salmonella Typhimurium). En humans, aquests dos serotips causen gastroenteritis mentre que en animals de granja, les infeccions subclíniques són molt comunes. Per aquesta raó, actualment, la indústria avícola destina molts esforços a la recerca d’estratègies nutricionals alternatives als AGP per a la seva inclusió en els programes de control de Salmonella. Així, el principal objectiu d’aquesta tesi ha estat determinar l’efecte del Salmosan® (S- βGM), un producte ric en mannan oligosacàrids (MOS), sobre la funció intestinal de barrera a l’intestí de pollastres d’engreix infectats amb S. Enteritidis, i en cultius de cèl·lules intestinals Caco-2 colonitzades per diferents serotips de Salmonella i en dos models d’inflamació intestinal amb cèl·lules Caco-2. En el pollastre, els resultats revelen que el S-βGM redueix la presència de bacteris adherits a l’epiteli i incrementa la producció de moc. Aquest moc constitueix una superfície d’adhesió i una barrera física per a l'accés dels bacteris a l'epiteli. En els cultius de cèl·lules intestinals Caco-2, el S-bGM té un efecte protector sobre la funció epitelial de barrera malmesa per la infecció amb S. Enteritidis, S. Dublin i S. Thyphimurium. En el cas de S. Enteritidis, aquest efecte es pot atribuir a una reducció de la invasió del cultiu i a la modulació de la permeabilitat paracel·lular. En els models d’inflamació intestinal, es va estudiar la capacitat del S-bGM per a protegir la funció epitelial de barrera independentment de la seva capacitat per a interaccionar amb Salmonella. Per aquesta raó, es varen utilitzar co-cultius de cèl·lules Caco-2 amb macròfags (cèl·lules dTHP1) estimulats amb LPS de S. Enteritidis i cultius de cèl·lules Caco-2 estimulats amb TNFa. En aquests models, es va estudiar l’efecte del S-bGM i el d’un probiòtic, Lactobacillus plantarum. Els resultats posen de manifest que en cap dels models estudiats, ni el S- bGM ni el probiòtic protegeix la funció epitelial de barrera. En canvi, la combinació d’ambdós reverteix els efectes sobre la funció epitelial de barrera. A més a més, també es va observar que el S-βGM estimula el creixement del L. plantarum. Aquests resultats permeten atribuir al S-βGM la capacitat per a interaccionar amb l’epiteli i que té com a resultat la modulació de la secreció de citocines. Així doncs, el S-βGM, un MOS ric en βGM, té propietats prebiòtiques i exerceix efectes beneficiosos sobre la funció intestinal de barrera que permeten considerar-lo un bon candidat com a alternativa als AGP, ja sigui sol o en combinació amb un probiòtic.<br>Antibiotics were widely used in the 50s in animal production for the prevention of infections and as growth promoters (AGPs). However, the increasing emergence of antibiotic-resistant bacteria meant that in 2006 the European Union banned the use of AGP in animal nutrition (EC No 1831/2003). Currently, the poultry industry devotes much effort finding nutritional strategies as alternatives to AGP to be included in Salmonella control programs. Thus, the aim of the thesis was to study the use of Salmosan® (S-βGM), a mannan oligosaccharide (MOS) rich product, in broilers and in in vitro (in intestinal Caco-2 cell cultures and in two models of intestinal inflammation). In chickens infected with Salmonella Enteritidis, the use of β-galactomannans (βGM) showed more presence of mucus associated with greater number of goblet cells, less M cells and longer villus in animals fed a diet supplemented with Cassia or Duraió gum. In intestinal cell cultures infected with Salmonella, S-βGM recovered the effects caused on paracellular permeability. This effect was associated with the reduction on reactive oxygen species production, the modulation of tight junction permeability and the capacity of S-βGM to agglutinate the bacteria. In the models of inflammation, in co-cultures of Caco-2 cells and macrophages stimulated with lipopolysaccharide (LPS) of S. Enteritidis and in Caco-2 cells stimulated with tumor necrosis factor α (TNFα), S-βGM or Lactobacillus plantarum did not prevent the disruption of paracellular pathway. However, the combination of both restored the values of non-stimulated cells. In stimulated co-cultures, S-βGM significantly increased interleukin (IL)-10 production whereas L. plantarum, significantly increased IL-6 production and the combination of both produced a significant reduction in TNFα and a significant increase in IL-10 and IL-6 levels. In addition, the S-βGM promoted L. plantarum growth. Thus, the results demonstrate that S-bGM has beneficial effects on intestinal morphology and protects epithelial barrier function by reducing Salmonella invasion, increasing probiotic growth, modulating the immune response and tight junction permeability. In conclusion, our data provide evidence of the positive effects of this product in animal nutrition as an alternative of AGP, alone or as a symbiotic.

El objetivo general de esta tesis doctoral fue el desarrollo de biosensores basados en aptámeros para la detección de bacterias enteropatógenas. Para ello se estudiaron diferentes estrategias de detección gracias a las propiedades únicas de los aptámeros, tales como su capacidad de adoptar diferentes estructuras conformacionales tras el enlace con la molécula diana, su estabilidad y la facilidad con que se pueden modificar químicamente, introduciendo marcajes o ciertos grupos funcionales. Las bacterias utilizadas en todas las estrategias de detección fueron: Salmonella typhimurium (diana del aptámero seleccionado, específicamente las proteinas transportador ABC, OmpA y precursor OmpD), Eschericia coli O157 Shiga, Shigella sonnei, Escherichia coli k5, Proteus mirabilis, Bacillus cereus y Kocuria lutea. En la primera fase de la tesis se realizó la caracterización de la bacteria Salmonella typhimurium y de la afinidad del aptámero por este microorganismo. La afinidad del aptámero por la bacteria se comprobó utilizando las técnicas de impresión por microcontacto (μCP), la microscopía de fuerza atómica (AFM) y la microscopía de fluorescencia. Posteriormente en la fase siguiente se desarrolló un sistema de detección basado en partículas magnéticas como plataforma de captura, preconcentración y detección basada en un ensayo competitivo indirecto. Se llevó a cabo la detección colorimétrica, por absorbancia, y electroquímica, por voltametría de pulso diferencial (DPV). En ambas alternativas de detección se observó reactividad cruzada de la bacteria Salmonella typhimurium con E. coli O157 Shiga y con Shigella sonnei, no presentándose dicha reactividad con las otras bacterias. El porcentaje de similitud de las proteinas diana análogas fue mayor en relación a las proteinas correspondientes a E. coli O157 Shiga y Shigella sonnei, y menor respecto a las proteinas análogas de las otras bacterias. En la tercera fase de la tesis se desarrollaron dos biosensores electroquímicos. Uno basado en la detección directa del enlace con la bacteria por espectroscopía de impedancia electroquímica (EIS). El otro biosensor se basó en la inhibición enzimática causada por el cambio conformacional del aptámero tras su enlace con la bacteria. En este caso se utilizó la técnica electroquímica DPV para medir la actividad de la enzima fosfatasa alcalina. Con el primero se lograron los mejores resultados, por cuanto los parámetros de comportamientos fueron los mejores (menor LOD, mayor sensibilidad, menor tiempo de análisis), pero además es una estrategia en la cual la construcción del aptasensor es simple puesto que consta de un solo paso. Finalmente los ensayos de selectividad llevados a cabo en todas las estrategias de detección muestran que todos los sistemas de detección fueron capaces de discriminar el grupo de bacterias clasificadas como enteropatógenos (Salmonella typhimurium, Escherichia coli O157 Shiga y Shigella sonnei) del resto de bacterias (Escherichia coli k5, Proteus mirabilis, Bacillus cereus y Kocuria lutea), demostrando que la afinidad del aptámero por Salmonella typhimurium no es específica para esta bacteria, sino que también existe una afinidad equivalente por E. coli O157 Shiga y Shigella sonnei.<br>The overall objective of this Thesis was to develop different biosensors based on aptamers to detect enteropathogen bacteria. This objective was accomplished through the study of different detection strategies which were proposed based on the unique properties of aptamers such as the capacity to present different conformational structures after linking to the target molecule, stability and ease of chemical functionalization. The affinity of the aptamer against the Salmonella typhimurium was confirmed by Microcontact Printing (μCP), Atomic Force Microscopy (AFM) and Fluorescence Microscopy. Then, a magnetic particle detection system was developed as a capture, pre-concentration and detection platform based on an indirect competitive test. Colorimetric and electrochemical detection, using differential pulse voltammetry (DPV) were performed. In both detection alternatives, cross-reactivity of Salmonella typhimurium with Escherichia coli O157 Shiga and Shigella sonnei was observed, with no cross- reactivity with Proteus mirabilis, Bacillus cereus, Kocuria lutea and Escherichia coli k5. In the next phase of the thesis, two electrochemical biosensors were developed, one based on the direct detection of the binding to the bacterium by electrochemical impedance spectroscopy (EIS) and the other based on the enzymatic inhibition caused by the conformational change of the aptamer after binding with the bacterium. In the second case the DPV technique was used to measure the activity of alkaline phosphatase. The best results were obtained with the impedance biosensor because as it showed improved behavior parameters (lower LOD, higher sensitivity and shorter analysis time). In addition, the construction of the aptasensor is simple as it consists of a single step. Selectivity tests carried out on all detection strategies showed that the developed detection systems were able to discriminate the bacterial group classified as enteropathogens (Salmonella typhimurium, Escherichia coli O157 Shiga and Shigella sonnei) from other bacteria (Escherichia coli K5, Proteus mirabilis, Bacillus cereus and Kocuria lutea). Hence, the affinity of the aptamer for Salmonella typhimurium is not specific for this bacterium and there is also an equivalent affinity for E. coli O157 Shiga and Shigella sonnei.

The symbiotic lifestyle represents a fundamental contribution to the diversity of marine ecosystems. Sponges are ideal models to study symbiotic relationships from evolutionary and ecological points of view since they are the most ancient metazoans on Earth, are ubiquitous in the marine benthos, and establish complex symbiosis with both prokaryotes and animals, which in turn harbour their own bacterial communities. In this thesis, we aim to go deeper into the mechanisms by which sponges establish symbiotic associations with members of the three domains of life, combining taxonomical, ecological, and molecular approaches. We study how sponges acquire their symbiotic microbes and whether these microbes contribute to shape the ecological distribution of their hosts. Moreover, we use the sponge-polychaete relationship as an example of multi-partner symbiosis and study the eukaryotic association from the microbial perspective. Finally, we focus on the less studied domain of life, the archaea, to gain insights into the composition and stability of these symbionts in sponges. To assess these goals, we characterized the sponge assemblages in two contrasting environments (well-preserved and impacted) of Nha Trang Bay (Vietnam) and selected the most abundant species for the study of their microbiomes. Additionally, four sponge species harbouring thousands of polychaetes were sampled to analyse the relationships sponge-microbes-polychaetes. Sponges and polychaetes were identified and their respective microbiones and the seawater bacterial communities were analysed by high-throughput sequencing of the 16S rRNA gene (V4 region). We first describe and illustrate the sponges collected to facilitate further taxonomic and faunistic studies in the area. Our samples belonged to 60 species (9 orders, 22 families, and 36 genera) of demosponges. A total of 24 species were added to the already known sponge fauna of Vietnam, from which, 11 species likely represent new species to science. The described species represent an increase of 8 % in the already known sponge list of Vietnam. Our results show that sponge assemblages were more diverse and rich in the well-preserved environments, being dominated by Neofibularia sp. and Aaptos suberitoides in the reefs, and by Monanchora unguiculata, Antho (Antho) sp., and Amphimedon sulcata in rocky habitats. On the other hand, impacted coral reefs were mainly dominated by two abundant species: Clathria reinwardti and Amphimedon paraviridis. Similar ecological metrics were shown by the sponge microbiomes according to the type of habitat, being more diverse in the well-preserved environments. Morever, the sponge microbiomes of the sponge assemblages from the impacted habitats showed higher intra-species dispersion and lower core size (shared ZOTUs across species replicates) than microbiomes of sponges from the well-preserved environments. In this sense, we propose that the Anna Karenina concept, which states that intraspecific variability is higher in dysbiotic than in healthy individuals, can also be applied at the community level for the study sponge assemblages. In our study sponges, bacterial communities were highly stable regardless of the environment, whereas some of their associated polychaetes varied depending on the sampling location. Environmental resilience to different habitat conditions was certainly true for bacterial communities of A. sulcata, the solely species that was found abundant in the two contrasting habitats explored. Moreover, the high overlap in bacteria composition between sponges and seawater suggest microsymbiont acquisition from the environment. In a similar manner, polychaetes were also able to specifically select and enrich some bacteria from their food sponge. Overall, most sequences were shared between biotypes, but at differential abundances, leading to highly specific and stable invertebrate microbiomes, acquired from the environment. Our results support the tenet “Everything is everywhere, but the environment selects”.<br>La vida en simbiosi representa una contribució fonamental a la diversitat dels ecosistemes marins. Les esponges són models ideals per l’estudi de les relacions simbiòtiques des del punt de vista evolutiu i ecològic, ja que són els metazous més antics de la Terra, són ubiqüistes al bentos marí, i estableixen simbiosis complexes amb procariotes i animals, que al seu torn, contenen les seves pròpies comunitats bacterianes. En aquesta tesis, volem aprofundir en els mecanismes pels quals les esponges estableixen associacions amb membres dels tres dominis de vida, combinant eines taxonòmiques, ecològiques i moleculars. Estudiem com les esponges adquireixen els seus microbis simbionts i com aquests microbis contribueixen a modelar la distribució ecològica de les esponges. A més, utilitzem la relació esponja-poliquet com a exemple de simbiosis multi-organisme i estudiem les associacions eucariotes des de un punt de vista microbià. Finalment, ens centrem en el domini de vida menys estudiat, les arqueas, per aprofundir en la composició i estabilitat d’aquests simbionts en esponges. Per assolir aquests objectius, vam caracteritzar els grups d’esponges de dos ambients diferenciats (impactat i ben preservat) de la badia de Nha Trang (Vietnam), i vam seleccionar les espècies més abundants per l’estudi del seu microbioma. Addicionalment, vam mostrejar quatre espècies d’esponges que contenien milers de poliquets per l’anàlisi de les relacions esponja-microbis-poliquets. Els nostres resultats mostren que les comunitats d’esponges eren més riques i diverses en els ambients ben preservats, i els seus microbiomes mostraven variables ecològiques similars, en els dos tipus d’ambients. La majoria de simbiosis estudiades mostraven una gran especificitat i estabilitat, independentment de l’ambient on vivia l’esponja. El gran solapament entre els bacteris de l’aigua i de l’esponja suggereix que hi ha adquisició microbiana de l’ambient. De forma similar, els poliquets també eren capaços d’adquirir específicament bacteris de les esponges de les quals s’alimentaven. En resum, la majoria de seqüències microbianes eren compartides entre els tres habitats estudiats (aigua/esponge/poliquet), però a diferents abundàncies, donant lloc a microbiomes específics i estables adquirits de l’ambient en els dos grups d’invertebrats estudiats .

The presence of agrochemical residues in both urban and agricultural water bodies has become ubiquitous, often producing deleterious effects in the impacted watershed including reductions in biodiversity, alterations in species interactions, and toxicity to non-target organisms. While these effects have been studied on metazoan consumers, the consequences of agrochemical contamination on microorganisms, such as bacteria, protozoa, and viruses, are poorly understood. Agrochemicals could act directly on microorganisms, including pathogens, by either facilitating their survival or decreasing their abundance. Further, a multitude of indirect effects of agrochemicals on microorganisms are possible, whereby agrochemicals alter predation, competition, or parasitism on or available nutrient to microbes. The primary method by which agrochemicals enter water bodies is through stormwater and agricultural runoff, which can also introduce agriculturally-associated zoonotic pathogens. Presently, regulatory standards utilize fecal indicator bacteria (FIB) to predict the presence of pathogens in contaminated watersheds. However, if agrochemicals have different effects on FIB and bacterial pathogens, then these regulatory standards might be confounded by the presence of pesticide residues in impacted water bodies. Additionally, if agrochemicals promote the survival of zoonotic pathogens, then the presence of pesticide residues could potentially increase risks to human health. The studies in this dissertation investigated both the direct and indirect effects of agrochemicals on the growth and survival of FIBs ( Escherichia coli and Enterococcus faecalis), zoonotic bacterial pathogens (E. coli O157:H7, and Salmonella enterica), and two virus groups (human polyomaviruses and adenoviruses). The agrochemicals utilized in these experiments are among the most prominently used in their respective pesticide classes and included the herbicide atrazine, the insecticide malathion, the fungicide chlorothalonil and inorganic fertilizer containing phosphate and fixed nitrogen. Initially, complex mesocosms containing zooplankton, phytoplankton, leaf litter, and vertebrate and invertebrate species were used to examine net (direct and indirect) effects of agrochemicals on FIB in sediments. Subsequent studies utilized experiments in simplified microcosms to detect direct or indirect effects (i.e., predation, competition or effects on nutrient resources) on FIBs and pathogens. In complex mesocosms, atrazine and fertilizer significantly increased FIB densities in the sediment; however, because of the complexity of the mesocosms, it was not possible to determine whether these results were the product of direct or indirect agrochemical effects. Simplified microcosms, limited to predominantly direct effects, as well as in vitro growth curves, revealed no direct effects of any agrochemical treatment on either growth or survival of FIB or bacterial pathogens. When algal communities were allowed to establish, however, atrazine significantly reduced both phytoplankton and E. coli densities in the water column, but increased E. coli densities within the sediments. These effects on E. coli were indirect because they required the presence of algal species. To investigate indirect effects of predation on FIBs and E. coli O157:H7, we manipulated the presence and absence of an obligate heterotroph, Tetrahymena pyriformis, a facultative heterotroph, Ochromonas danica, and natural protozoan populations. In both laboratory and greenhouse microcosm experiments, the fungicide chlorothalonil significantly reduced all protozoan populations, which resulted in increased densities of FIBs and E. coli O157:H7 because of reduced predation. Atrazine was not found to have any significant direct effect on the densities of T. pyriformis or natural protozoans; however, atrazine did significantly reduce O. danica densities in greenhouse experiments. In laboratory experiments with O. danica, atrazine treatments resulted in decreased densities of E. coli O157:H7. Presumably, atrazine prevented or reduced photosynthesis forcing O. danica to increase its predation on E. coli thus shifting its trophic level. These studies reveal that agrochemicals can have a significant effect on microbial communities, but that these effects are often indirect and mediated through alterations of nutrient resources and predation. Atrazine application reduced FIB and pathogen densities in the water column via reduction of phytoplankton and increased predation by O. danica. These data suggest that the net effects of atrazine is deleterious to FIB survival in the water column and that application of this herbicide could result in an ecosystem service, reducing the abundance of zoonotic pathogens and lessening the risk to human health. However, elevation of FIB densities was observed in the sediments when atrazine was applied. The potential resuspension of increased sediment bacteria may negate or out-weigh the deleterious effects of atrazine on bacteria in the water column. Chlorothalonil application decreased protozoan densities, lessening the stress of predation on the bacterial targets and increasing FIB and E. coli O157:H7 densities. The use of chlorothalonil may therefore have negative implications for human health risks, as the reduction in predation seems to facilitate the survival of zoonotic waterborne pathogens. Understanding the net effects of agrochemicals is important for public health, as pesticide applications can act to either maintain or diminish potential bacterial and protozoan pathogens of humans. These studies show that indirect effects of agrochemicals on non-target microbes tend to be more prominent than direct effects and can significantly impact the fate of bacterial pathogens in aquatic environments.

Les aminoglycosides (AG) sont une famille d’antibiotiques qui ciblent le ribosome bactérien. À titre d’exemple il s’agit de la néomycine, la gentamicine et la streptomycine. Quand ces antibiotiques se fixent au ribosome, ils provoquent des erreurs de lectures ou inhibent la synthèse des protéines, ce qui conduit à la mort cellulaire. Même s’ils ont été découverts il y a plus d’un demi-siècle, de nombreux aspects de leur mode d’action restent inconnus. L’accumulation des AG dans les bactéries se passe en trois étapes. La première consiste en une interaction électrostatique avec la membrane. La deuxième est une phase I énergie-dépendante (EDPI). Les antibiotiques rentrent dans le cytoplasme, atteignent les ribosomes, ce qui cause des erreurs de lecture et donc la production de protéines mal repliées. EDPI dépend du niveau énergétique de la cellule et le mécanisme d’entrée à travers les membranes reste inconnu. La troisième étape est la deuxième phase énergie-dépendante (EDPII), où l’antibiotique pénètre dans le cytoplasme en grande quantité par des membranes endommagées lors de la phase I. Le but de cette thèse était de créer de nouveaux outils afin d’étudier l’interaction des AG avec les bactéries et d’appliquer la méthodologie à des bactéries en phase rapide de croissance ou bien en état de persistance. Nous avons synthétisé des conjugués fluorescents des AG aux propriétés bactéricides. Avec ces conjugués nous avons analysé l’interaction des AG avec les bactéries à l’échelle de la cellule unique par microscopie de fluorescence. Nous avons combiné cette technique avec la cytométrie de flux (FACS) pour évaluer la cinétique d’accumulation. Cette étude démontre qu’il y a deux types d’accumulation : une à la périphérie avec interaction à la membrane et une deuxième où l’antibiotique est localisé dans le cytoplasme. Notre analyse démontre aussi que de faibles niveaux d’antibiotiques dans le cytoplasme sont tolérés et n’inhibent pas la croissance cellulaire. En utilisant un inhibiteur des phases EDPI et EDPII nous démontrons que cette technique permet de distinguer les différentes étapes de l’accumulation. Au cours d’ajustements du protocole, nous avons découvert que les AG peuvent entrer dans le cytoplasme par mechano-sensation et activation de canaux mécanosensibles (MS). Ces canaux sont connus pour avoir une affinité pour les AG. Ici pour la première fois nous montrons qu’une manipulation mécanique ouvre les canaux et stimule une entrée massive d’antibiotiques. Ce résultat inattendu pourrait permettre de mieux comprendre le mécanisme d’entrée des AG dans le cytoplasme. Après avoir étudié l’accumulation des AG dans les cellules en croissance nous avons étudié la tolérance aux AG pour les bactéries en phase de dormance : les cellules persistantes. Elles forment une sous-population parmi une population sensible. Elles sont en dormance et tolèrent de fortes doses d’antibiotiques. En absence d’antibiotique elles sortent de l’état de dormance pour reformer une population sensible à l’antibiotique. Par microscopie de fluorescence, nous montrons que les cellules persistantes ont une accumulation périphérique d’AG. Grâce à notre méthodologie, nous avons un outil performant pour identifier les différents états d’accumulation des AG. Avant cette étude il était seulement possible de connaître les niveaux d’accumulation mais la localisation de l’antibiotique demeurait inaccessible. Nous avons avec cette méthode étudié deux mutants d’E. coli, qui sont moins tolérants aux AG et identifié leurs caractéristiques d’accumulation. Enfin, nous avons développé un système de microfluidique adapté à l’étude de nos conjugués fluorescents pour étudier en temps réel l’accumulation par les cellules persistantes<br>Aminoglycoside (AG) is a family of antibiotic which target bacterial ribosome. Few examples of this family are neomycin, gentamicin and streptomycin. When these antibiotics bind to ribosomes, they cause miscoding or inhibit protein synthesis which consequently leads to cell death. Although discovery of these antibiotics was more than half a century ago, there are many facts about AGs’ action mechanism which remain unknown. AG accumulation in the bacterial cells happens in three steps. First step is cell membrane attachment. This step is driven by an electrostatic interaction with the cationic AGs. Second step is an energy dependent phase I (EDPI). In EDPI, the antibiotic enters into the cytoplasm and reaches ribosomes, causing miscoding and production of misfolded proteins. EDPI depends on cellular energy level, however to date the mechanism by which AGs pass through membranes and enter cytoplasm is unknown. The third step is energy dependent phase II (EDPII) in which the antibiotic enters into the cytoplasm in larger amount due to damages in the membrane that resulted from EDPI. The aim of this PhD was to create new tools to study the interaction of AGs with bacteria and apply the methodology to study fast growing bacteria as well as persister cells. We have made fluorescently-tagged AGs with preserved bactericidal properties. We used these conjugates to track down the interaction of AG at single cell level by fluorescence microscopy. We combined fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis to measure AGs accumulation in the cells at different time points to capture the kinetics of antibiotic penetration. This study showed that there are two accumulations patterns for the drug in cells: in the first step there is a peripheral accumulation, which corresponds to specific binging to cell membrane. Next there is a cytoplasmic accumulation in which the antibiotic in entering into the cytoplasm. According to microscopy time laps study, low levels of cytoplasmic accumulation is tolerated by cells and did not cause cell death. Using FACS analysis, we used an inhibitor of EDPI and EDPII and proved that with this technique we can distinguish different steps of AGs accumulation. During protocol adjustment steps we found that AGs can enter into the cytoplasm as a result of mechanosensation and activation of mechanosensitive (MS) channels. These channels have already been shown to have affinity to AG and here this is a first time that we observed that mechanical manipulation of cells lead to opening of MS channel causing massive cytoplasmic accumulation. This unpredictable result may lead us to a better understanding of the mechanism of AG entrance into the cytoplasm. After studying AG accumulation in fast growing cells, we studied AG tolerance for non-growing cells, which are called persisters. Persisters are antibiotic tolerant sub-population among susceptible bacterial cell population. Persisters are non-growing, dormant cells which tolerate high concentrations of antibiotic. In the absence of antibiotic, they exit this dormant state and grow into an antibiotic susceptible population. By fluorescence microscopy we showed that persister cells have peripheral accumulation of AG. Thanks to our methodology, we have a powerful tool by which we can determine the patterns of AG accumulation. Prior to this study, it was only possible to know the levels of accumulation and not the corresponding patterns. We applied the method to investigate AG accumulation in two mutants of E. coli, which are less tolerant to AG and defined their pattern of accumulation. Finally, we developed a coated microfluidic system, which is adapted to our antibiotics for studying in real time drug accumulation by persister cells

Diversity analysis of Escherichia coli have routinely utilised isolates obtained by culture of faeces on MacConkey selective media, under the assumption that the diversity identified in faecal isolates are representative of similar diversity in E. coli in the gastrointestinal tract (GIT). This study has addressed this important issue by specifically isolating E. coli from different regions of the gut in pigs and subjecting them to enzymatic multilocus enzyme electrophoresis (MLEE) and molecular virulence factor (VF) analysis to ascertain whether E. coli populations inhabiting different regions of the gut are different from each other. Combination of these results showed that on average, E. coli strains isolated from the upper GIT region (small intestine) of the pig are distinctly different from the E. coli strains isolated from the lower GIT region (large intestine). An important aspect of the finding that faecal E. coli are not truly representative of the diversity in the GIT is the mechanism used by specific clonotypes that have adapted to different geographical habitats to survive challenge from incoming strains.

Thesis (Ph.D.)--University of Western Sydney, Hawkesbury, 2004.<br>A thesis presented to the University of Western Sydney, Hawkesbury, Centre for Advanced Food Research, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.

The adenylate energy charge values of twenty bacteria isolated from soil and cultured in a medium consisting of soil and distilled water were determined by the luciferin-luciferase bioluminescense method. The purpose of this study was to examine the growth and energy charge values of these organisms in soil extract medium, and to determine what effect the addition of glucose has on their energy charge values. Three of the organisms employed in this study showed energy charge values similar to those reported for bacteria grown in enriched media. The remainder of the isolates demonstrated low energy charge values, and scant growth in the soil medium.

Aquesta recerca centrada en els bacteris processos relacionats amb enològic, com ara bacteris d'àcid làctic (BL) i bacteris d'àcid acètic (BA). Treballa principalment amb tècniques de cultiu independent per a la identificació de bacteris en una fermentació malolàctica fermentació (MLF) i en un acetificacion tradicional. En el cas de la BL, va dissenyar una estratègia d'identificació mitjançant PCR-RFLP. Perfils característics pels 4 gèneres BL presents en un FML s'aconsegueix. L'aplicació de nitrogen és comú per aconseguir un bon desenvolupament del responsable de llevats fermentació alcohòlica. Aportacions de nitrogen pot generar problemes més tard com és la producció d'amines biògenes. Més buscats amina és histamina. Els resultats de les proves d'aplicació de fosfat cristal·lí en diferents dosis, que va llançar en dosis baixes i es recomana, es generen les concentracions d'histamina en intervals ja descrit en la literatura. Superior a la dosi necessària de fosfat cristal·lí generats gairebé 5 vegades la concentració d'histamina en la dosi recomanada. Trobar en aquestes laboratori FML eren Leuconostoc i Oenococcus, principalment en les etapes finals de la MLF aquest últim. Per l'acidificació, després d'anàlisi d'identificació mitjançant tres tècniques diferents, es va identificar com Acetobacteris pasteurianus i algunes espèciess del gènere Gluconacetobacter com les espècies principals responsables de la producció tradicional de vinagres a Xile.<br>Esta investigación se centró en las bacterias relacionadas con los procesos enológicos, como son las bacterias lácticas (BL) y las bacterias acéticas (BA). Se trabajó principalmente con técnicas independientes de cultivo para lograr una identificación de las bacterias presentes en una fermentación maloláctica (FML) y en una acetificación tradicional. Para el caso de las BL, se diseñó una estrategia de identificación usando PCR-RFLP. Se logró obtener perfiles distintivos para los 4 géneros de BL presentes en una FML. La aplicación de nitrógeno es común para lograr un buen desarrollo de las levaduras responsables de la fermentación alcohólica. Los aportes de nitrógeno pueden generar problemas posteriores como es la producción de aminas biógenas. La amina más buscada es histamina.Los resultados de los ensayos de aplicación de fosfato diamonio (FDA) en diferentes dosis, arrojaron que en la dosis bajas y recomendada, se generan concentraciones de histamina dentro de rangos ya descritos en literatura. Dosis de FDA mayores a las requeridas, generaron casi 5 veces la concentración de histamina de la dosis recomendada. Las BL encontradas en estas FML fueron Leuconostoc y Oenococcus, esta ultima principalmente en las etapas finales de la FML. En el proceso de acetificación, tras el análisis mediante tres técnicas diferentes, se identificó como Acetobacter pasteurianus y algunas especies del género Gluconacetobacter como las principales especies responsables de la producción tradicional de vinagres en Chile.<br>This research focuses on the bacteria related to oenological practices, such as lactic acid bacteria (LAB) and acetic acid bacteria (BAA). He worked primarily with independent farming techniques to achieve identification of bacteria present in a malolactic fermentation (MLF) and traditional acidification. In the case of BAL, identification strategy using PCR-RFLP was developed. It was possible to obtain distinctive profiles for the 4 BAL genres present in an FML. The application of nitrogen is common for a good development of yeasts responsible for alcoholic fermentation. The nitrogen aplication can generate further problems such as the production of biogenic amines. The most wanted amine is histamine. Results of application trials diammonium phosphate (DAP) in different doses, showed that in the low and recommended dosage, levels of histamine are generated within ranges already described in literature. FDA doses greater than those required, generated almost 5 times the concentration of histamine than the recommended dose. The BAL were found in these FML Leuconostoc and Oenococcus, the latter mainly in the final stages of MLF. In the process of acetification, after identification analysis using three different techniques, was identified as Acetobacter pasteurianus and some species of Gluconacetobacter as the main species responsible for the traditional vinegar production in Chile.

Thesis (Master)--İzmir Institute of Technology, İzmir, 2005<br>Keywords: Lactococcus lactis ssp. lactis, chromosome profiling, pulsed field gel electrophoresis, plasmid profiling, plasmid stability. Includes bibliographical references (leaves 58-63)

A semi-synthetic medium for use in determining adherent growth with Staphylococcus epidermidis and Staphylococcus saprophyticus was developed. Production of an adherent biofilm was dependent upon the presence of hematin in the growth medium. Clinical strains of Staphylococcus epidermidis were tested for production of an adherent biofilm in trypticase soy broth, the semi-synthetic medium and the hyperalimentary nutrient solution used in the neonatal hospital unit. An adherent biofilm was obtained when Staphylococcus epidermidis was cultured m hematin supplemented hyperalimentary solution. Growth in the hyperalimentary nutrient solution diluted with fetal calf serum showed the same growth rate as when the nutrient solution was diluted with water. The final growth yield was always higher in serum diluted nutrients. There was no effect of hematin on the growth rate of the organisms.

The fruit production is threatened by several bacterial diseases, such as fire blight of apple and pear, bacterial canker of kiwifruit, bacterial spot of stone fruits, and angular leaf spot of strawberry. The conventional pesticides that are available for the control of these diseases are mainly copper compounds and they have a limited efficacy and negative impact on environment. Therefore, there is a need to develop alternative and sustainable management tools. This Ph.D. Thesis contributes to the development of a novel microbial biopesticide based on lactic acid bacteria. Two Lactobacillus plantarum strains were selected due to their broad-spectrum activity. In order to improve the epiphytic survival of both strains in plants and to get more consistency in their biocontrol efficacy, a physiological adaptive strategy was defined to increase the water-stress tolerance. Also, a monitoring method was developed to evaluate the population dynamics of a L. plantarum strain.<br>La producció de fruita està afectada per diferents malalties bacterianes de quarantena com el foc bacterià de les pomeres i pereres, el xancre bacterià del kiwi, la taca bacteriana dels fruiters de pinyol i la taca angular de les fulles de maduixera. Els plaguicides disponibles pel seu control són principalment compostos cúprics els quals tenen una eficàcia limitada i un impacte negatiu en el medi ambient. Existeix la necessitat de desenvolupar eines de control alternatives i més sostenibles. Aquesta tesi contribueix en el desenvolupament d’un bioplaguicida microbià basat en bacteris de l’àcid làctic. Es van seleccionar dues soques de Lactobacillus plantarum amb activitat d’ampli espectre i es va definir una estratègia fisiològica d’adaptació per incrementar la tolerància a l’estrès per manca d’aigua i així millorar la supervivència epifítica a la planta. També es va desenvolupar un mètode de monitoratge per avaluar les dinàmiques poblacionals d’una soca de L. plantarum.

Bacterial infections of the central nervous system (CNS), though uncommon, are associated with very high rates of morbidity and mortality. Recent research has also highlighted the correlation between pathogens and chronic diseases of the CNS, such as neurodegenerative disorders, particularly Alzheimer’s disease. Whilst some bacteria can cross the blood-brain/blood-cerebrospinal fluid barriers, to date, other pathways by which bacteria enter the CNS remain largely unknown. Identifying alternative paths by which pathogens can enter the CNS is thus important for developing novel strategies preventing CNS infection and potential long-term sequelae. Novel evidence suggests some bacterial species (as well as certain viruses and amoebae) can enter the brain via the cranial nerves innervating the nasal cavity, particularly the olfactory nerve that mediates the sense of smell and connects the nasal cavity with the olfactory bulb in the forebrain. The trigeminal nerve also innervates the nasal cavity and constitutes another invasion path. Only a handful of pathogens are thought to use cranial nerves to reach the brain; certain Chlamydia species (spp.) being amongst these. Chlamydia pneumoniae is to date the bacterium with the strongest established link to Alzheimer’s disease. Previous research by our laboratory has also demonstrated that the bacterium causing the tropical disease melioidosis, Burkholderia pseudomallei, can invade both the olfactory and trigeminal nerves, travel along these nerves, to then infect the CNS (the olfactory bulb and brainstem, respectively). We have also previously shown that in outbred mice, the olfactory nerve is resistant to B. pseudomallei infection. The nasal mucosa contains both innate and adaptive immune components and prevents many infections. If pathogens penetrate the mucosal barrier and reach nerves, glial cells of the nerves can also combat the infection. Whilst only a few macrophages are present inside the olfactory nerve fascicles, olfactory nerve glial cells, termed olfactory ensheathing cells (OECs), are powerful phagocytes with innate immune functions. Thus, in addition to the immune cells and other components of the immune system in the nasal mucosa, cranial nerve glia are thus thought to be key for preventing CNS infection, explaining why such infections are relatively rare. Some pathogens, however, can evade destruction by these cells and invade the nerves, however, it remains largely unknown which pathogens are capable of doing so. Furthermore, injuries to the nasal epithelium are common, and if the mucosal barrier is removed by injury, perhaps it is easier for pathogens to infect the underlying nerves (in particular the olfactory nerve) and then reach the CNS. With the exception of one bacterium (Staphylococcus aureus) for which injury has been shown to allow infection of the olfactory nerve, it also remains unknown whether epithelial injury increases the risk of pathogens invading the CNS via these paths. Thus, we need to determine which pathogens are capable of invading the CNS via nerves connecting the nasal cavity and the brain, and whether epithelial injury increases the risk of infection. Furthermore, determining the cellular mechanisms that protect against microbial invasion of the CNS via nerves, as well as why certain pathogens can evade destruction of the immune system may pave the way for the development of novel therapies preventing and treating CNS infections. The key aims of this thesis were to determine (1) whether prior injury to the nasal epithelium could allow B. pseudomallei to invade the olfactory nerve and bulb in the mouse strain where this nerve is usually resistant to this infection, (2) whether the bacterium Chlamydia muridarum (which infects mice and is commonly used to study Chlamydia spp. infection in rodents) can utilise cranial nerves that innervate the nasal cavity to invade the CNS and, if C. muridarum can invade the CNS, to then determine whether the bacteria remained viable and (3) whether C. muridarum can infect OECs, and how OECs respond to C. muridarum in vitro. This thesis demonstrated that injury to the olfactory epithelium allowed the invasion of the olfactory nerve and bulb by B. pseudomallei in S100β-DsRed Quackenbush mice, in which the olfactory nerve is otherwise typically resistant to infection. This work also showed that C. muridarum can rapidly (within 48 h) reach the CNS (olfactory bulb and cerebral cortex) via the olfactory nerve, as well as infect the trigeminal nerve, in mice. Immunohistochemistry showed the presence of C. muridarum inclusion bodies (membrane-bound components inside which the bacteria replicate intracellularly) and viable C. muridarum bacteria were also isolated from these regions. C. muridarum was shown to readily infect OECs in vitro, which led to the upregulation of a range of cytokines. The outcomes from this project will contribute to an increased understanding of how bacteria can reach the CNS and has revealed that injury to the nasal epithelium may increase the risk of CNS bacterial invasion via the olfactory nerve. The outcomes also include an increased understanding of how olfactory nerve glia become infected by and respond to bacteria. This work may also contribute towards the growing body of knowledge regarding the link between pathogens and certain diseases of the CNS, such as Alzheimer’s disease. Furthermore, with an increased understanding of how glial cells respond to bacteria, new therapies may be developed that stimulate bacterial degradation by the glia. Such therapies may provide valid future alternatives to antibiotics, also combating the growing problem of antibiotic resistance. Thus, this work may contribute to the foundation required to develop therapies to treat diseases that are currently not curable, as well as to better diagnose and identify susceptibilities to certain conditions.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Medical Science<br>Griffith Health<br>Full Text

Bacteria are ubiquitous to marine living surfaces, taking on a broad spectrum of roles from mutualistic to pathogenic. Despite their universality, much remains unknown about their basic ecology and interactions with higher organisms. To address this gap, this thesis firstly examines the bacterial communities associated with three co-occurring marine eukaryote hosts from temperate Australia: the demosponge Cymbastela concentrica, the subtidal red macroalga Delisea pulchra and the intertidal green macroalga Ulva australis. Molecular characterisation of the bacterial communities was undertaken using 16S rRNA gene library analysis to compare within-host (alpha) and between-host (beta) diversity for the three microbial communities. This study highlights the potentially substantial contribution host-associated microorganisms could have on marine microbial diversity. The remaining focus for this thesis was on the bacterial community associated with D. pulchra. This alga produces a suite of biologically active secondary metabolites (furanones) that non-toxically inhibit acyl homoserine lactone (AHL)-driven quorum sensing in bacteria, affecting a range of phenotypes including colonisation and virulence traits. The ecology of D. pulchra???s epiphytic bacteria was investigated using a mechanistic approach to explain bacterial colonisation patterns. In particular, concepts and models of ecological succession founded in eukaryote ecology were investigated. The thesis concludes with a study investigating the effect of furanones and elevated temperature on bacteria-induced disease and thallus bleaching of D. pulchra. In the presence of furanones colonisation and infection of two Roseobacter isolates from D. pulchra???s epiphytic bacterial community were inhibited. Ruegeria strain R11 was demonstrated to have temperature regulated virulence, which caused thallus bleaching in furanone-free algae. The implications of elevated sea temperatures resulting from global warming for algal health are discussed.

Floodplain lakes are wetlands which receive flood waters from nearby rivers or other sources. Water samples were taken from floodplain lakes near the Illinois River, the Mississippi River, and the Cache River in Southern Illinois. Fluorescence in situ hybridization (FISH), spectrophotometry, and gene probes were used to investigate the effect of nutrient and chemical concentrations on the abundance of nitrifying bacteria; specifically ammonia-oxidizing Nitrosococcus and Nitrosomonadales and nitrite-oxidizing Nitrospira and Nitrobacter. Nitrosococcus was the dominant ammonia-oxidizing bacteria at each river system. Nitrospira and Nitrobacter had similar average abundances. Nitrosococcus abundances showed a significant positive correlation with nitrate (NO3-) (R2= 0.247, P=0.05, 95% confidence R2≥0.199) and a positive trend with nitrite (NO2-) (R2= 0.194, P=0.10, 90% confidence R2≥0.125). Nitrosomonadales abundance positively correlated with temperature (R2= 0.530, P=0.05, 95% confidence R2≥0.510). Nitrospira abundances positively correlated with ammonium (NH4+) (R2= 0.265, P=0.05, 95% confidence R2≥0.199), NO2- (R2= 0.372, P=0.05, 95% confidence R2≥0.199), and NO3- (R2= 0.482, P=0.05, 95% confidence R2≥0.199). None of the target bacterial abundances significantly correlated with pH or dissolved inorganic phosphate.

Made available in DSpace on 2014-06-11T19:25:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T20:13:38Z : No. of bitstreams: 1 paro_mlc_me_araca.pdf: 3376248 bytes, checksum: 3cb7c10029858de2655c594466804a68 (MD5)<br>As doenças infecciosas representam uma das principais causas de perda precoce dos dentes, podendo levar a seqüelas graves. Os microrganismos normalmente envolvidos nessas patologias quase sempre pertencem a microbiota autóctone da cavidade bucal e, quase invariavelmente, são de baixa virulência. Assim, o objetivo desse estudo foi avaliar a susceptibilidade a antimicrobianos de bactérias anaeróbias obrigatórias e anaeróbias facultativas isoladas de processos infecciosos da cavidade bucal, procurando verificar a existência de padrões de susceptibilidade à fármacos nas diferentes espécies e gêneros microbianos. As amostras microbianas foram obtidas de 4 casos de osteomielite crônica da mandíbula, 3 lesões periapicais refratárias ao tratamento endodôntico, 30 infecções endodônticas, 7 casos de periodontite agressiva localizada e 2 casos de periodontite agressiva generalizada. O isolamento dos microbiano foi realizado em meios de cultura seletivos e a identificação dos isolados foi realizada de acordo com suas características morfológicas e bioquímico-fisiológicas. Os isolados, uma vez identificados, foram mantidos em nitrogênio líquido (- 196 oC). Nos testes de susceptibilidade, empregou-se o método de diluição em ágar e o meio de cultura empregado foi o ágar infuso de cérebro coração acrescido de extrato de levedura. Os resultados evidenciaram resistência natural dos anaeróbios facultativos ao metronidazol e níveis moderados de resistência às penicilinas, enquanto a cefoxitina, a associação de amoxicilina/ácido clavulânico e imipenem foram quase que universalmente eficazes. A lincomicina e a clindamicina também se mostraram eficazes, particularmente sobre os anaeróbios obrigatórios. O principal mecanismo de resistência aos b-lactâmicos foi a produção de compostos capazes de degradar essas drogas.<br>Infections diseases represent one of the major causes of early tooth loss, and they can lead to sequels. The microorganisms involved oftenly in these pathologies belong to oral microflora and almost all of them are of virulence potential. Thus, the objective of this study was to evaluate the susceptibility to antimicrobials of obligate and facultative anaerobes recovered from infections in head and neck area, trying to verify the existence of susceptibility patterns to those drugs the different species and microbial goods. Microbials samples were obtained of 4 cases of chronic osteomyelitis, 3 refractary periapical lesions, 30 endodontics infections, 7 cases of localized aggressive periodontitis, 2 cases of generalized aggressive periodontitis. The isolation microbial was accomplished in selective culture means and the identification of the isolated ones was accomplished in agreement with its morphologic and biochemical-physiologic characteristics. The isolates, after identification, were maintained in liquid nitrogen (- 196°C). The susceptibility tests, were carried out throught na agar dilution method and the culture medium employed was brain heart infusion agar supplemented with yeast extract. The results evidenced natural resistance of facultative anaerobes to metronidazole and moderate levels of resistance to penicillin, while cefoxitin, amoxycillin/clavulanic acid and imipenem were almost universally effective, particularly on obligate anaerobes. The main mechanisms of resistance to b-lactams was the production of compounds capable to destroy these drugs.

Thesis (M.S.)--West Virginia University, 2008.<br>Title from document title page. Document formatted into pages; contains ix, 98 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 66-70).

Leafy greens such as spinach have been the object of several recent food-borne pathogen outbreaks. The purpose of this study was to isolate bacteria spinach epiphytic bacteria that inhibit growth of E. coli O157:H7, which we describe as antagonism. The mechanism of antagonism was investigated and we attempted to improve the antagonistic potential in vitro and on spinach leaves when cellobiose, a carbon source utilized by the antagonists but not E. coli O157:H7, was added. There were larger culturable populations of bacteria on the leaves of savoyed cultivars compared to flat. From the isolated colonies, 47 displayed antagonism towards E.coli O157:H7, and were identified as members of 11 different genera and sixteen species. A representative isolate from each species was evaluated for three possible mechanisms of antagonism: acid production, secretion of an inhibitory compounds or secreted protease. The majority (14/16) produced at least a moderate level of acid. Two of these strains, Paenibacillus polymyxa and Pseudomonas espejiana, were found to secrete a non- protease antagonistic compound. These antagonists varied in their reduction of E.coli O157:H7 numbers in vitro, but all significantly reduced numbers in 48 hours of co-culturing in nutrient rich media. Five antagonists resulted in a significant reduction in E.coli O157:H7 populations when co-cultured on spinach leaves. Application of cellobiose did not improve the amount of antagonism in vitro or on the leaf surface after 24 hours.<br>Master of Science

Sources of E. coli were determined from impaired waterways in the Appomattox River watershed (in the lower Piedmont and South-Central Virginia) for the development of bacterial Total Maximum Daily Loads (TMDLs). The Appomattox River watershed is primarily undeveloped with 70.8% of the land forested, 17.0% used for agriculture (mainly livestock production), and 7.7% classified as water, wetland or barren land. The remaining 4.5% is developed for residential, commercial, and industrial land uses (mainly within the city of Petersburg). Using Antibiotic Resistance Analysis, a known source library of 1,280 E. coli isolates (320 isolates per source) was constructed. Water samples were collected monthly for between eleven and fourteen months (11/02-12/03) from 40 locations throughout the Appomattox watershed and analyzed for fecal coliforms, E. coli, and resistance to 7 antibiotics of varying concentrations. A total of 486 water samples (9,907 isolates) were analyzed during the study. The objectives of this study were verify that each sampling site exceeded state bacterial count standards (using fecal coliform data), to compare the Discriminate Analysis and Logistic Regression statistical models for use in the classification of isolates, and finally to determine the source of contamination at each site. The fecal coliform and E.coli data was used to determine if each site exceeded state standards during the assessment period. Thirty-eight of the sites exceeded the fecal coliform standard at least 10% of the time, and thirty-three exceeded the E.coli standard at least 10% of the time. Discriminate Analysis (DA) is typically used to classify isolates, but the results obtained from the DA model were unrealistic based on the watershed land uses. By statistically analyzing the original 1,280 E.coli isolates six different ways, a more appropriate classification of isolates was determined. The six analyzing methods were Regular DA and Logistic Regression (LR); DA and LR where each isolate whose probability fell below 80% was deleted; DA and LR where each isolate whose probability fell below 80% was used to create an Unknown category. The Logistic Regression model with an Unknown category proved to be the most appropriate. By using the Logistic Regression model, with Unknown category, to classify isolates, twenty five of the forty sites were discovered to be contaminated predominately with Livestock and fourteen of the sites predominately by Wildlife. One site was equally divided between these two categories. Human and Pet contamination were not dominant at any of the forty sites. This comparison of the DA and LR statistical methods could change the analysis standard for Bacterial Source Tracking and suggests that the model required to classify isolates depends on the watershed characteristics.<br>Master of Science