Vibrio cholerae é uma bactéria autóctone em ecossistemas aquáticos, os fatores responsáveis pela virulência podem contribuir com a patogenicidade, influenciados por fatores genéticos e ambientais. Considerando a importância de conhecer e monitorar o V. cholerae, o estudo pretende caracterizar isolados da especie e padronizar uma metodologia para detecção em amostras de água. Os isolados foram avaliados por metodologias clássicas e moleculares, para confirmar espécie. Também, foi avaliada a presença de genes de virulência, susceptibilidade aos antibióticos e resposta em modelo invertebrado. Tres marcadores moleculares foram avaliados por PCR quantitativa. Observou-se que setenta dos isolados pertenciam a espécie V. cholerae e mostraram variação na prevalência dos genes de virulência e ao perfil de suscetibilidade ao antibióticos. Mostrou uma influencia da temperatura e concentração do inoculo no modelo invertebrado. Os marcadores moleculares selecionados mostraram a viabilidade da metodologia proposta neste estudo pela alta especificidade e sensibilidade. / Vibrio cholerae is an autochthonous bacterium in aquatic ecosystems, factors responsible for virulence may contribute to pathogenicity, influenced by genetic and environmental factors. Considering the importance of knowing and monitoring V. cholerae, the study pretend to characterize selected isolates and to standardize a methodology for detection in water samples. The isolates were evaluated by classical and molecular methodologies to confirm species. Also, the presence of factors associated with virulence, antibiotics susceptibility and response in invertebrate model were evaluated. Three molecular markers were evaluated by quantitative PCR. It was observed that seventy of the isolates belonged to the V. cholerae species and showed a variation in the prevalence of the virulence genes and the antibiotic susceptibility profile. Also, showed an influence of the inoculum temperature and concentration on the invertebrate model. The selected molecular markers showed the viability of the methodology proposed in this study for the high specificity and sensitivity.

L’augmentation de température que subit la planète ces dernières décennies a de nombreuses conséquences dont la recrudescence de maladies infectieuses aussi bien chez l’homme que chez les animaux. Certaines populations de l’ormeau européen Haliotis tuberculata, vivant dans les zones les plus chaudes de Bretagne et de Normandie ont ainsi subi de très importantes mortalités depuis 1997, dues à la bactérie Vibrio harveyi. Cependant, certaines des populations les plus sévèrement touchées se sont aujourd’hui reconstruites et les mortalités semblent s’être arrêtées dans certaines de ces zones. La question se pose donc de l’apparition d’une résistance de l’ormeau face à cette maladie émergente. Pour répondre à cette question, les réponses à l’infection de plusieurs populations naturelles par cette bactérie ont été analysées. Une population présentant une forte résistance à la maladie a été identifiée.La voie d’entrée de la bactérie (ie. les branchies) a été identifiée comme jouant un rôle dans la résistance à l’infection. Par ailleurs, des infections successives ont permis de démontrer un effet d’amorçage immunitaire. Suite à une première exposition, une protection durant jusqu’à deux mois intervient contre l’effet d’inhibition de la phagocytose, provoquée normalement par une infection à V. harveyi. La différence d’expression de gènes des hémocytes d’ormeaux sensibles et résistants a été quantifiée par RNAseq pendant une infection expérimentale. Cette comparaison a montré une reconnaissance plus efficace du pathogène chez les résistants, par des récepteurs tels que les TLR ou les PGRP. La forte surexpression chez la population résistante, d’un gène impliqué dans la synthèse de mucine qui est l’un des composants principaux du mucus renforce l’hypothèse d’une forte implication des branchies dans la résistance. Enfin, une analyse in silico des séquences obtenues en RNAseq a permis d’apporter des preuves de l’existence d’un système de méthylation de l’ADN chez H. tuberculata ainsi qu’une possible implication de ce système dans l’adaptation de l’ormeau à son milieu. / Increasing global temperatures have numerous consequences for marine ecosystems, including the rise of infectious diseases. Certain populations of the European abalone Haliotis tucerculata have suffered from severe and recurrent mortality since 1997 due to infection caused by the bacterium Vibrio harveyi, particularly in areas with higher average summer temperatures. Given the spatial heterogeneity in mortalities, and the observation that the historically most severely impacted populations have recovered in recent years, the question of the emergence of resistance to the disease was addressed. The mortality rate in response to infection by V. harveyi was quantified experimentally in abalone originating from three natural populations, and one population exhibiting resistance to the disease was identified. In a subsequent experiment, the immune response of abalone was compared between infected individuals from a resistant and from a susceptible population. The portal of entry of the bacterium (ie. gills) was identified as playing a role in resistance. Furthermore, successive exposures of abalone to the bacterium demonstrated an immune priming effect, such that following a first exposure, phagocytosis was no longer inhibited by infection with V. harveyi, and that this improved protection against the disease lasted for at least two months. Differences in gene expression was quantified by RNAseq in the hemocytes of resistant and susceptible abalone following exposure to the pathogen. This comparison showed that resistant abalone had more effective recognition of the bacterium by receptors as the TLR or PGRP. The substantial over-expression of a gene involved in the synthesis of mucin, the main component of mucus, (UDP-GalNAC) in the resistant population, supports the interpretation of a strong involvement of gills in the resistance. Finally, an in-silico analysis of the sequences obtained from RNAseq indicate the existence of a DNA methylation system in H. tuberculata and suggested an involvement of epigenetic mechanisms in the adaptation of abalone to its environment.

L’ostréiculture constitue l'une des principales composantes de l’aquaculture. Cependant, ce secteur est confronté à des épisodes de mortalités anormales survenant aussi bien en écloseries que dans le milieu naturel, affectant les huîtres diploïdes et triploïdes et à différents stades de leur vie. Pendant les épisodes de mortalité des mollusques bivalves en France, des bactéries, initialement classées dans le groupe de V. harveyi, ont été régulièrement isolées à coté des virus de type herpès, V. splendidus ou de V. aestuarianus. Afin d'affiner l’affiliation taxonomique de ces isolats, une caractérisation génotypique et phénotypique a été réalisée. Les isolats bactériens, initialement classés dans le groupe de V. harveyi, se sont révélés génétiquement plus proches de souches du groupe V. tubiashii, reconnues comme agents pathogènes affectant larves et juvéniles de mollusques aux Etats-Unis et en Angleterre. Des outils de diagnostic ont été élaborés pour évaluer la propagation de cette espèce lors des périodes de mortalité depuis 2007, supportant cette première description de V. tubiashii en France. La virulence des isolats et la toxicité de leurs produits extracellulaires (ECPs) ont été confirmés par infections expérimentales sur des larves et des juvéniles de C. gigas. Les essais in vitro ont révélé la capacité des ECPs de V. tubiashii à perturber des fonctions immunitaires hémocytaires probablement via la dégradation de certaines protéines structurales. Finalement, des analyses protéomiques et transcriptomiques ont révélé la conjonction de multiples facteurs de virulence, y compris les métalloprotéases dans la virulence des souches françaises de V. tubiashii. / The oyster farming constitutes one of the major components of the global aquaculture. However, this sector is facing abnormal mortalities outbreaks that affect diploid and triploid oyster at their different life stages, in the hatcheries and in the field. During bivalve molluscs mortality events in France, bacteria initially classified into Harveyi group, were regularly isolated along with herpes virus, V. splendidus or V. aestuarianus. In order to fine tune the taxonomic affiliation of those isolates, a genotypic and phenotypic approach was used. The bacterial isolates, initially misclassified into the Harveyi clade, were shown to be genetically closed to V. tubiashii strains already recognized as the main causative agents of larvae and juvenile mollusc mortalities in America and in England. A diagnostic tool was developed to evaluate its spread in mortality events since 2007, supporting this first description of V. tubiashii in France. Moreover, the virulence of isolates and the toxicity of their extracellular products (ECPs) were confirmed on C. gigas larvae and juveniles by experimental infections. Using in vitro assays, French V. tubiashii ECPs revealed their ability to alter some hemocytes immune defense probably through the degradation of matrix structural proteins. Finally, proteomic and transcriptomic analyses revealed the conjunction of multiples virulence factors including metalloproteases in the virulence of the French V. tubiashii strains.

Travaux de recherche effectués sous la supervision de la docteure Marylise Duperthuy (directrice) et de la docteure Catherine Paradis-Bleau (codirectrice). / L’augmentation de l’incidence des infections bactériennes par des souches résistantes, multirésistantes, voire même ultrarésistantes, aux antibiotiques, combinés à la crise de découverte de nouvelles molécules depuis les années 1960 et du sous-investissement chronique de certains états dans la recherche publique, pourrait coûter la vie à 10 millions d’êtres humains par an d’ici 2050. L’écrasante majorité des souches bactériennes résistantes aux antibiotiques sont des bactéries à Gram négatif. Ceci est notamment dû à la composition intrinsèque de leur enveloppe, leur permettant d’être insensibles à de nombreuses molécules pourtant létales pour d’autres types de bactéries. L’étude des composants et des mécanismes de biosynthèse de l’enveloppe, qui sont essentiels au maintien de l'intégrité des bactéries et peuvent être impliqués dans leur virulence, devrait permettre l’identification de nouvelles cibles thérapeutiques. Dans le premier chapitre, nous allons tout d’abord faire un tour d’horizon des éléments composant l’enveloppe des bactéries à Gram négatif, des mécanismes de résistance aux antibiotiques et du danger que représentent les bactéries à Gram négatif. Mes résultats de recherches, présentés sous forme de quatre articles aux chapitres deux à cinq, sont précédés d’une mise en contexte des recherches propres aux deux laboratoires, portant d’une part sur les biofilms et la mobilité de Vibrio cholerae et d’autre part sur l’enveloppe de Escherichia coli. Dans le premier article, nous avons déterminé par quel mécanisme la polymyxine B en concentration sous-inhibitrice affecte la formation de biofilm chez Vibrio cholerae. Nous avons observé que la polymyxine B affecte principalement le flagelle par une action mécanique. La formation de biofilm nécessitant le flagelle dans ses premières étapes de formation, nous avons conclu que l’action de la polymyxine B prévenait ce changement d’état. Dans le second article, nous avons conçu un protocole d’évolution expérimentale qui nous a permis d’identifier des mutations dans différents gènes permettant à Vibrio cholerae de se déplacer plus rapidement en présence de concentration sous-inhibitrice de polymyxine B. Nous avons alors identifié chez plusieurs souches différentes mutations ayant probablement réduit ou anéanti la fonction des protéines IhfA, DacB, VacJ (MlaA) et MlaF. En nous basant sur la littérature, nous proposons que la perte de fonction de ces 5 protéines induisant l’augmentation de la mobilité en présence de polymyxine B puisse s’expliquer selon trois mécanismes, impliquant la stabilité de l’enveloppe, la sécrétion de vésicules de membrane ou une altération de l’expression de différents gènes. Dans le troisième article, nous avons étudié l’implication du stress oxydatif dans l’arrêt de la synthèse de la paroi qui est fatal au mutant ∆elyC de Escherichia coli. Nous avons alors démontré que la lyse du mutant ∆elyC est causée par la surproduction de radicaux hydroxyles dans son enveloppe. Cette molécule cause des dommages dans l’enveloppe suffisamment importants pour provoquer l’arrêt de synthèse de la paroi. Le mécanisme provoquant cette surproduction de radicaux toxiques reste encore à déterminer. Par le biais de l’étude de la fonction du facteur ElyC, nous avons découvert une nouvelle vulnérabilité dans l’homéostasie de l’enveloppe qu’il nous tarde de pouvoir exploiter. Dans le dernier article, nous avons étudié l’importance de la voie de synthèse de l’antigène commun aux entérobactéries dans le processus létal apparaissant en l’absence du facteur ElyC. Nous avons découvert que le mutant ∆elyC accumule un élément commun aux voies de synthèse de la paroi et de l’antigène commun aux entérobactéries, l’undécaprényl pyrophosphate. Nous avons également découvert que l’augmentation du recyclage de cette dernière via la surexpression du gène codant pour la protéine PgpB permettait de prévenir la lyse du mutant. Notre hypothèse est que l’absence de ElyC induit une mauvaise répartition de l’undécaprényl phosphate entre les voies de synthèse de la paroi et de l’antigène commun aux entérobactéries. La suractivité de la voie de synthèse de l’antigène commun aux entérobactéries induirait l’accumulation d’undécaprényl pyrophosphate provoquant l’inhibition de Pbp1b, empêchant l’ajout de nouvelles sous-unités à la paroi conduisant ainsi à la lyse du mutant ∆elyC. Ainsi l’ensemble de ces travaux a permis de mieux comprendre et d’identifier des vulnérabilités de l’enveloppe de Vibrio cholerae et Escherichia coli, de préciser certains mécanismes ainsi que d’entrevoir le rôle de divers facteurs impliqués dans l’homéostasie de leur enveloppe. / The increased incidence of bacterial infections by resistant, multiresistant and even extensively drug-resistant strain, combined with the crisis of new molecules discovery since the 1960s and the chronic underinvestment of certain states in public research, could cost 10 million human lives per year by 2050. The overwhelming majority of resistant bacteria are the Gram-negative one. This is mainly due to the intrinsic composition of their envelope, allowing them to be insensitive to many molecules yet lethal for other types of bacteria. The study of envelope components and biosynthetic mechanisms, which are essential for the maintenance of bacterial integrity and could be involved in their virulence, should lead to the identification of new therapeutic targets. The first chapter gave an overview of the different elements composing Gram-negative bacteria envelope, mechanisms of resistance and the threat that Gram-negative bacteria represent. The presentation of my work, divided in four articles from chapter two to chapter five, was preceded by a contextualization of the research projects specific to both laboratories, on biofilm and motility of Vibrio cholerae on one hand and on Escherichia coli envelope from the other hand. In the first paper, we determined the mechanism by which a sub-inhibitory concentration of polymyxin B affects biofilm formation in Vibrio cholerae. We observed that polymyxin B mainly affected the flagellum by a mechanical action. Since biofilm formation requires the flagellum in the early stages of its formation, we concluded that the action of polymyxin B prevented this change in Vibrio cholerae lifestyle. In the second paper, we designed an experimental evolution protocol which allowed us to identify mutations in different genes that increase Vibrio cholerae motility in the presence of sub-inhibitory concentration of polymyxin B. We then identified that different mutations had altered ihfA, dacB, vacJ (mlaA) and mlaF genes in different mutants. These mutations have probably induced reduction or loss of function of the proteins for which they respectively code. Based on literature, we hypothesize that the loss function of these proteins inducing increase in mobility in the presence of polymyxin B could be explained by three 7 mechanisms involving envelope stability, secretion of membrane vesicles or altered expression of various genes. In the third paper, we investigated the involvement of oxidative stress in the fatal peptidoglycan synthesis arrest occurring in the ∆elyC mutant of Escherichia coli. We then demonstrated that lysis of the ∆elyC mutant is caused by the overproduction of hydroxyl radicals in its envelope. This molecule causes multiple and sufficiently important damages in the envelope to stop the synthesis of the wall. The mechanism causing this overproduction of toxic radicals has yet to be determined. By studying the function of the ElyC factor, we have discovered a new weakness in envelope homeostasis that we are eager to exploit. In the last paper, based on a previously formulated hypothesis, we investigated the importance of the enterobacterial common antigen synthesis pathway in the lethal process occurring in the absence of ElyC. We found that the ∆elyC mutant accumulates common element to the cell wall and enterobacterial common antigen synthesis pathways, undecaprenyl pyrophosphate. We also observed that the ∆elyC mutant lysis phenotype could be suppressed by increasing undecaprenyl pyrophosphate recycling via the overexpression of the gene coding for the phosphatase PgpB. We propose that the absence of ElyC induces a misallocation of undecaprenyl phosphate between the cell wall and enterobacterial common antigen synthesis pathways, in favors the latter pathway. Overactivation of the enterobacterial common antigen pathway would induce undecaprenyl phosphate accumulation, causing inhibition of Pbp1b, thus blocking addition of new subunits to the cell wall and leading to its lysis. Ultimately, all the original data generated by my research project has led to a better understanding of envelope vulnerabilities of Vibrio cholerae and Escherichia coli, to clarify certain mechanisms but also to glimpse the role of various factors involved in the homeostasis of their envelope. / L'aumento dell’incidenza delle infezioni da batteri resistenti, multi-resistenti e anche estremamente resistenti agli antibiotici, combinata con la rarefazione della scoperta di nuove molecole fra gli anni 1960 e il sotto investimento cronico di certe nazioni nella ricerca pubblica, potrebbe costare la vita a 10 milioni di essere umano all'anno nel 2050. La stragrande maggioranza delle specie resistenti agli antibiotici sono batteri Gram- negativi. Questo è dovuto principalmente alla composizione intrinseca dei loro involucri che permette loro di essere insensibili a molte molecole che sono letali per altri tipi di batteri. Lo studio delle componenti e i meccanismi di sintesi dell’incurvo, che sono essenziali nell’integrità dei batteri e che possono essere coinvolti nella loro virulenza, dovrebbe permettere l'identificazione di nuovi bersagli terapeutici. Nel primo capitolo, avremo una panoramica dei diversi elementi che compongono l'involucro dei batteri Gram-negativi, dei meccanismi di resistenza e della minaccia che i batteri Gram-negativi rappresentano. La presentazione del mio lavoro, diviso in quattro articoli dal capitolo due a cinque, sarà preceduta da una contestualizzazione dei progetti di ricerca specifici di ciascuno dei due laboratori, sul biofilm e la motilità di Vibrio cholerae da un lato e sull'involucro di Escherichia coli dall'altro. Nel primo articolo, abbiamo determinato il meccanismo con cui la polimixina B in concentrazione sub-inibitoria influenza la formazione de biofilm in Vibrio cholerae. Abbiamo osservato che la polimixina B danneggia principalmente il flagello attraverso un'azione meccanica. Poiché la formazione del biofilm richiede il flagello nelle prime fasi della sua formazione, abbiamo concluso che l'azione della polimixina B ha impedito questo cambiamento nello stile di vita di Vibrio cholerae. Nel secondo articolo, abbiamo messo a punto un protocollo di evoluzione sperimentale che ci ha permesso di identificare diversi geni che aumentano la motilità di Vibrio cholerae in presenza di una concentrazione sub-inibitoria di polimixina B. Abbiamo quindi identificato vari mutazioni in diversi varianti che hanno probabilmente provocato la riduzione o il soppresso della funzione delle proteine IhfA, DacB, VacJ (MlaA) e MlaF. Sulla base della letteratura, proponiamo che la perdita di funzione di queste proteine inducendo un aumento della 9 mobilità in presenza di polimixina B può essere spiegato attraverso tre meccanismi coinvolgendo la stabilità dell'involucro, la secrezione di vescicole di membrana o l’alterazione dell’espressione di diversi geni. Nel terzo articolo, abbiamo studiato il coinvolgimento dello stress ossidativo nell'arresto della sintesi del peptidoglicano che è fatale al mutante ∆elyC di Escherichia coli. Abbiamo poi dimostrato con numerosi esperimenti che la lisi del mutante ∆elyC è causata dalla sovrapproduzione di radicali idrossilici nel suo involucro. Questa molecola causa importanti danni nell’involucro provocando l’inibendo della sintesi del peptidoglicano. Il meccanismo che causa questa sovrapproduzione di radicali tossici deve ancora essere determinato. Studiando la funzione del fattore ElyC, abbiamo scoperto una nuova vulnerabilità nell'omeostasi dell’involucro che siamo in fretta di sfruttare. Nel nostro ultimo articolo, basandoci su un'ipotesi precedentemente formulata, abbiamo studiato l'importanza della via di sintesi dell'antigene comune agli enterobatteri nel processo letale che si svolge in assenza di ElyC. Abbiamo scoperto che il mutante ∆elyC accumula un elemento comune alle vie di sintesi del peptidoglicano e dell'antigene comune agli enterobatteri, l'undecaprenil pirofosfato. Abbiamo anche scoperto che l'aumento del riciclaggio di quest'ultimo attraverso la sovraespressione del gene pgpB previene la lisi del mutante. Proponiamo che l'assenza di ElyC induce una allocazione diffetosa del undecaprenil fosfato tra le vie di sintesi del peptidoglicano e dell'antigene comune agli enterobatteri. L'iperattivazione della via dell'antigene comune agli enterobatteri provoca l'accumulo di undecaprenil pirofosfato, causando l'inibizione di Pbp1b e dunque dell’aggiunta di nuove unità al peptidoglicano, provocandone quindi la lisi. Quindi, questo lavoro ha portato a una migliore comprensione o identificazione delle vulnerabilità dell'involucro di Vibrio cholerae ed Escherichia coli, a chiarire certi meccanismi essenziali ma anche a intravedere il ruolo di vari fattori coinvolti nell'omeostasi del loro involucro.

Cholera is a water-borne disease that continues to ravage resource poor communities around the world especially those in developing countries. The disease is caused by Vibrio cholerae microorganisms whose natural habitat is the aquatic ecosystem. It is believed that this microorganism prior to becoming the primary cause of cholera acquired virulence factors expressed by two separate genetic elements. These genetic elements are known as VPIФ and CTXФ were acquired in that order for known physiological reasons. However only V. cholerae in possession of the CTX genetic element are capable of causing cholera disease. At present only two serotypes are known to have the ability to cause cholera and these are V. cholerae serotypes O1 and O139. SODIS (Solar disinfection) is an extremely low cost refined technology that can be used for the disinfection of water especially in areas where there is a considerable amount of sunshine. Although this technology is a composite of various factors the underlying principle is the use of solar ultraviolet radiation (SUVR). The preliminary target of SUVR is the cytoplasmic membrane and this was confirmed by flow cytometric analysis. The consequences of leaky cytoplasmic membrane include cellular death to the microorganism as well as an increase in cholera toxin secretion. The main objective of this study was to investigate the effect of solar ultraviolet radiation on the growth of V. cholerae and on the secretion of cholera toxin and to provide supporting information for the use of SODIS in South Africa while observing the possible role that climate may play in the onset of cholera disease. The initial part of the study evaluated the culturability, biomass increase and cholera toxin secretion in both a nutrient poor and a nutrient rich media by two toxigenic and one non toxigenic strain of V. cholerae. A series of pH and temperature combinations were used to achieve this objective. The result revealed that the microorganisms survived in both media. An increase in biomass was observed for all the bacteria grown in the nutrient rich media whereas in the poor nutrient media the bacteria remained culturable but no increase in biomass was observed. Interestingly lower temperatures seemed to provide more optimal growth conditions while high temperature on most occasions favoured cholera toxin secretion, in both media.The second part of the study required the exposure of the microorganisms to SUVR. A SODIS approach was used with a few modifications. The V. cholerae strains were exposed to solar radiation during all the seasons of the year. Evaluation of the viability, the increase in biomass and the detection of cholera toxin secretion was determined after each exposure to solar radiation. The results seem to suggest that the effect of SUVR depended on the season of the year, the nature of the media, strain, solar conditions and in the duration of solar exposure, in no particular order. The secretion of cholera toxin was mainly dependent on the media used, the season of the year and on the serotype of the strain. This study represents the first report on the evaluation of SUVR for the disinfection of water under South African conditions (Pretoria area) during all seasons of the year with variations in solar radiation levels and temperature. Furthermore what actually happened to V. cholerae during solar exposure in terms of cell morphology, cell viability and secretion of cholera toxin is also reported and this can give an insight of the possible role that SUVR may play in the onset of cholera. The main recommendation emanating from this study is the sensitisation of communities worldwide about the capacity that, SUVR carries to lighten the burden of communicable water borne diseases especially, in resource limited areas through the implementation of SODIS. / Life and Consumer Sciences / M. Sc. (Life Science)

Made available in DSpace on 2015-03-04T18:50:21Z (GMT). No. of bitstreams: 1 thesis.pdf: 4253837 bytes, checksum: 15abaa1d981d9b5cd585cfb3e8da60c9 (MD5) Previous issue date: 2010-07-22 / Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior / The discovery of the non-coding RNA (ncRNA) has been primarily focused on the genomes of eukaryotes and pathogenic bacteria. In vibrios, all that is known up to now regarding ncRNAs involves V. cholerae N16961 e V. campbellii ATCC BAA-1116. In this study, ncRNA candidate genes were identified in the genomes of V. campbellii ATCC BAA-1116, V. alginolyticus 40B, V. communis 1DA3 e V. mimicus VM573. V. alginolyticus 40B and V. campbellii ATCC BAA-1116 are abundant in brazilian corals, whereas V. mimicus VM573 is a toxic strain (carrying the Vibrio pathogenicity island) atypical to this species. In order to identify the ncRNAs in silico, the tools Infernal and Rfam database were used. Perl programs were developed in the present work. The Infernal tool and the Rfam database are based on the Covariance Model (CM), a special case of Stochastic Context Free Grammars (SCFG). Up to 38 ncRNAs were identified per species. They were classified into seven classes according to their regulatory function and/or structural (1. riboswitches, 2. modulators of protein activity, 3. RNA's antisensus of trans action, 4. RNA's antisensus of cis action, 5. ribonucleoproteins, 6. regulation by transcription termination and 7. unknown classification). The most abundant group was the riboswitch, whereas the less abundant group was the ribonucleoprotein. This work demonstrated that the ncRNAs show a great diversity in functional classes, possibly associated with the regulation of different cellular processes in vibrios, including regulation of pathogenicity. / A descoberta de RNA não-codificante (ncRNA) tem focado principalmente nos genomas de eucariontes e de bactérias patogênicas. Em vibrios, o que se conhece até o momento sobre ncRNAs envolve V. cholerae N16961 e V. campbellii ATCC BAA-1116. Neste estudo, são identificados genes candidatos de ncRNAs no genoma de V. campbellii ATCC BAA-1116, V. alginolyticus 40B, V. communis 1DA3 e V. mimicus VM573. V. alginolyticus 40B e V. campbellii ATCC BAA-1116 são abundantes em corais brasileiros, enquanto que V. mimicus VM573 é uma linhagem toxigênica (CT e TCP positiva) atípica desta espécie. Para identificar os ncRNAs através de análise in silico foram utilizadas ferramentas já disponíveis (Infernal e a base de dados Rfam) e programas em Perl desenvolvidos no presente trabalho. A ferramenta Infernal e a base de dados Rfam são baseados em modelo de covariância (CM), um caso especial de gramáticas estocásticas livres de contexto (SCFG). Foram identificados até 38 ncRNAs por espécie, os quais foram classificados em sete classes de acordo com sua função regulatória e /ou estrutural (riboswitches, moduladores da atividade de proteínas, RNAs antisenso de ação trans, RNAs antisenso de ação cis, ribonucleoproteinas, regulação por término de transcrição e classificação desconhecida). O grupo mais abundante foi o riboswitch, enquanto que o grupo menos abundante foi o ribonucleoproteina. Este trabalho demonstrou que os ncRNAs apresentam uma ampla diversidade de classes funcionais, estando possivelmente associados com a regulação de diferentes processos celulares em vibrio, incluindo a regulação da patogenicidade.

Made available in DSpace on 2016-04-28T20:17:28Z (GMT). No. of bitstreams: 1 2008 - Ronaldo Leao Guimaraes.pdf: 352046 bytes, checksum: 7639df2176f027af3722d0b90e93a856 (MD5) Previous issue date: 2008-12-01 / The aim of this study was to evaluate the presence of Vibrio sp., Aeromonas sp. and Enterobacteriaceae in marine and farmer shrimp. For this, 20 samples of marine shrimp (Penaeus sp.) were collected from Itajuru channel, Cabo Frio, RJ, and other 20 samples of farmer shrimp were acquired in municipal market of Cabo Frio, RJ between January to August 2007. These samples were processed using standard methodology and inoculated in selective media for the research of these microorganisms. The isolates identification was performed by specific biochemical tests. 88 strains were isolated; 51.1% belonging to Vibrio sp., 28.5% to Aeromonas sp. and 20.4% to Plesiomonas shigelloides. It was not detected the presence of E. coli or Salmonella. Among the Vibrio sp., there was a prevalence of Vibrio alginolyticus with 45% and Vibrio parahaemolyticus with 10.5%. Aeromonas hydrophila contributed with 30% of the isolates of this genus and 83.3% of the isolates from Plesiomonas shigelloides were obtained from marine shrimp while 16.7% were obtained from farmer shrimp. The results show a high frequency of potentially pathogenic strains in these shrimps, emphasizing the importance of monitoring these organisms not only to shrimp industry but also to public health. / Este trabalho teve como objetivo pesquisar em camar?es de vida livre e criados em cativeiro a presen?a de microrganismos dos g?neros Vibrio, Aeromonas e representantes da fam?lia Enterobacteriaceae. Para tal, foram coletadas 20 amostras de camar?o de vida livre (Penaeus sp.) do canal de Itajuru, Cabo Frio, RJ; e outras 20 amostras de camar?o de cativeiro do com?rcio local deste munic?pio, no per?odo de Janeiro a Agosto de 2007. Estas amostras foram processadas seguindo metodologia padr?o e inoculadas em meios seletivos para a pesquisa dos microrganismos citados. A identifica??o dos isolados foi realizada atrav?s de testes bioqu?micos espec?ficos. 88 cepas foram isoladas, das quais, 51,1% pertencente ao g?nero Vibrio, 28,5% ao g?nero Aeromonas e 20,4% a Plesiomonas shigelloides. Em nenhuma amostra foi detectada a presen?a de E. coli ou Salmonella spp. Dentre os Vibrios, houve predomin?ncia de Vibrio alginolyticus com 45% e Vibrio parahaemolyticus com 10,5%. Aeromonas hydrophila contribuiu com 30% dos isolados deste g?nero e 83,3% dos isolados de Plesiomonas shigelloides foram obtidos de camar?o de vida livre enquanto 16,7% foram obtidos de camar?o de cativeiro. Os resultados apresentam uma grande freq??ncia de cepas potencialmente patog?nicas ao homem presentes nestes camar?es; ressaltando a import?ncia do monitoramento destes organismos n?o s? para a ind?stria de carcinicultura como tamb?m para a sa?de p?blica.

Le contrôle des pathologies est un élément primordial pour réduire les risques en élevage larvaire de mollusques bivalves. Il requiert une connaissance précise des maladies, des pathogènes et de leur mode d'action. Une nouvelle espèce de vibrio pathogène pour les larves de P. maximus, Vibrio pectenicida a été décrite par des caractères phénotypiques et génotypiques. Elle appartient au groupe de V. splendidus dans lequel se retrouvent d'autres nouvelles espèces pathogènes. La pathogénie de V. pectenicida a été étudiée en histologie et par des infections expérimentales sur des élevages larvaires conventionnels et axéniques. Ces études ont confirmé le caractère pathogène des souches de V. pectenicida et l'absence de symptômes spécifiques de la maladie. En ultrastructure, la localisation des bactéries à l'intérieur des tissus et de cellules peut être expliquée par un phénomène de translocation. L'activité toxique des vibrios pathogènes se manifeste vraisemblablement par des facteurs internes à la bactérie. En effet, une action létale de lysats bactériens de Vibrionaceae, et principalement de la fraction cytoplasmique, a été mise en évidence sur des larves axéniques. L'existence de facteurs toxiques internes a été confirmée par l'utilisation d'un test de chimioluminescence (CL) sur des hémocytes d'adultes. En effet, l'activité inhibitrice observée avec les bactéries entières, a été retrouvée dans la fraction cytoplasmique des vibrios pathogènes. D'après des essais de purification partielle, un des facteurs toxiques doit être une petite molécule (moins de 3kDa), hydrophile, résistante aux protéases, aux acides et à la chaleur. Cette toxine est éloignée des toxines bactériennes déjà décrites. Les relations entre l'immunité des adultes, très résistants aux attaques bactériennes, et celles des larves ont été abordées à partir de deux expériences: une infection expérimentale provoquée par une souche proche de V. splendidus sur des P. maximus adultes et l'injection de V. pectenicida à des animaux adultes entraînant la baisse de réactivité des hémocytes. Enfin quelques applications pratiques ont été proposées pour éviter l'utilisation d'antibiotiques durant la phase larvaire.

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

La culture de la palourde japonaise, Ruditapes philippinarum, en Bretagne s'est estompée à partir des années 80 après l'apparition de la vibriose appelée Maladie de l'Anneau Brun (MAB). Cette maladie est causée par la présence de la bactérie Vibrio tapetis qui provoque un dépôt caractéristique de conchyoline sur le bord interne des valves des palourdes. Les animaux parviennent à réparer la coquille au niveau des dépôts de conchyoline par un phénomène de recalcification. Des géniteurs et des familles de palourdes ont été étudiés afin de différencier les individus les moins sensibles à cette maladie. Ainsi, ces études ont permis de discriminer deux familles montrant des caractères de défense immunitaires différents au niveau des paramètres hémocytaires et au niveau du développement de la MAB. De plus, il a été démontré que le V. tapetis induit une production de l'oxyde nitrique in vitro dans les hémocytes de la palourde japonaise. Enfin, les approches moléculaires ont permis de mettre en évidence l'expression spécifique de deux transcrits de gènes (carbonique anhydrase like et inhibiteur de sérine protéases (serpine)) dans le manteau en lien avec le développement de la MAB.