Publicación a texto completo no autorizada por el autor / La incidencia de las mareas rojas y la presencia de las especies de vibrios durante la ocurrencia de estos eventos constituyen una amenaza al desarrollo de la maricultura y a los bancos naturales de recursos bentónicos, sobre todo en las áreas someras. En el litoral peruano, las mareas rojas ocurren de manera recurrente durante la primavera y verano, y su tiempo de permanencia está sujeto a los cambios de las condiciones ambientales. Durante estos eventos se presentan algunas especies de Vibrio sp. con características patogénicas y toxigénicas que afectan a los organismos acuáticos y la salud humana, por lo que durante 2010 y 2012 se estudió la distribución temporal y espacial de vibrios marinos durante los eventos de mareas rojas en las bahías de Sechura, Callao, Pisco y San Nicolás-Marcona; esta última, considerada como control, porque son escasos los reportes de mareas rojas. Se observaron densidades bajas (<106 cel/L) de organismos que causan mareas rojas nocivas. El dinoflagelado Akashiwo sanguinea y el ciliado Mesodinium rubrum fueron observados en la zona del Callao; Akashiwo sanguinea, Mesodinium rubrum y Prorocentrum minimum, en Pisco; la diatomea Pseudo-nitzchia pungens y el dinoflagelado Dinophysis rotundata, en Sechura. También se observó la predominancia de la especie cosmopolita Vibrio alginolyticus en todas las áreas. En menor número, las especies V. vulnificus y V. parahaemolyticus en Pisco y Callao asociadas a temperaturas cálidas; estas últimas son conocidas por ser patógenas y provocar severas intoxicaciones en los seres humanos, por el consumo de mariscos o pescado crudos. El índice de correlación R varió entre 0,5 y 0,6, lo que indica la relación significativa entre mareas rojas, Vibrio sp. y factores ambientales. / Tesis

This study was conducted to examine the rate of contamination and molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred and eighty raw food samples were tested and 60.8% of meat and 18.0% of shellfish samples were found to be contaminated with Salmonella spp. which belonged to variety of serogroups and serotypes. More than 90% of all food sources contained Escherichia coli and 32% of 50 shellfish samples were contaminated with Vibrio parahaemolyticus. PFGE was used to determine the degree of relatedness of Salmonella spp. There were 33 distinct PFGE patterns from 51 Salmonella spp. isolates tested, indicating that PFGE could be used as an alternative method for serotyping for use in epidemiology of Salmonella spp. Susceptibility of the isolates to 15 antimicrobial agents was investigated. Moderate to high frequencies of resistance to antibiotics were observed in Salmonella spp. and E. coli isolates and multi-resistance, defined as resistance to at least 4 antibiotics, was observed. All of the V. parahaemolyticus isolates were resistant to ampicillin/amoxicillin but not to other antibiotics. Betalactam TEM gene and tetracycline resistance tetA, tetB genes were widely distributed in both E. coli and Salmonella spp. isolates. Other resistance genes, including sulI, cmlA, aadA, aphA1, dhfrV, and aac(3)-IV were also present at high to moderate levels. Identification and characterisation of the mobile genetic elements, including identification of class 1 integrons and plasmids were carried out for multi-resistance isolates. The integrons harboured varying gene cassettes, including aadA1, aadA2, aadA5, aacA4, dhfrXII, drfA1 and dhfrA17, blaPSE1 and catB3. Thirty-five percent of Salmonella spp. isolates and 76% of E. coli isolates harboured plasmids of more than 95 kb. Transfer of resistance phenotypes between the isolates via conjugation and phage transduction was also demonstrated. Salmonella genomic island 1 (SGI1), a 43-kb genomic region contains a 13-kb antibiotic resistance gene cluster, has been identified in an isolate of S. Albany from chicken. The presence of Salmonella spp. virulence genes was investigated to examine the pathogenicity potential of the isolates. The invA gene was present in all Salmonella spp. isolates and the plasmid virulence gene spvC was detected in one S. Typhimurium isolate only, on a 95 kb virulence plasmid. Invasion assays performed in vitro demonstrated that all Salmonella isolates were capable of invading human intestine INT407 cells. In addition, the investigation for the presence of 58 selected virulence genes showed that all the tested isolates contained at least one virulence gene and there were 16 genes which are associated with different pathotypes detected. The data obtained in this study indicates that raw food in Vietnam is a potential reservoirs for many pathogenic organisms, and confirms the role of food animals as a reservoir of multidrug resistant E. coli and Salmonella spp.

L'ostréiculture, dominée par l'élevage de l'huître creuse Crassostrea gigas, représente plus de 70% du chiffre d'affaire réalisé par l'aquaculture française. Au sein des écosystèmes aquatiques, les bactéries appartenant au genre Vibrio forment l'un des groupes bactériens les plus abondamment représentés. Deux espèces, Vibrio splendidus et Vibrio aestuarianus, sont fréquemment associées et de façon récurrente, à des mortalités sévissant dans les élevages d'huître creuse Crassostrea gigas, le plus souvent en période estivale. Ce travail de thèse avait pour objectifs d'étudier des interactions Vibrio-huître et leurs modulations en fonction de la virulence des pathogènes et des paramètres génétiques et physiologiques de l'hôte. Le développement d'outils de détection et de quantification sensibles et spécifiques et la maîtrise de protocoles d'infection expérimentale à Vibrio ont permis d'explorer des mécanismes de virulence, d'étudier la variabilité de la sensibilité des huîtres à ces Vibrio et de caractériser la pathogenèse. L'étude de la diversité spécifique des souches bactériennes isolées dans un contexte de mortalité estivale sur une large échelle de temps et d'espace a permis de montrer la prédominance épidémiologique du groupe V. splendidus et de l'espèce V. aestuarianus associée aux épisodes de mortalité estivale de C. gigas en France. Une corrélation ayant été observée entre pouvoir pathogène et activité métalloprotéasique, un test phénotypique prédictif de la virulence des souches a été proposé. L'exploration du phénomène de synergie dans la pathogénicité des deux souches observé en co-injection expérimentale a conduit à la mise en évidence de l'existence d'un système de quorum sensing régulant aux niveaux intraspécifique (V. splendidus) et interspécifique (V. splendidus/V. aestuarianus) la production et l'expression au niveau transcriptionnel des gènes codant les métalloprotéases Vsm et Vam des deux souches étudiées. L'analyse statistique des cinétiques de mortalité obtenues chez des familles de demi-frères diploïdes et triploïdes soumises à un protocole de co-infection standardisée révèle une sensibilité accrue des huîtres à cette vibriose expérimentale, en période de gamétogenèse active. Les huîtres triploïdes soumises à cette même infection expérimentale n'ont présenté aucun avantage significatif. L'existence d'une base génétique de la sensibilité des huîtres aux vibrioses expérimentales a été illustrée par l'évaluation des sensibilités de quatorze familles de la cinquième génération (G5) issue du programme de sélection divergente réalisée dans le cadre de MOREST. Cette étude a également permis la description de co-infections à herpès virus OsHV-1 et V. aestuarianus suggérant une multi-étiologie des phénomènes de mortalité estivale. Une étude de pathogenèse à V. splendidus et V. aestuarianus réalisée par cohabitation a visé l'exploration des interactions liant l'huître creuse C. gigas et les Vibrio virulents, V. splendidus et V. aestuarianus, ou non virulents présents naturellement dans la flore endogène de l'hémolymphe ou dans l'eau des aquariums. Cette nouvelle approche a permis de mettre en évidence la rapidité de transmission des Vibrio virulents des huîtres infectées aux huîtres sentinelles en moins de deux heures, accompagnée d'une perturbation significative, précoce et transitoire de la réponse immunitaire de l'hôte au niveau transcriptionnel au cours des six premières heures de cohabitation. La prise en charge différentielle des Vibrio pathogènes et des Vibrio commensaux par l'huître suggère l'existence de mécanismes conduisant à une spécificité des réponses de l'huître visant l'élimination des Vibrio pathogènes et le maintien d'une flore vibrionacée endogène probablement bénéfique pour C. gigas.

La Nouvelle-Calédonie présente de nombreux atouts pour la pénéiculture. Le ralentissement brutal de son développement en 1993 s'explique par l'émergence d'une pathologie saisonnière qui affecte les crevettes juvéniles en grossissement, mettant en cause des bactéries du genre Vibrio et qui a reçu le nom de Syndrome 93. Vibrio penaeicida et V. nigripulchritudo sont deux espèces au pouvoir pathogène particulièrement élevé qui affectent les élevages. Une étude de typage moléculaire a permis de préciser leur épidémiologie et de proposer quelques conseils d'ordre zoosanitaire. Un suivi épidémiologique par amplification génique est en cours pour V. penaeicida, qui montre l'importance des facteurs écologiques dans l'apparition des épisodes pathologiques. La pathogénicité de V. penaeicida a été étudiée en fonction du stade de développement de l'hôte, et des facteurs toxiques ont été mis en évidence dans les surnageants de culture. Une hypothèse a été émise sur la voie d'entrée du pathogène dans l'hôte. L'hypothèse d'un support plasmidique de la virulence a été émise et est actuellement étudiée. Des moyens de lutte ont été étudiés, des aspects de gestion zoosanitaires discutés. La sélection de lignées de crevettes à moindre sensibilité à l'infection apparaît comme une piste prometteuse. Des perspectives de recherches sont proposées, notamment l'étude de l'écologie de ces Vibrio pathogènes dans l'environnement d'élevage, de la pathogénie des vibrioses engendrées en terme de facteurs de virulence, des relations hôte – pathogène. Ces travaux pourront s'appuyer sur les nombreuses connaissances acquises depuis plusieurs années pour d'autres espèces du genre Vibrio.

The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

Many bacteria produce signal molecules that serve in a cell-to-cell communication system termed quorum sensing. This signalling system allows a bacterial population to co-ordinately regulate functions according to their cell number in a defined environment. As bacterial growth progresses towards the stationary phase, signalling molecules accumulate in the growth medium and, above a certain threshold level, regulate the expression of genes involved in diverse functions. Most of the functions monitored by quorum sensing are most beneficial when they are performed as a population than by single cells, such as virulence factor production, biofilm formation, conjugation and bioluminescence. Vibrio anguillarum is a bacterial pathogen that causes terminal hemorrhagic septicaemia in marine fish. V. anguillarum possesses multiple quorum sensing circuits similar to the LuxI/LuxR and the V. harveyi-type systems. In this study, a characterisation of the quorum sensing-regulated transcriptional activator VanT was made. VanT belongs to the V. harveyi LuxR family of transcriptional regulators, which play a central role in quorum sensing signalling in Vibrio species. VanT was shown to regulate serine, metalloprotease, pigment, exopolysaccharide (EPS) and biofilm production. VanT repressed an EPS locus that plays a critical role in bacterial colonization of the fish integument and virulence. The V. harveyi-like quorum sensing systems were shown to limit rather than induce vanT expression throughout growth in V. anguillarum. In contrast to homologous proteins in other Vibrio spp., the quorum sensing phosphorelay protein VanU and the response regulator VanO had antagonistic roles in the regulation of vanT expression. Unlike other members of the luxR family, vanT was expressed at low cell density and no significant induction due to quorum sensing regulation was seen. Interestingly, VanT expression was induced by the alternative sigma factor RpoS as the cells entered stationary phase. RpoS was shown to regulate VanT expression post-transcriptionally by promoting vanT mRNA stability. VanT and RpoS were important for bacterial survival under stress conditions, indicating that VanT is likely an essential factor of V. anguillarum stress response.

Vibrio cholerae, the causal agent of cholera typically encodes two critical virulence factors: cholera toxin (CT), which is primarily responsible for the diarrhoeal purge, and toxin-co-regulated pilus (TCP), an essential colonisation factor. Nontoxigenic strains expressing TCP can efficiently acquire the CT gene through lysogenic conversion with CTXΦ, a filamentous phage that encodes CT and uses TCP as a receptor.  V. cholerae is a Gram-negative bacterium and a natural inhabitant of estuarine and coastal waters throughout both temperate and tropical regions of the world. In the aquatic environment, V. cholerae encounters several environmental stresses, such as change in salinity, UV stress, nutrient limitation, temperature fluctuations, viral infections and protozoan predation. To fully understand the pathogenic and virulence potential of V. cholerae, knowledge is required of its interactions with, not only human, but also environmental factors. By using the nematode Caenorhabditis elegans as host model, we were able to identify a previously uncharacterised protein, the extracellular protease PrtV. PrtV was shown to be required for the killing of. elegans and also necessary for survival from grazing by the ciliate Tetrahymena pyriformis and the flagellate Cafeteria roenbergensis. The PrtV protein, which belongs to a M6 family of metallopeptidases was cloned and purified for further characterisations. The purified PrtV was cytotoxic against the human intestinal cell line HCT8. By using human blood plasma, fibrinogen, fibronectin and plasminogen were identified as candidate substrates for the PrtV protease. Outer membrane vesicles (OMVs) are released to the surroundings by most Gram-negative bacteria through “bulging and pinching” of the outer membrane.  OMVs have been shown to contain many virulence factors important in pathogenesis. Therefore, we investigated the association of PrtV with OMVs. PrtV was not associated with OMVs from the wild type O1 strain. In contrast, in an LPS mutant lacking two sugar chains in the core oligosaccharide PrtV was found to be associated with the OMVs. The OMV-associated PrtV was shown to be proteolytically and cytotoxically active. V. cholerae strains are grouped into &gt;200 serogroups. Only the O1 and O139 serogroups have been associated with pandemic cholera, a severe diarrhoeal disease.  All other serogroups are collectively referred to as non-O1 non-O139 V. cholerae. Non-O1 non-O139 V. cholerae can cause gastroenteritis and extraintestinal infections, but unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content and their potential to become human pathogens. We analysed clinical and environmental non-O1 non-O139 isolates for their putative virulence traits. None of them carry the genes encoding CT or the TCP, but other putative virulence factors were present in these isolates. The incidence of serum resistance was found to vary considerably and was independent of encapsulation. Three strains were strongly serum-resistant, and these same strains could also kill C. elegans.

Carbohydrates are known to play important roles in a large number of physiological and pathological processes. Conceivably, “binders” of carbohydrates of biological importance could be used as diagnostic and therapeutic agents. Currently, lectins are the major available tools in research for carbohydrate recognition. However, the available lectins often have cross-reactivity issues, along with the high costs and stability issues. Therefore, there is a critical need to develop alternatives (lectin mimics). In this regard, there have been very active efforts in developing different “binders”, such as small molecule lectinmimics and aptamers. Among all the small molecule lectinbmimics developments, boronic acid stands out as the most important building blocks of the sensors design for carbohydrates biomarkers due to its intrinsic binding affinities with diols. To address a fundamental question that whether boronic acid also binds to six-membered ring sugars, with very limited precedents, we provided a concrete experimental evidence of the binding. Specifically, a series of isoquinolinylboronic acids were found to have remarkably high binding affinities with fluorescence change upon binding to representative sugars. Most importantly, these isoquinolinylboronic aicds showed weak but very encouraging bindings with six-membered sugar model. All these promising results paves the way of using boronic acids, especially isoquinolinylboronic acid as building blocks for chemosensors design for biological carbohydrates biomarkers, which universally contain six-membered ring and liner diols. Aptamer provides another alternative way for sensors development for carbohydrates biomarkers as lectin mimics. Compared to lectins, they are normally cheaper and more stable. However, there is much less options. Another challenging area for aptamer-based lectin mimics development is the difficulty to differentiate changes in glycosylation patterns of a glycoprotein, which affect the function of a glycoprotein and thus recognized as biomarkers. To address this major challenge, our group first demonstrated that the incorporation of a boronic acid into DNA would allow for the aptamer selection process to gravitate towards the glycosylation site. To examine the generality of boronic acid incorporation, increase the structural diversity, and broaden the application of boronic acid-modified DNA, a series of B-TTP analogues with simplified structures were designed, synthesized, and successfully incorporated into DNA. A simple route was also developed using 1,7-octadiyne as a linker for both Sonogashira coupling with thymidine and CuAAC tethering of a boronic acid moiety. This paves the way for the preparation of a large number of B-TTPs with different structural features for aptamer selection or array analysis. Finally, bacterial quorum sensing has received much attention in recent years because of its relevance to pathological events such as biofilm formation. As one of the very first groups that developed a series of antagonists for AI-2 mediated quorum sensing, we herein designed and synthesized a series of analogues based on the structures of two lead inhibitors identified through virtual screening. Besides, we also examined their inhibitory activities, twelve of which showed equal or better inhibitory activities compared with the lead inhibitors. The best compound showed an IC50 of about 6 mM in a whole cell assay using Vibrio harveyi as the model organism. This encouraging results and SAR discuss also paves the way for the finding of more potent compound through further structure optimization.

The bacterial pathogen Vibrio cholerae is responsible for ongoing cholera outbreaks in Haiti and elsewhere. Association of V. cholerae with the human host is responsible for fatal disease, but the bacteria also reside as natural inhabitants of aquatic environments, commonly attaching as biofilms to chitinous surfaces of copepods and crabs. Prior studies in V. cholerae demonstrated that competence for genetic transformation, a mechanism of horizontal gene transfer (HGT), requires the TfoX regulator protein that is triggered by chitin, and the HapR transcription factor that is made in response to quorum sensing (QS) signals produced by V. cholerae and Vibrios. To define regulatory components connecting extracellular signals to natural competence, I first demonstrated that QS molecules produced by Vibrios within multi-species chitinous biofilms are required for DNA uptake by V. cholerae, confirming the critical role of QS signals in HGT. Second, I identified by transposon-mutagenesis a new positive regulator of competence, CytR (cytidine repressor), only studied prior in E. coli as a regulator of nucleoside scavenging. Specific mutations in V. cholerae CytR impaired expression of competence genes and halted DNA uptake; and the addition of exogenous cytidine had similar affects as predicted in E. coli. V. cholerae and other competent Vibrios encode TfoX, HapR, and CytR, although none of these regulators directly controls genes coding for the DNA uptake apparatus. Thus, these results have uncovered a regulatory network, likely used by many Vibrios, that contains additional factors linking several extracellular chemical molecules (cytidine, chitin, and QS signals) to DNA uptake. My study has begun to define a molecular mechanism by which both environment and genetics contribute to genome evolution for this important marine pathogen.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 162-167). Also available via World Wide Web.