Aquatic animals, such as fish and shellfish, provide important economic and nutritional benefits for human society. Due to overexploitation of natural fish sources through traditional wild-caught fisheries, aquaculture (generally described as fish farming or culturing) has grown into an economically important industry. A major focus area for the aquaculture field is related to sustainability by ensuring the health and welfare of the aquatic animals. Communities of microorganisms inhabiting the various niches of a given host comprise its microbiome and provide several key health benefits. The microbiome impacts nutrient acquisition, gut homeostasis, protection against pathogens, and immune system modulation. Therefore, much attention has been placed on studying how various culturing conditions and host factors impact the microbiomes of aquatic animals. Here, multiple studies were conducted to elucidate the impacts of various parameters on the microbiomes of rainbow trout, steelhead trout, and Nile tilapia, including dietary supplementation, administration of probiotics and animal age. Though there is a significant correlation between the diet fed to fish and their microbiome communities, small dietary changes such as the inclusion of a dried and lysed yeast product, acting as a protein source alternative to unsustainable fishmeal did not significantly alter the intestinal adherent microbiome of rainbow trout. Moreover, an optimal percentage of yeast replacement that did not negatively impact weight gain for the aquaculture-raised fish was identified, suggesting its efficacy for the industry. Similarly, the intestinal adherent microbiomes of steelhead trout were not significantly altered by diet supplementation with a Bacillus subtilis probiotic. The total microbiome of steelhead trout (mucosa combined with digesta) was instead significantly changed when they were only fed the probiotic additive at an early stage of intestinal development. This change in the microbiome of steelhead trout correlated with a significant increase in weight gain compared to fish only fed the probiotic during later stages of intestinal development. These findings also corroborate previous observations wherein the intestinal microbiome of fish varies during their developmental stages but then stabilizes over time. Determining the core set of bacteria present in fish microbiomes, independent of treatment variables, is another important factor when considering attempts to manipulate the microbiome. To that end, a literature review was conducted in which the phyla Firmicutes, Proteobacteria and, to a lesser extent, Actinobacteria, Bacteroides, and Tenericutes were identified as likely members of the rainbow trout core microbiome. Bacterial families identified as part of the core phyla included Lactobacilliaceae that are commonly used as probiotics and Mycoplasmataceae that lack cell walls. Preventing dysbiosis of the rainbow trout microbiomes will be crucial to ensuring the health of the fish hosts and increasing longevity and profitability of the aquaculture industry. Another important aquaculture-raised species is the Eastern oyster. This animal is critical for the ecological health of the Chesapeake Bay, and it is also an important source of revenue. A significant portion of the revenue flow is the harvest and sale of live oysters for consumption. Unfortunately, consumption of raw or undercooked oysters is the most common route of infection by the human pathogen Vibrio parahaemolyticus (VP) as oysters are a natural reservoir for VP. This bacterium is responsible for a debilitating acute gastroenteritis with potential to cause fatal septicemia. Despite efforts to mitigate infection by this CDC-reportable pathogen, cases continue to increase. The understudied host-microbe relationship between the Eastern oyster and VP has been implicated as a path to research for potential future therapeutics. A novel culturing system for oysters was created using fermentation jars within a BSL-2 ready biosafety cabinet. Using this system, the effect of harvest season was tested against the inoculation efficiency of VP. It was found that higher native Vibrio levels within the oysters were present during the summer compared to the winter. Moreover, addition of the bacteriostatic antibiotic chloramphenicol (Cm) enabled a higher inoculation efficiency by VP during both the summer and winter compared to oysters not exposed to the antibiotic. During the winter, exposure to Cm led to the highest inoculation efficiency (~100%). These findings confirm the importance of the existing microbial communities against exogenous inoculation. Therefore, a year-long study was conducted to investigate the microbiome of oysters during each season. This pan-microbiome study identified a significant impact of harvest season on the microbiome structure. An increased diversity, including higher levels of Cyanobacteriaceae, was observed during the summer. Whereas an increase in Arcobacteriaceae was observed during the winter. Bacteria that persisted throughout the year included Mycoplamataceae and Spirochaeteacae; these families may represent potential members of the Eastern oyster core microbiome. Further work is needed to study the localization patterns of VP within oysters. Such work includes further optimization of immunohistochemistry (IHC) and intracellular colonization assay methods under development here. Collectively, studies of the oyster-microbe interactions will help improve aquaculture methods and identify mitigation targets to reduce VP-related clinical infections. / Doctor of Philosophy / Fish and shellfish provide important economic and nutritional benefits for human society across the globe. Unfortunately, over-fishing of traditional sources of fish and shellfish has led to a reduced supply for world markets, even as the human population increases. Aquaculture, or fish farming, has been around for centuries, but its role in society has significantly increased in the past 50 years. It currently provides about half of fish and other aquatic products on the market today. To better maintain and increase the sustainability and profitability of this industry, more focus is being placed on the health of the fish. The microbiome is the collection of communities of microorganisms, including bacteria, fungi, and archaea, that inhabit various environments including animal hosts. The majority of this dissertation focuses on the impact of factors like diet and age on the microbiomes of aquaculture-raised animals, especially fish. Dietary changes such as the addition of dried yeast-products had a significant impact on fish health but not on the microbiome communities. However, a common probiotic, Bacillus subtilis, did significantly increase not only the growth rate of trout but it also significantly altered the total intestinal microbiome found in the feces and the intestinal mucosal layer. Moreover, it was found that early exposure of the animals to the probiotic had enhanced benefits even though the microbiome appeared to stabilize over time as the fish developed. Maintaining or improving the microbiomes of fish, paying close attention to the microbes that exist as part of a core group of bacteria always present, is vital to ensuring fish health and understanding vertebrate host-microbe relationships. Thus, an analysis of the core microbiome of trout was performed. The final set of projects within this dissertation focused on the relationship between the Eastern oyster, a mollusk native to the Chesapeake Bay, and the bacterial human pathogen Vibrio parahaemolyticus (VP). VP is the leading cause of seafood-borne acute gastroenteritis worldwide, and efforts are needed to mitigate the increasing rate of human infections. Therefore, a simple system using fermentation jars within the laboratory biosafety cabinet was designed to enable safe culture of oysters that were exposed to VP under experimentally controlled conditions. Oysters harvested during the summer naturally harbored higher amounts of native Vibrio organisms in contrast to the winter oysters that harbored much lower levels. A separate microbiome analysis revealed large shifts in the oyster microbiome between summer and winter, although some microbes were continually present. The lower levels of existing Vibrio species detected in winter oysters may have allowed for the higher efficiency of inoculation of winter animals by VP. In fact, these winter animals had Vibrio microbiomes that were completely dominated by the inoculated strain which will enable future work to observe the pattern by which VP localizes, or colonizes, the oysters. Ultimately, these efforts may lead to the development of future disease mitigation strategies against VP.

The objective of this project was to examine the effects of various storage temperatures and times on the microbiological quality, safety, and viability of hard shelled quahog clams. Samples were stored at four different incubation temperatures (3.3, 7.2, 10.0, and 12.S0C) for a period of three weeks, following their harvest from growing waters during the summer when the water temperatures were ~ SO°F, and during the winter when water temperatures were ~ 40°F. With regards to safety, clams were analyzed for two naturally-occurring pathogens, Vihrio parahaemolyticus and Vihrio vulnificus. Aerobic plate counts (APCs), coliforms, and fecal coliforms were determined to define the level of quality. During the summer, mesophilic APCs containing 2% NaCI ranged from 104 to 108 CFU/g, and during the winter, mesophilic APCs with salt ranged from < 100 ESPC to 104 CFU/g. Comparatively, during the summer, mesophilic APCs containing no added NaCI ranged from < 100 ESPC to 105 CFU/g, and during the winter, APCs without salt ranged from < 100 ESPC to 102 CFU/g. Coliform and fecal coliform counts ranged from < 0.3 to 61.1 MPN/g and < 0.3 to 24.4 MPN/g, respectively. During the summer, V. parahaemolyticus was isolated from 56% of the samples, with the highest concentration, 24,000 MPN/g, occurring on day 12 at 12.8°C. / Master of Science

Vibrio parahaemolyticus is a Gram-negative, halophilic pathogen that occurs naturally in coastal and estuarine environments. This human pathogen is frequently isolated from a variety of seafood, particular oysters, and is the leading cause of gastroenteritis associated with seafood consumption. Several outbreaks of V. parahaemolyticus infections linked to consumption of raw oysters have been documented. Contamination of oysters with V. parahaemolyticus is a concern for public health. This study investigated the efficacy of high pressure processing (HPP) in inactivating V. parahaemolyticus in raw Pacific oysters (Crassostrea gigas) and identified a process condition capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in raw oysters for commercial application. Raw Pacific oysters were inoculated with a clinical strain of V. parahaemolyticus 10293 (O1:K56) to levels of 10⁴⁻⁵ cells per gram and processed at 293 MPa (43K PSI) for 90, 120, 150, 180 and 210 s. Populations of V. parahaemolyticus in oysters after processes were analyzed with the 5-tube most probable number (MPN) method. A minimum HPP of 293 MPa for 120 s at groundwater temperature (8±1 °C) was identified capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in Pacific oysters. The HPP (293 MPa for 120 s at 8±1 °C) was validated at a commercial scale according to the FDA's National Shellfish Sanitation Program Post Harvest Processing (PHP) Validation/Verification Interim Guidance for Vibrio vulnificus and Vibrio parahaemolyticus. Negative results obtained by the MPN method were confirmed with a multiplex PCR detecting genes encoding thermolabile hemolysin (tl), thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). Oysters processed at 293 MPa for 120 sec had a shelf life of 6-8 days when stored at 5 °C or 16-18 days when stored in ice. This validated HPP was accepted by the FDA as a post harvest process to eliminate V. parahaemolyticus in raw oysters. / Graduation date: 2012

A análise da expressão gênica em larga escala é de fundamental importância para a melhor compreensão do funcionamento celular e dos mecanismos de regulação gênica. Ela possibilita a medida dos níveis de expressão de milhares de genes simultaneamente, o que torna possível uma visão mais abrangente do sistema biológico. Dentre as principais técnicas experimentais disponíveis para esta finalidade, a tecnologia de microarranjo tem sido amplamente utilizada. O objetivo desta dissertação foi determinar os genes de V. parahaemolyticus que têm sua expressão induzida ou reprimida na presença do aminoaçúcar N-acetilglucosamina (NAG). V. parahaemolyticus é uma bactéria marinha, comumente encontrada na água e em associação com organismos marinhos. O NAG é um dos aminoaçúcares mais abundantes no meio marinho. Para isso, Vibro parahaemolyticus RIMD2210633, foi cultivada em dois meios como fontes de energia. O primeiro meio composto por maltose e NAG e o segundo apenas por NAG. O cultivo bacteriano foi feito em condições aeróbicas, sob baixa agitação, a 28C. Foram retiradas duas amostras do cultivo no tempo de 24 horas após o início do experimento a fim de realizar a extração de mRNA e a preparação do cDNA. Os experimentos foram feitos em três replicas. As misturas de cDNA preparadas a partir do RNA extraído de cada réplica foram utilizadas em hibridizações em lâminas de microarranjo contendo um total de 4832 ORFS do genoma de V. parahaemolyticus RIMD2210633. A análise comparativa da expressão gênica de V. parahaemolyticus nas duas condições de cultivo resultou na detecção de 59 genes com expressão induzida, 38 genes reprimidos, e 4245 sem expressão modificada (aumentada ou diminuída) na presença de NAG. No total, 523 genes foram excluídos da comparação pois a hibridização não foi satisfatória. Ocorreu uma ordenação dos genes seguindo a classificação funcional do banco de dados TIGR-CMR e KEGG. Os genes com expressão induzida, pertencem principalmente às classes de funções regulatórias, metabolismo de energia, e proteínas de transporte. Foram também encontradas proteínas PilA e de quimiotaxia, sugerindo um papel da NAG na transformação. Já os genes de expressão reprimida compreendem principalmente as funções de metabolismo de energia, endereçamento celular, e proteínas hipotéticas. O presente estudo demonstrou que NAG interfere na regulação de diferentes processos celulales, incluindo a capacidade de captura de DNA do meio por V. parahaemolyticus. / Large scale gene expression analysis has fundamental importance for understanding cellular function and gene regulation mechanics. It enables the measurement of expression levels of thousands of genes simultaneously, and makes possible a wider understanding of the biological system. Among the main experimental techniques available for this purpose, microarray technology has been widely used. The objective of this work was to determine the genes of Vibrio parahaemolyticus which have their expression induced or repressed in presence of amino-sugars N-acetylglucosamine (NAG). V. parahaemolyticus is a marine bacterium, commonly found in water and in association with marine organisms. NAG is one of the most abundant amino sugars in the marine environment. For this, V. parahaemolyticus RIMD2210633, was cultivated in two media as sources of energy. The first medium consists of maltose and NAG (control) and the second only by NAG (treatment). Bacterial culture was done under aerobic conditions and low agitation at 28C. Two samples were drawn from the medium 24 hours after the experiment beginning in order to perform the extraction of mRNA and preparation of cDNA. Three replicas of the experiments were made. Mixtures of cDNA prepared from RNA extracted from each replicate were used in hybridizations in microarray slides containing a total of 4832 ORFS from the V. parahaemolyticus RIMD2210633 genome. Comparative analysis of gene expression of V. parahaemolyticus in two culture conditions resulted in detection of 59 genes with expression induced, 38 repressed genes, and 4245 without modified expression (increased or decreased) in presence of NAG. In total, 523 genes were excluded from this comparison because the hybridization was unsatisfactory. There was a gene ordination following the functional classification of the database TIGR-CMR and KEGG. The genes with induced expression mainly belong to classes of regulatory functions, energy metabolism, and transport proteins. PilA and Chemotaxis proteins were found, suggesting a role of NAG in the transformation. Repressed expression genes are mainly included in the functions of energy metabolism, cell address, and hypothetical proteins. This study demonstrated that NAG interfere in regulation of different cell processes, including the ability to capture DNA from the medium by V. parahaemolyticus.

Dans les écosystèmes marins côtiers, l‘activité anthropique et les paramètres naturels induisent chez lesorganismes aquatiques des situations de «multistress». Parmi ces paramètres, deux d‘entre eux ont été étudiés:La contamination métallique et l’infection bactérienne. Dans ce contexte la compréhension des réponsesmoléculaires et physiologiques de l’ormeau rouge Haliotis rufescens a été étudiée, en particulier, face l’impactmétallique (cuivre), et au pathogène (Vibrio parahaemolyticus) en interaction avec des bactéries probiotiquespendant le stade juvénile de ce mollusque en conditions contrôlées. Dans un première temps, une évaluation surl’effet d’un consortium probiotique formé de trois souches bactériennes: Vibrio sp C21, Agarivorans albus F1et Vibrio sp F15 a été testée et les paramètres de survie et de croissance des juvéniles de l’ormeau ont été suivis.Les résultats montrent que les probiotiques augmentent les performances de ces deux paramètres de trait de viecomparés aux individus témoins sans probiotiques. Dans un second temps, une approche génomique parbanques soustractives a été réalisée afin d’identifier les transcrits régulés par l’impact métallique. Cetteméthode a permis d’identifier 108 gènes potentiellement impliqués dans la réponse cellulaire et physiologiquedes organismes face au stress. La cinétique de l’expression génique a été suivie à l’aide de 14 transcrits et leurvariation temporelle pendant la phase critique révèle des régulations parfois complexes et différentielles chezles organismes. Ensuite, les réponses immunitaires des hémocytes d’ormeau face l'exposition au cuivre ont étérecherchées. Les résultats montrent que l’exposition au métal provoque des disfonctionnement importants desactivités hémocytaires. Enfin, l’interaction entre les bactéries pathogènes et probiotiques ont étés étudiées etleurs effets sur le mollusque ont été également recherchés. La présence des bactéries pathogènes régulel’expression génique des transcrits impliqués dans des fonctions physiologiques clés des organismes (lesdéfenses immunitaires et l’état énergétique). De plus, les résultats de cette interaction nous renseignent sur lerôle capital accompli par les bactéries probiotiques sur les performances en termes d’amélioration de la survieet de la croissance des jeunes ormeaux en condition de stress. / In coastal ecosystems, anthropogenic activity and natural factors induce “multi-stress” situations. Inthis study, we focused on two such stress-inducing factors: metal contamination and bacterial infection.Specifically, under controlled conditions, we examined the molecular and physiological responses of redabalone Haliotis rufescens toward the impact of a metal (copper) and a pathogen (Vibrio parahaemolyticus)with respect to this mollusk’s interaction with probiotic bacteria during the juvenile stage. First, we assessed theeffect of a consortium of three probiotic bacterial strains—Vibrio sp. C21, Agarivorans albus F1, and Vibrio sp.F15—on two abalone lifecycle-related parameters, namely, survival and growth. The results showed that,compared to the control treatment (no probiotics), the probiotics improved the performance of these twolifecycle parameters. In a second step, the molecular mechanisms underlying the juvenile abalone response tocopper stress were studied under experimental conditions, using a suppression subtractive hybridizationmethod. This method identified 108 partial sequences of genes involved in the cellular and physiologicalresponses to stress. The kinetics of gene expression were followed using 14 of these transcripts, and theirtemporal variations during the critical phase showed complex and differential regulation exerted by copper inthese organisms. We then investigated the immune response of abalone hemocytes to copper exposure. Theresults showed that exposure to the metal causes a significant dysfunction of hemocyte activities. Finally, theinteraction between probiotics and pathogenic bacteria (transcriptomic and cellular aspects) was studied and theeffects of these bacteria on mollusk performance were also investigated. The presence of pathogenic bacteriawas found to regulate gene expression of transcripts involved in key physiological functions, including thosethat regulate immune responses and energy metabolism. Examination of these interactions thus indicates therole of probiotic bacteria in performance in terms of the improved survival and growth of juvenile abaloneunder stress conditions.

Vibrio parahaemolyticus (Vp) é uma bactéria naturalmente presente em regiões estuarinas, sendo a principal causa de gastrenterite de origem bacteriana associada a pescados, principalmente ostras cruas. Nesta pesquisa, foi desenvolvida uma avaliação quantitativa de risco para avaliar a probabilidade de Vp causar doença após o consumo de ostra crua, produzida e comercializada no Estado de São Paulo. O estudo incluiu a identificação e caracterização do perigo, a avaliação da exposição e a caracterização do risco. Um modelo matemático foi desenvolvido. Este modelo leva em consideração o comportamento de Vp em ostras na cadeia produtiva, em cada estação do ano, além da relação entre a dose de Vp ingerida e a probabilidade de desenvolver a doença. A avaliação da exposição foi desenvolvida em três etapas: cultivo, pós-coleta e consumo. Na etapa de cultivo foram considerados os fatores que influenciam a prevalência e o número de Vp em ostras no momento da coleta. Na etapa pós-coleta, foram descritas as práticas da indústria e foram considerados os fatores associados ao processamento, transporte e manipulação. Já na etapa de consumo foram considerados os fatores como a quantidade de ostras consumidas por porção, o peso médio por ostra consumida e a população de Vp patogênico no momento do consumo. O resultado do modelo quantitativo da avaliação da exposição foi, então, integrado ao modelo dose-resposta, Beta-Poisson, para se obter uma estimativa do risco. Esta estimativa expressa o impacto da exposição humana a Vp, sobre a saúde pública, associada ao consumo de ostras. A simulação de Monte Carlo foi utilizada para avaliar o efeito da variabilidade e incerteza das variáveis do modelo sobre a estimativa do risco. O modelo prediz uma probabilidade de ocorrência de doença de 4,6x10-4, por porção de ostra, consumida ao longo do ano. As variáveis que possuem maior influência sobre o risco de ocorrência de doença são a população de Vp em ostras no cultivo, a temperatura de transporte das ostras até o varejo e a porcentagem de Vp patogênico em ostra, no momento do seu consumo. O modelo evidencia que uma das maneiras de reduzir o risco de ocorrência de doença seria intervir nas condições de transporte de ostras até o varejo por meio da sua refrigeração. Com o modelo é possível identificar fatores e simular cenários para avaliar o comportamento de V. parahaemolytícus como um perigo microbiológico, ao longo da cadeia produtiva de ostra até o momento do seu consumo. Também é possível avaliar o impacto de medidas de intervenção na cadeia produtiva. As suposições adotadas limitam a aplicabilidade do modelo. Portanto, é necessário que o modelo seja validado, particularmente com relação ao número de casos de doença causados por Vp, cujos dados de vigilância epidemiológica inexistem no Brasil. / Vibrio parahaemolyticus (Vp) is naturally present in estuarine regions and is the main cause of gastroenteritis associated with the consumption of bivalve molluscan shellfish, specially raw oysters. In this research, a quantitative risk assessment was developed to evaluate the probability of Vp causing disease after consumption of raw oyster, produced and commercialized in the state of Sao Paulo. The study included the identification and characterization of the hazard, exposure assessment and risk characterization. A mathematical model was developed. This model takes into account the behavior of Vp in oysters in the productive chain, for each season of the year, besides the relationship between the number of cells of Vp ingested and the probability of developing the disease. The exposure assessment was done in three steps: farming, after harvesting and consumption. At the farming step, the factors that influence the prevalence and the population of Vp at the time of harvesting were considered. At the after harvesting step, the factors associated with transportation, handling and processing were considered. At the consumption step, factors related to the amount of oysters and the average weight per oyster consumed and the density of pathogenic Vp at the time of consumption were considered. Then, the quantitative model of exposure assessment was integrated to the dose-response model, BetaPoisson, in order to obtain a risk estimate. This calculation expresses the impact of the human exposure to Vp associated with the consumption of oysters on public health. The Monte Carlo simulation was used to evaluate the effect of variability and uncertainty of variables of the model in the risk estimation. The model predicts a probability of occurrence of the disease of 4,6x10-4 per serving of oyster consumed during one year. The variables showing the greatest influence on the risk of occurrence of disease are the density of Vp in oyster in the farming step, the temperature during transportation of oysters to the retail market and the percentage of pathogenic Vp strains in oysters,\' at the moment of consumption. The model indicates that the use of refrigeration during transportation of oysters to retail could reduce the risk of disease. The model allows the identification of factors and the simulation of scenarios in order to evaluate the behavior of V. parahaemolyticus, as a microbiologícal hazard, in the productive chain of oyster to the consumption. It is also possible to evaluate the impact of intervention measures in the productive chain. The assumptions Iimit the application of the model. Therefore, it is necessary to validate the model, particularly in relation to the number of cases of dísease caused by V. parahaemolyticus of which the data on epidemiologic surveillance do not exist in Brazil.

The gram-negative marine bacterium Vibrio parahaemolyticus is a pathogen and a common worldwide cause of seafood-associated gastroenteritis. When grown on a surface, V. parahaemolyticus undergoes a dramatic differentiation to an elongated, highly flagellated swarmer cell from the short rod typical of swimming cells. Swarming motility is a complex form of adaptation to growth on a surface, and we developed a set of microarray experiments to examine the global gene expression changes that occur upon differentiation to the swarmer cell. We hypothesized that growth on a surface would elicit a specific response involving genes for motility and surface colonization and not the broad changes in physiology suggested by others to be co-regulated with swarming motility. By taking advantage of the two known signals required for swarmer cell induction (inhibiting polar flagellar rotation and limiting iron), the swarming response was artificially induced in liquid and used to define the set of genes associated with surface sensing by transcriptome analysis. This approach avoided the confounding physiological differences between growth in liquid and growth on a surface. Fifteen microarrays performed with different strains and growth conditions were used to define a concise set of about 70 genes that comprise the core set of surface-induced genes. This set includes genes encoding the surface motility system lateral flagella and virulence factors including a type three secretion system (T3SS1). I showed a biological consequence of the increased expression of T3SS1 genes, as surface-induced cells were more toxic in a tissue culture infection than either liquid-grown or surface-grown non-swarming mutants. I explored the role of calcium signaling in regulating the surface sensing network, as calcium seemed a pertinent signal to a marine organism and low calcium is a known inducing signal for T3SS in other organisms. Calcium was shown to enhance swarming motility and lateral flagellar gene expression. Microarrays were used to analyze the transcriptome response to growth with EGTA (a cation chelator commonly used to generate low calcium) or calcium. Surprisingly, both low and high calcium induced T3SS1 gene expression. The EGTA effect was determined to be the result of iron limitation, which was thus shown to be a new inducing signal for T3SS1. I overexpressed the master transcriptional regulator of the T3SS system, encoded by exsA, to define the entire set of T3SS1-associated genes. I found that ExsA was also a new regulator of the surface sensing regulon, which was repressed when exsA was overexpressed. Microarray analysis showed that calcium is a global regulator, controlling transcription of about 50 genes under the conditions tested. I characterized a new calcium-regulated transcription factor that we named CalR, and showed that CalR repressed swarming motility and T3SS1 gene expression. The transcription factor OpaR was previously known to repress swarming genes and control colony opacity. It is homologous to the output regulators of the quorum sensing pathway in other Vibrio species. I used microarray analysis and mutant strains to explore the functionality of the quorum sensing cascade in V. parahaemolyticus and define the OpaR regulon during growth on a surface. I showed that the quorum sensing regulator LuxO when active silences opaR as it does in other Vibrios, using a translational reporter fusion in opaR. I used microarray analysis to show that 323 genes are induced or repressed by OpaR. The surface-sensing regulon is repressed by OpaR. Many genes encoding proteins involved in virulence, signal transduction, and modulation of the signaling molecule cyclic dimeric GMP are regulated by OpaR. The quorum sensing controlled network of gene expression in V. parahaemolyticus is quite distinct from other Vibrios, with respect to both the specific nature as well as the direction of regulation of genes controlled by OpaR.

Vibrio spp., natural inhabitants of aquatic environments, are one of the most common causes of bacterial gastroenteritis in the world, being spread to humans via the ingestion of seafood, contaminated drinking water or exposure to seawater. The majority of Vibrio spp. are avirulent, but certain strains may sporadically be human pathogenic. Vibrio cholerae may cause cholera and fatal wound infections, Vibrio parahaemolyticus may cause gastroenteritis and Vibrio vulnificus may cause wound infections and sepsis. To expand current knowledge of the occurrence, ecological niche and persistence of potential human pathogenic Vibrio spp. in aquatic environments, occurrence and laboratory studies were performed. The seasonal variation of Vibrio spp. in clams and mussels from Mozambique and Sweden were studied, with isolated strains characterized and compared with those isolated from water samples collected in India. Results showed that the numbers of Vibrio spp. in Mozambican clams peaked during the warmer rainy season and that the dominating species was V. parahaemolyticus. Biochemical fingerprinting and virulence screened by PCR revealed a high similarity among strains from the different aquatic environments. However, isolate functional hemolytic analyses and antibiotic resistance patterns differed between strains; Swedish and Indian strains were less sensitive to the tested antibiotics and had a lower hemolytic capacity than those from Mozambique. Molecular analysis of bacterial DNA from Swedish mussels showed the presence of the three Vibrio spp. most commonly linked with human illness, as well as their associated virulence genes. The strains isolated from marine and clinical environments were equally and highly harmful to the tested eukaryotic cells. The persistence of clinical V. cholerae in aquatic environments was investigated in vivo. Strains were exposed to mussels, with bacterial uptake and elimination then examined. The mussels were able to avoid the most potent strain by complete closure of shells. The less potent strain was accumulated in mussel tissue in low levels and one marine control strain to a higher degree. Mussels eliminated the pathogenic strain less efficiently than they did the marine strain. One clinical and one marine strain were then exposed to 4°C for 21 days, with the temperature then increased to 20°C. The clinical strain was more prone to lose culturability than the marine strain at 4°C, the former performed significantly better in regaining culturability after the temperature up-shift. Subsequently, the persistence of the clinical strain in natural bottom sediment, incubating as above, was studied and results showed a similar decrease in culturable numbers in the sediment as in the water. As the clinical V. cholerae strains did not carry any of the standard set of virulence genes, the ability to change from non-culturable to culturable may be of great importance to strain pathogenicity. The results also show that natural bottom sediment may be a potential reservoir of human pathogenic Vibrio spp.

Oysters are filter-feeding bivalves, which filter water for nutrients and often accumulate contaminants and human pathogens such as Vibrio parahaemolyticus and Vibrio vulnificus naturally occurring in the marine environment. These naturally occurring pathogens have been frequently isolated from raw shellfish, particularly oyster, in the United States and are recognized as the leading causes of human gastroenteritis associated with seafood consumption. Human illness caused by consumption of raw oyster contaminated with V. parahaemolyticus and Vibrio vulnificus typically results in reduced sales of oysters and a consequent significant financial burden for the producers. The United States produces more than 27 million pounds of oysters each year with a large portion of them being produced from the coastal water of the Gulf of Mexico. It is estimated that 20 million Americans eat raw shellfish and consumption of raw oyster is responsible for about 95% of all deaths associated with seafood consumption in the U.S., making raw oysters one of the most hazardous seafoods. Several post-harvest processes, including low temperature pasteurization, freezing, high pressure processing and irradiation, have been reported capable of reducing Vibrio contamination in raw oysters. However, most of them require either a significant amount of initial investment or operation costs, and oysters are often killed during processing. Cost-effective post-harvest processing for reducing V. parahaemolyticus in raw oysters without significant adverse effects on the oysters remains to be developed. This study was conducted to determine impacts of low-temperature (15, 10 and 5°C) depuration and frozen storage on reducing V. parahaemolyticus and V. vulnificus in raw oysters. Depuration of the Gulf oyster (Crassostrea virginica) with electrolyzed oxidizing (EO) water (chlorine, 30 ppm; pH 2.82; oxidation-reduction potential, 1,131mV) containing 3% NaCl was found ineffective on reducing both V. parahaemolyticus and V. vulnificus in the oysters. Reductions of V. parahaemolyticus and V. vulnificus in oyster after 48 h of EO water depuration at 22°C were limited to 0.7 and 1.4 log MPN/g, respectively. Depuration with EO water at lower temperatures did not enhance reductions of Vibrio in the oysters. Greater reductions of V. parahaemolyticus (1.2 log MPN/g) and V. vulnificus (2.0 log MPN/g) were observed when the oysters were depurated with artificial seawater (ASW) at room temperature (22°C) for 48 h. Decreasing temperature of ASW to 15°C for depuration significantly increased the reductions of V. parahaemolyticus and V. vulnificus to 2.1 and 2.9 log MPN/g, respectively, after 48 h of process. However, depuration of oyster in ASW at 10 and 5°C were found less effective than at 15°C in reducing Vibrio in the Gulf oysters. An extended depuration with ASW at 15°C for 96 h was capable of achieving 2.6 and 3.3 log MPN/g of reductions of V. parahaemolyticus and V. vulnificus, respectively, in the Gulf oysters. Study of effects of frozen storage at -10, -23 and -30°C on reducing V. parahaemolyticus in raw half-shell Pacific oyster (Crassostrea gigas) found that the population of the bacterium decreased faster in oysters stored at -10 than at -23 or -30°C. Holding half-shell Pacific oyster at -10°C for three months or at -23°C for four months was capable of achieving a greater than 3-log (MPN/g) reduction of V. parahaemolyticus in the Pacific oyster. / Graduation date: 2008

Thesis (M.S.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 80-96). Also available on the World Wide Web.