<p>This research examines three potential mechanisms by which bacteria can adapt to different temperatures: changes in strain-level population structure, gene regulation and particle colonization. For the first two mechanisms, I utilize bacterial strains from the Vibrionaceae family due to their ease of culturability, ubiquity in coastal environments and status as a model system for marine bacteria. I first examine vibrio seasonal dynamics in temperate, coastal water and compare the thermal performance of strains that occupy different thermal environments. Our results suggest that there are tradeoffs in adaptation to specific temperatures and that thermal specialization can occur at a very fine phylogenetic scale. The observed thermal specialization over relatively short evolutionary time-scales indicates that few genes or cellular processes may limit expansion to a different thermal niche. I then compare the genomic and transcriptional changes associated with thermal adaptation in closely-related vibrio strains under heat and cold stress. The two vibrio strains have very similar genomes and overall exhibit similar transcriptional profiles in response to temperature stress but their temperature preferences are determined by differential transcriptional responses in shared genes as well as temperature-dependent regulation of unique genes. Finally, I investigate the temporal dynamics of particle-attached and free-living bacterial community in coastal seawater and find that microhabitats exert a stronger forcing on microbial communities than environmental variability, suggesting that particle-attachment could buffer the impacts of environmental changes and particle-associated communities likely respond to the presence of distinct eukaryotes rather than commonly-measured environmental parameters. Integrating these results will offer new perspectives on the mechanisms by which bacteria respond to seasonal temperature changes as well as potential adaptations to climate change-driven warming of the surface oceans.</p> / Dissertation

Bacterial infections are an increasing problem for human health. In fact, an increasing number of infections are caused by bacteria that are resistant to most antibiotics and their combinations. A new solution to fight bacteria and infectious diseases, without promoting antimicrobial resistance, is required. A promise strategy is the disruption or attenuation of bacterial Quorum Sensing (QS), a refined system that bacteria use to communicate. In a QS event, bacteria produce and release specific small chemicals, signal molecules - autoinducers (AIs) - into the environment. AIs regulate gene expression as a function of cell population density. Phenotypes mediated by QS (QS- phenotypes) include virulence factors, toxin production, antibiotic resistance and biofilm formation. In this work, two polymeric materials (linear polymers and molecularly imprinted nanoparticles) were developed and their ability to attenuate QS was evaluated. Both types of polymers should be able to adsorb bacterial signal molecules, limiting their availability in the extracellular environment, with expected disruption of QS. Linear polymers were composed by methyl methacrylate as backbone and itaconic acid or methacrylic acid as functional monomer. IA and MAA monomers were identified by computer modelling to have strong interactions with the AIs produced by Gram-negative bacteria. Cont/d.

Wood is a natural raw material, and would hardly be considered a risk to the environment. However, the handling of wood materials on an industrial scale has been shown to have a negative impact on water bodies that run-off from industrial sites that process wood-based materials. Most investigations related to water pollution from wood-based industries have focused on large industrial sites such as pulp and paper mills. The goal of the present thesisis to understand better such environmental problems and treatment solutions that occur at most wood handling sites. Where there are large outdoor storage areas for logs and sawdust that are exposed to rainfall or irrigation, such as the wooden-floor and bio-energy sectors. Leachate generated by the contact of water with wood in storage areas contains a large amount of organic matter that is potentially hazardous to water bodies that receive run-off from the site. It has been found that different wood species yield different leachate compositions, with leachate from oak having the highest pollutants content, followed by leachate from pine. This investigation shows that oak has the potential to leach about 10 times the amount of polyphenols compared with other investigated species (i.e., pine, beech and maple). Furthermore, oak leachates have the lowest ratio of biological oxygen demand at 7 days tochemical oxygen demand (0.12), which suggests a potential problem with the biological degradation of this leachate. It has also been shown that leachate from wood are potentially toxic to aquatic organisms. Treatability studies with the aim of reducing the environmental impact of wood leachate were conducted on a pilot scale as part of the scope of this thesis. The results showed, among other options, the possibility of using constructed wetlands to treat leachate. It was found that plants and aeration can affect the performance of a wetland. However, the most important factor is the time water spends in the wetland. Filter material that could be used to absorb leachate was also studied. A filter consisting of a mixture of peat and ash (from incinerated organic matter), was used to absorb a specific chemical group (polyphenols) in the leachate. It was also shown that polyphenols are vulnerable to ozone, representing a third viable treatment process. / Trä är ett naturligt råmaterial som knappast anses vara en risk för miljön. Däremot har hanteringen av trämaterial i industriell skala visat sig ha negativ inverkan på vattendrag. De flesta utredningar avseende vattenföroreningar inom träbaserade industrier har fokuserat på stora industrier med stor vattenförbrukning så som massa- och pappersbruk. Lakvatten som alstras vid kontakten mellan vatten och trä innehåller höga halter av organiska ämnen som är potentiellt farliga för vattendrag. Målet med denna avhandling är att få kunskap om ovanstående miljöproblem och möjlig behandlingar av dessa. Förorenat lakvatten skapas av industrier, där stora mängder timmer och sågspån är placerade utomhus och utsätts för regn och bevattning, såsom inom trägolv och bioenergisektorn. Man har funnit att lakning från olika träslag ger olika sammansättningar på lakvattnet, där lakvatten från ek har den högsta halten av föroreningar följt av furu. Denna undersökning visar att ek har potential att laka ut tio gånger så mycket polyfenoler jämfört med andra undersökta arter (tall, bok och lönn). Dessutom har lakvatten från ek den lägsta andelen av biologisk syreförbrukning efter 7dagar per kemisk syreförbrukning (0,12), vilket tyder på ett potentiellt problem med den biologiska nedbrytningen av detta lakvatten. Man har även påvisat att lakvattnet från trä är potentiellt giftigt för akvatiska organismer. Studier för att försöka minska miljöpåverkan av lakvatten från denna trädsort gjordes i pilotskala inom ramen för denna avhandling. Dessa studier visade bland annat på möjligheterna att använda sig av våtmarker som reningsmetod, där växter och luftning påverkar hur bra en våtmark fungerar. Men den viktigaste faktorn var den ökade uppehållstiden som vattnet får i våtmarken. Utöver studier av våtmarker har också ett filtermaterial studerats, filtret bestod av en blandning av torv och aska, som användes för att absorbera en specifik kemisk grupp i lakvattnet (polyfenoler). I en tredje typ av behandlingsprocess visade det sig också att polyfenoler effektivt kan brytas ned av ozon.

Made available in DSpace on 2015-03-04T18:58:03Z (GMT). No. of bitstreams: 1 thesisMarianaCampeao.pdf: 13162328 bytes, checksum: ed5b7096328fd1404655a6f926f0f292 (MD5) Previous issue date: 2014-04-25 / Vibrios sao bacterias amplamente distribuidas no meio aquatico e podem ser encontradas em associacao com organismos marinhos, tanto como causadores de doencas quanto como simbiontes. O advento das tecnicas de sequenciamento de nova geracao e de alto desempenho tem possibilitado o acesso cada vez mais amplo a dados genomicos microbianos, incluindo vibrios. Tal quantidade e disponibilidade de dados permitem analises in silico, que podem compreender desde caracteristicas genomicas ate fenotipicas. A taxonomia microbiana e fundamentada na abordagem polifasica, que mede as relacoes evolutivas a partir do uso de sequencias de genes, especialmente o RNAr 16S, similaridade genomica, por meio de hibridizacao de DNA, e ampla caracterizacao fenotipica. A caracterizacao fenotipica requer testes experimentais, que muitas vezes sao demorados, caros e requerem grande experiencia. Neste estudo propomos o uso de genomas para a analise da diversidade e identificacao fenotipica de vibrios. Para tanto, foram avaliadas caracteristicas basicas de vibrios (tais como tamanho do genoma, conteudo genico e posicao logenetica); analisaram-se genes unicos e suas possiveis funcoes ecologicas; e desenvolveu-se uma ferramenta prototipo para identificacao de fenotipos diagnosticos de vibrios, denominada vibriophenotyping 1.0. A logenia construida a partir do genoma minimo recuperou os diferentes generos e clados descritos na literatura para o grupo vibrio, bem como posicionou as especies consideradas irmas em relacao a um ancestral comum proximo. Os genes unicos, por sua vez, puderam ainda revelar peculiaridades entre especies irmas. Por m, o programa de identificacao fenotipica desenvolvido foi testado com genomas de linhagens tipo de vibrios e apresentou uma media de similaridade superior a 70% entre os fenotipos obtidos in vitro e in silico, sendo alcançada uma similaridade de 100% para genomas integros. Dessa forma, concluiu-se que analises do pangenoma permitem a recontrucao logenetica dentro do grupo vibrios e a identificacao de genes unicos relevantes para o papel ecologico da especie no ambiente de origem, e, ainda, que a identificacao fenotipica atraves da automatizacao por uma ferramenta computacional e possivel a partir da analise de genomas.

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.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-10T13:14:13Z No. of bitstreams: 1 Emanuell Felipe Beserra da Silva.pdf: 411741 bytes, checksum: bc52ae73b30a1e2f1b60236283006192 (MD5) / Made available in DSpace on 2017-02-10T13:14:13Z (GMT). No. of bitstreams: 1 Emanuell Felipe Beserra da Silva.pdf: 411741 bytes, checksum: bc52ae73b30a1e2f1b60236283006192 (MD5) Previous issue date: 2010-02-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This dissertation consisted of two studies and was divided into two papers, the first evaluated the influence of the addition of probiotic Bacillus spp. in the larviculture of white shrimp Litopenaeus vannamei, while the second compared the use of probiotic and antibiotic in the postlarvae culture of the species. For the first study, three experiments were performed: (I) Nauplios4-5 to Zoea3, where they were exposed to probiotic through water (Pw), microalgae (Pm), water and microalgae (Pwm) and control without probiotic (C); (II) Mysis1 to Mysis3, exposed to probiotic through the water (Pw), Artemia (Pa), water and Artemia (Pwa) and control (C) and (III) PL1 to PL10,subject to the same treatments of experiment II. The water quality parameters showed no significant differences among treatments of each experiment. In general, the weight and survival of shrimp in the treatments with probiotic were significantly higher compared with controls in experiments I and II, but this difference was not observed among treatments of experiment III. The concentration of Vibrio spp. both in the water and postlarvae in experiment III was significantly higher in control compared with treatments with the use of probiotic. Already in experiments I and II, the concentration of Vibrio spp. in the water did not differ among treatments, however, Mysis3 larvae treated with probiotic had a lower concentration of Vibrio spp. compared with the control. In the second study, postlarvae (PL1 to PL10) were cultured in three treatments, corresponding to a biological treatment (TB), which was added probiotic Bacillus spp., chemical treatment (TQ) with the addition of antibiotic (erythromycin) and a control (TC), without both products in the culture water. The water quality parameters, as well as the zootechnical performance of postlarvae (weight, total length and survival) did not differ among treatments. However, the final concentrations of Vibrio spp. both in the culture water and postlarvae were significantly reduced with the use of probiotic (TB) in relation to other treatments. Based on the results obtained in the two studies, suggested that the use of probiotic Bacillus spp. in the larviculture of shrimp L. vannamei can provide an increase in the growth and survival of animals, but mainly provides a reduction in the concentration of Vibrio spp. in the culture system. Furthermore, the use of probiotic has great potential to replace the use of antibiotic in the culture of L. vannamei postlarvae. / A presente dissertação foi composta por dois estudos e dividida em dois artigos científicos, o primeiro avaliou a influência da adição do probiótico Bacillus spp. na larvicultura do camarão branco Litopenaeus vannamei, enquanto o segundo comparou a utilização de probiótico e antibiótico no cultivo de pós-larvas da espécie. Para o primeiro estudo, foram realizados três experimentos: (I) Nauplios4-5 a Zoea3, onde foram expostos ao probiótico através da água (Pw), microalga (Pm), água e microalga (Pwm) e controle sem probiótico (C); (II) Mísis1 a Mísis3, expostos ao probiótico através da água (Pw), Artemia (Pa), água e Artemia (Pwa) e controle (C) e (III) PL1 a PL10, submetidos aos mesmos tratamentos do experimento II. Os parâmetros de qualidade de água não apresentaram diferenças significativas entre os tratamentos de cada experimento. Em geral, o peso e sobrevivência dos camarões nos tratamentos com probiótico foram significativamente superiores em comparação aos controles nos experimentos I e II, porém esta diferença não foi observada entre os tratamentos do experimento III. A concentração de Vibrio spp. tanto na água quanto nas pós-larvas no experimento III foi significativamente maior no controle comparado com os tratamentos com uso de probiótico. Já nos experimentos I e II, a concentração de Vibrio spp. na água não diferiu entre os tratamentos, entretanto, as larvas Mísis3 tratadas com probiótico apresentaramu ma menor concentração de Vibrio spp. comparadas com o controle. No segundo estudo, póslarvas (PL1 a PL10) foram cultivadas em três tratamentos, correspondendo a um tratamento biológico (TB), onde foi adicionado probiótico Bacillus spp., tratamento químico (TQ), com adição de antibiótico (eritromicina) e um tratamento controle (TC), sem ambos os produtos na água de cultivo. Os parâmetros de qualidade de água, assim como o desempenho zootécnico das pós-larvas (peso, comprimento total e sobrevivência) não diferiram entre os tratamentos.Entretanto, as concentrações finais de Vibrio spp. tanto na água de cultivo como nas póslarvas foram reduzidas significativamente com a utilização de probiótico (TB) em relação aos outros tratamentos. Com base nos resultados obtidos nos dois estudos, sugere-se que o uso do probiótico Bacillus spp. na larvicultura do camarão L. vannamei pode proporcionar um incremento no crescimento e sobrevivência dos animais, mas principalmente possibilita uma redução na concentração de Vibrio spp. no sistema de cultivo. Além disso, o emprego do probiótico tem grande potencial para substituir o uso de antibiótico no cultivo de pós-larvas de L. vannamei.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-15T13:16:21Z No. of bitstreams: 1 Juliana Rangel de Aguiar Interaminense.pdf: 435666 bytes, checksum: fc8f9ada1887395801298c79901d84cc (MD5) / Made available in DSpace on 2017-02-15T13:16:21Z (GMT). No. of bitstreams: 1 Juliana Rangel de Aguiar Interaminense.pdf: 435666 bytes, checksum: fc8f9ada1887395801298c79901d84cc (MD5) Previous issue date: 2012-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study aimed to evaluate the antibacterial effects of different supplements on Artemia sp. hatching and enrichment. The supplements were added to the water used for Artemia hatching of capsulated and decapsulated cysts and for Artemia enrichment water. The experiment consisted in the addition of the diatom Chaetoceros calcitrans, a commercial probiotic (Bacillus spp.), antimicrobial Florfenicol to the hatching water and control without supplements. The enrichment experiment was performed by the application of C. calcitrans, probiotic and commercial emulsion DHA / EPA rich to enrichment water and control constituted by newly hatched nauplii. The enriched Artemia were offered for the PL7 to PL19 of Litopenaeus vannamei stages. The Vibrio spp. load of hatching water and newly hatched nauplii were quantified at the end of hatching. The Vibrio spp. quantification of postlarvae, enriched Artemia and water of enrichment and postlarvae rearing was also performed. The Vibrio presumptive colonies isolated from newly hatched nauplii were identified. Furthermore, Vibrio spp. present in nauplii subjected to freezing and Bacillus spp. colonies of Artemia and postlarvae of Probiotic treatment were quantified. Assessing the overall results of the study, the decapsulation process did not shown to be effective in reducing the Vibrio spp load of nauplii and water in all treatments. The C. calcitrans addition in Artemia hatching water has proven to be an effective alternative to antibiotic use. The probiotic use must also be considered to control Vibrio spp. load in Artemia nauplii. However, the supplements use to Artemia enrichment process may promote a bacterial increase and other procedures for its control must be evaluated. / O presente trabalho teve por princípio avaliar os efeitos antibacterianos de diferentes suplementos na eclosão e no enriquecimento de Artemia sp. Os suplementos foram adicionados na água utilizada para eclosão de cistos de Artemia capsulados e descapsulados e à água de enriquecimento de metanáuplios de Artemia. O experimento de eclosão consistiu no acréscimo da diatomácea Chaetoceros calcitrans, de probiótico comercial (Bacillus spp.), do antimicrobiano Florfenicol e controle sem adição de agentes. O experimento de enriquecimento foi realizado pela aplicação de C. calcitrans, de probiótico comercial e de emulsão comercial rica em DHA/EPA à água de cultivo de metanáuplios e controle constituído por náuplios recém eclodidos. Os metanáuplios foram oferecidos para os estágios de PL7 a PL19 de Litopenaeus vannamei. A carga de Vibrio spp. da água de eclosão e náuplios recém eclodidos foram quantificadas no final do período de eclosão. A quantificação de Vibrio spp de pós larvas, metanáuplios, água de cultivo das pós larvas e enriquecimento de Artemia também foi realizada. As colônias presuntivas de Vibrio oriundas de náuplios recém eclodidos foram identificadas. Além disso, Vibrio spp. presente em náuplios submetidos ao congelamento e colônias de Bacillus spp. em amostras de Artemia enriquecida e pós-larvas do tratamento Probiótico foram quantificadas. Avaliando dos resultados gerais do estudo, o processo de descapsulação não demonstrou ser eficiente na redução da carga de Vibrio spp. nos náuplios e na água de todos os tratamentos. A adição de C. calcitrans na água de eclosão de Artemia provou ser uma alternativa eficaz para em alternativa a utilização de antibióticos. A utilização de probiótico deve ser também considerado para controlar a carga de Vibrio spp em náuplios de Artemia. No entanto, a utilização de suplementos para o processo de enriquecimento de Artemia pode favorecer o aumento da carga bacteriana e outros procedimentos para o seu controle deve ser avaliada.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Marine shrimp culture is an agribusiness activity with great economic importance to Brazil, especially to the states of Rio Grande do Norte and Cearà that have an outstanding production. Over the last years, arising diseases and infections contributed to the decline in carciniculture development. The usustainable use of antibiotics as preventive measures and in the treatment of shrimp illness initiated an outbreak in drug-resistant bacterial strains that can cause damage to human health and therefore jeopardize exportation. This research aimed to evaluate the phenotypic resistant profile of the species Vibrio, isolated from samples of culture pond water and cultured marine shrimp hepatopancreas (Litopenaeus vannamei) in three shrimp farms in the state of CearÃ, Brazil. Amongst the total of 31 Vibrio isolates, it was analyzed the resistance to ampicillin (46.15% and 44.44%), cefoxitin (7.69% and 27.78%) and to the tetracycline group (53.84% and 38.89%) in samples collected in culture pond water (N=13) and shrimp (N=18), respectively. The majority (80.64%) of isolated Vibrio sp. strains presented resistance to at least one tested antimicrobial drug, some been commonly used in human clinics. A significant percentage (44%) of resistant strains presented a multiresistant profile. Seven multiresistant profiles were identified in 32.26% of strains, where 5 strains were resistant to 2 antibiotics, 4 were resistant to 3 antibiotics and 2 were resistant to 4 antibiotics. A correlation amongst the presence of plasmid and antibiotic resistance was present in 81.8% of the multiresistant isolates. The Minimum Inhibitory Concentration (MIC) was calculated for the isolates that presented resistance to oxytetracycline, indicating maximum values of 697 mg/L and minimum of 79 mg/L. The qualitative behavior of antimicrobial resistance was not influenced by the origin of the isolates as for the type of sample or the estuary origin. The presence of antimicrobial resistant bacteria, commonly used in human clinics, is a concerning factor once it tends to hamper infection treatment in shrimp cultures, possibly causing damage to human health. / O cultivo de camarÃo marinho à uma atividade do agronegÃcio de grande importÃncia econÃmica para o Brasil, principalmente, para os Estados do Rio Grande do Norte e Cearà que se destacam pela sua produÃÃo. Nos Ãltimos anos o surgimento de doenÃas e infecÃÃes contribuÃram para uma queda nos Ãndices de desenvolvimento da carcinicultura. O uso desordenado de antibiÃticos como medida preventiva e no tratamento das enfermidades dos camarÃes implicou no surgimento de estirpes bacterianas mais resistentes aos medicamentos, podendo provocar danos à saÃde humana e comprometer a exportaÃÃo. Esta pesquisa objetivou avaliar o perfil fenotÃpico de resistÃncia das espÃcies de Vibrio isoladas da Ãgua de viveiro e hepatopÃncreas de camarÃo marinho (Litopenaeus vannamei) cultivados em trÃs fazendas do Estado do CearÃ. Dentre o total de 31 isolados de Vibrio analisados foi observada uma maior incidÃncia de resistÃncia aos antibiÃticos: ampicilina (46,15% e 44,44%), cefoxitina (7,69% e 27,78%) e ao grupo das tetraciclinas (53,84% e 38,89%) para as amostras oriundas de Ãgua de viveiro (N=13) e de camarÃo (N=18) respectivamente. A maioria (80,64%) das estirpes de Vibrio sp. isoladas apresentou resistÃncia a pelo menos um dos antimicrobianos testados, alguns dos quais sÃo drogas utilizadas na clÃnica humana. Um percentual significativo (44%) dentro das cepas resistentes apresentou perfil de multi-resistÃncia. Foram observados 7 perfis de resistÃncia mÃltipla, onde 5 cepas foram resistentes a 2 antibiÃticos, 4 resistentes a 3 antibiÃticos e 2 cepas resistentes a 4 antibiÃticos. A relaÃÃo entre a presenÃa de plasmÃdios e a resistÃncia aos antibiÃticos foi verificada para 81,8% dos isolados multi-resistentes. A ConcentraÃÃo InibitÃria MÃnima (CIM) foi obtida para os isolados que se apresentaram resistentes à oxitetraciclina, indicando valores mÃximo de 697mg/L e mÃnimo de 79mg/L. O comportamento qualitativo de resistÃncia aos antimicrobianos nÃo foi influenciado pela origem dos isolados quanto ao tipo de amostra e estuÃrio de origem. A presenÃa de bactÃrias resistentes aos antimicrobianos comumente utilizados em clÃnica humana à um fato preocupante, uma vez que dificultam o tratamento de infecÃÃes dentro do cultivo, podendo trazer danos à saÃde humana.

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.

La investigación tuvo como objetivo incorporar una metodología de identificación rápida conocida como ARDRA (Amplified Ribosomal DNA Restriction Analysis) para identificar especies del género Vibrio. Se estandarizó la técnica con cepas referenciales. Luego, se aislaron cepas bacterianas asociadas con el cultivo de Litopenaeus vannamei. Posteriormente, se realizaron pruebas bioquímicas para encontrar cepas candidatas de pertenecer al género Vibrio. Al finalizar esta primera etapa, la técnica ARDRA estandarizada, fue aplicada en las cepas candidatas, confirmando de esta manera la factibilidad de la metodología bajo las condiciones estudiadas. En una segunda etapa, se secuenció la región 16S rDNA para confirmar e identificar las cepas candidatas por análisis filogenético. Se reportaron tres especies diferentes con alta similitud pertenecientes al Vibrio core group (Vibrio communis, Vibrio harveyi y Vibrio parahaemolyticus). Con estos resultados, fue posible diseñar una identificación rápida por ARDRA para identificar el Vibrio core group y la especie Vibrio communis. La metodología de diseño del ARDRA fue soportado por una valoración diagnóstica bioinformática, obteniendo de esta evaluación, una sensibilidad y una especificidad de 97,1 y 76,9%, respectivamente para la identificación del Vibrio core group, mientras que para identificar la especie Vibrio communis, se obtuvo una sensibilidad y una especificidad de 100 y 97,4%, respectivamente. Finalmente, se ha demostrado que es posible identificar ciertas especies del genero Vibrio asociados con la acuicultura de Litopenaeus vannamei, por ARDRA y esta metodología de identificación, tiene la ventaja de ser mucho más rápido y económico en comparación con la identificación por análisis filogenético, teniendo a su vez la desventaja de ser dependiente del uso del secuenciamiento en un primer momento para el diseño del ARDRA. Palabras clave: Litopenaeus vannamei, ARDRA, Vibrio communis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio core group, evaluación diagnóstica bioinformática. / --- The research aimed to incorporate a quick identification methodology known as ARDRA (Amplified Ribosomal DNA Restriction Analysis) to identify Vibrio species. Technique was standardized with reference strains. Then, bacterial strains were isolated associated with the cultivation of Litopenaeus vannamei. Subsequently, biochemical tests were performed to find candidate strains belonging to the genus Vibrio. Upon completion of this first stage, the standard technique (ARDRA) was applied for candidate strains, thus confirming the feasibility of the method under the conditions studied. In a second step, the 16S rDNA region sequenced to confirm and identify candidate strains for phylogenetic analysis. Three different species were reported with high similarity belonging to the Vibrio core group (Vibrio communis, Vibrio harveyi and Vibrio parahaemolyticus). With these results, it was possible to design a quick identification by ARDRA to identify the Vibrio core group and Vibrio communis. The design methodology ARDRA was supported by a bioinformatics diagnostic assessment, obtaining this evaluation, a sensitivity and specificity of 97,1 and 76.9% respectively for the identification of Vibrio core group, while identifying the species Vibrio communis, yielded a sensitivity and specificity of 100 and 97,4%, respectively. Finally, it has proved possible to identify certain Vibrio species associated with aquaculture Litopenaeus vannamei, by ARDRA identification and this methodology has the advantage of being much faster and cheaper compared with the identification by phylogenetic analysis, having in turn, the disadvantage of being dependent on the use of the sequencing at first for ARDRA design. Keywords: Litopenaeus vannamei, ARDRA, Vibrio communis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio core group, bioinformatic diagnostic evaluation. / Tesis