Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2019-09-30T17:15:59Z No. of bitstreams: 1 2014 - Greiciane Fran?a Bronzato de Almeida.pdf: 964195 bytes, checksum: 940138c70922a9f9fdd5a243fa6bfa7e (MD5) Made available in DSpace on 2019-09-30T17:15:59Z (GMT). No. of bitstreams: 1 2014 - Greiciane Fran?a Bronzato de Almeida.pdf: 964195 bytes, checksum: 940138c70922a9f9fdd5a243fa6bfa7e (MD5) Previous issue date: 2014-02-24 CAPES Mytiliculture is the most globally expanded branch of the aquaculture practice. The mussel Perna perna has been considered the most economically relevant species. However, microbiological contamination of their tissues can turn them unsuitable for human consumption. Vibrio alginolyticus can be noticed among the microrganisms associated to marine animal diseases as an oportunistic pathogen for fish, crustaceans and mussels. The present study aimed to identify V. alginolyticus associated with mussels (Perna perna) and to assess the pheno-genotypic virulence profile of this microorganism in samples from different locations of the Rio de Janeiro coast. A total of 49 isolates were obtained. After phenotypic analysis, the isolates were subjected techniques for detection of pyrH gene by polymerase chain reaction (PCR) and MALDI-TOF MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) techniques. All 49 isolates tested positive for pyrH gene. Sequencing of the 541pb amplicon is used to differentiate Vibrio species. MALDI- TOF MS confirmed 43 isolates as V. alginolyticus (87,8%), four were identified as Shewanella putrefaciens (8,2%), only one was identified as V. parahaemolyticus (2%). Just one isolate was not identified by this technique (2%). After MALDI-TOF analysis, eight amplicons of pyrH gene of distinct profiles were sento to sequencing. The phenotypic evaluation of collagenase production, a virulence marker of V. alginolyticus was assayed using the gelatin hydrolysis test. All V. alginolyticus and S. putrefaciens were positive. Additionally, collagenase, ompK and toxR genes were used to genotypically assess the virulence of the V. alginolyticus species. Out of the 49 isolates, 47 were positive for the collagenase gene. The ompK gene expression was observed in 46.9% (23/49) of the isolates as following: 91,3% of V. alginolyticus (n = 21) and 4,3% (n = 1) of V. parahameolyticus and S. putrefaciens, respectively. Seven isolates (14.3%) produced amplicons of the toxR gene, all characterized as V. alginolyticus. Phenotypic tests showed significant correlation with genotypic and proteomics results, in the identification of V. alginolyticus. A mitilicultura ? o ramo da aquicultura que mais tem se expandido mundialmente. O mexilh?o Perna perna tem sido considerado o organismo de maior relev?ncia econ?mica nesse contexto, por?m a contamina??o microbiol?gica de seus tecidos desses animais pode inviabilizar seu consumo. Vibrio alginolyticus pode ser destacado entre os micro-organismos associados a quadros de infec??o em animais marinhos como pat?geno oportunista causador de vibriose em peixes, crust?ceos e moluscos. O presente trabalho objetivou identificar V. alginolyticus associados a mexilh?es Perna perna, bem como avaliar o perfil fenogenot?pico da virul?ncia deste microrganismo, em amostras coletadas em diferentes pontos da costa do Rio de Janeiro. Foram obtidos 49 isolados. Ap?s a an?lise fenot?pica, os isolados foram submetidos as t?cnicas de detec??o do gene pyrH por Rea??o em Cadeia de Polimerase (PCR) e MALDI-TOF MS (Espectrometria de Massa por Tempo de V?o de Ioniza??o/Desor??o por Laser Assistida por Matriz). Todos os isolados testados amplificaram o gene pyrH. O sequenciamento do fragmento de 541pb deste gene ? utilizado para diferencia??o de esp?cies de Vibrio. Atrav?s do MALDI-TOF MS, confirmou-se 43 isolados como V. alginolyticus (87,8%), 4 isolados como Shewanella putrefaciens (8,2%) e apenas 1 foi identificado como V. parahaemolyticus (2%). Apenas um isolado n?o foi identificado pela t?cnica (2%). Ap?s o resultado do MALDI-TOF, oito produtos de amplifica??o do gene pyrH representativos de diferentes perfis foram enviados para sequenciamento. Para a an?lise fenot?pica da produ??o de colagenase, um marcador de virul?ncia de V. alginolyticus, foi realizado o teste da hidr?lise da gelatina. Todos isolados de V. alginolyticus e S.putrefaciens foram positivos. Al?m disso, os genes collagenase, ompK e toxR foram utilizados para avaliar a virul?ncia genot?picamente das esp?cies estudadas. Dos 49 isolados estudados, os 47 isolados positivos a prova fenot?pica amplificaram o gene collagenase. A express?o do gene ompK pode ser observada em 46,9% (23/49) dos isolados sendo 91,3% (n = 21) de V. alginolyticus e 4,3% (n = 1) de V. parahameolyticus e S. putrefaciens, respectivamente. Sete isolados (14,3%) produziram bandas para amplifica??o do gene toxR, sendo todos caracterizados como V. alginolyticus. Os testes fenot?picos realizados demonstraram correla??o significante na identifica??o de V. alginolyticus quando comparados com os resultados genot?picos e de prote?mica.