Caracterização de Vibrio alginolyticus associados aos mexilhões Perna perna (LINNAEUS, 1758) através de técnicas fenogenotípica e proteônica e avaliação do seu perfil de virulência.

Abstract

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.