Isolation and genotyping of potentially pathogenic Vibrio alginolyticus associated with Ruditapes decussatus larva and juvenile mass mortalities

摘要

Recently, a series of mass mortalities of cultured Ruditapes decussatus have occurred in the bivalve hatchery of the National Institute of Marine Sciences and Technologies situated on the eastern littoral of Tunisia, North Africa. Vibrio alginolyticus species was predominantly isolated from dead and moribund veliger larva and juvenile. As far as we know, this is the first report of the involvement of this bacterium in clam epizootics in southern Mediterranean coastal areas. All bacterial isolates were biochemically identified, molecularly characterized by amplification of the Hsp-40 gene and analyzed by enterobacterial repetitive intergenic consensus-based PCR to evaluate genotypic relationships between the isolated strains. The antibiotics sensitivity, the lysozyme antibacterial effect and the in vitro cytotoxic activity (HeLa, Vero and Hep-2 cell lines) were also carried out. Furthermore, V. alginolyticus pathogenicity to R. decussatus larva was investigated using an experimental infection model. The majority of Vibrio isolates expressed high rates of antimicrobial resistance. The minimum inhibitory concentration results revealed that tetracycline and gentamycin were the most effective antibiotics against the tested strains. The phylogenetic analysis revealed relatedness between pathogenic isolates recovered from different locations. Vibrio cells were also able to resist to the bactericidal effect of the R. decussatus larva lysozyme. The extracellular products from V. alginolyticus isolates (L(5)11, PL(5)20 and N(3)85) displayed significant cytotoxic effects against Vero and HeLa cells. The experimental challenge showed that all Vibrio strains exhibited high rates of mortality (up to 90 %) in R. decussatus larva after 24-h exposure to high bacterial concentration (10(7) ufc mL(-1)).