Molecular characterization and phylogenetic analysis of highly pathogenic Vibrio alginolyticus strains isolated during mortality outbreaks in cultured Ruditapes decussatus juvenile.

In the summer of 2008 and 2009, a series of mortalities in growing out seeds of R. decussatus juveniles were occurred in the eastern Tunisian littoral. Nine predominant bacterial strains were isolated from dead and moribund juveniles and characterized as Vibrio alginolyticus. These isolates were subjected to biochemical and molecular characterization. All the Vibrio strains were tested for their susceptibility against the most widely used antibiotic in aquaculture as well as, the assessment of the presence of erythromycin (emrB) and tetracycline (tetS) resistance genes among the tested bacteria. The degree of genetic relatedness between V. alginolyticus strains was evaluated on the basis of the Entero-Bacterial Repetitive Intergenic Consensus (ERIC) and the Random Amplification of Polymorphic DNA-PCR (RAPD-PCR) approaches. We also looked for siderophore activity and the ability to grow under iron limitation. Furthermore, the pathogenic potential of the tested isolates was evaluated using R. decussatus larva and juveniles as infection models. On antimicrobial susceptibility test, Vibrio strains exhibited total resistance to at least four antibiotics. The MICs data revealed that flumequine and oxolinic acid were the most effective antibiotics to control the studied bacteria. Results also showed that studied antibiotics resistance genes were widely disseminated in the genome of V. alginolyticus strains. Both ERIC and RAPD-PCR fingerprinting showed the presence of genetic variation among Vibrio isolates. However, RAPD typing exhibited a higher discriminative potential than ERIC-PCR. Besides, we reported here for the first time the co-production of catechol and hydroxamte by V. alginolyticus species. The challenge experiment showed that most of Vibrio isolates caused high mortality rates for both larva and juveniles at 48-h post-exposure to a bacterial concentration of 106 CFU/ml.

Sediment contact assay using the carpet shell clam, Ruditapes decussatus L., embryos and larvae for assessing contamination levels of four sites in Tunisian coast.

This study assessed sediment pollution along the Tunisian coast using the sediment contact assay with embryo and larval life-stages of the clam Ruditapes decussatus. Sediment was collected from four sites for testing: Mahres, Monastir lagoon, Zarat and Chebba. Embryo development was significantly (p < 0.05) inhibited in sediment at concentrations ≥5 g L(-1) from the Mahres and Monastir sites, and ≥25 g L(-1) with sediment from the Zarat site. Sediment from the Chebba site had no effect at concentrations up to 125 g L(-1). Compared to the control, the percentages of larval mortality were significantly higher in sediment from Monastir, Mahres and Zarat at sediment concentrations ≥25 g L(-1); mortality increased with increasing sediment concentration to 50.1%, 39.5% and 31.2% respectively at 125 g L(-1). Chebba sediment did not affect the survival of larvae.

Genotypic diversity, antimicrobial resistance and screening of Vibrio cholerae molecular virulence markers in Vibrio alginolyticus strains recovered from a Tunisian Ruditapes decussatus hatchery.

In this study, a total of 54 Vibrio alginolyticus strains were analyzed. The isolates were recovered from different compartments of the Ruditapes decussatus hatchery in the National Institute of Marine Sciences and Technologies, Monastir, Tunisia. All isolates were biochemically identified (API 20E and API ZYM strips), characterized by amplification of the Hsp-40 gene polymerase chain reaction (PCR) and analyzed by enterobacterial repetitive intergenic consensus (ERIC)-based genotyping to evaluate genetic relationship between the isolated strains. We also looked for the presence of ten V cholera virulence genes (toxRS, toxR, toxT toxS, tcpP, tcpA, ace, vpi, zot and ctxA) in the genomes of Vibrio isolates. The antibiotics susceptibility, exoenzymes production and in vitro cytotoxic activitiy against HeLa cell line were also carried out for all tested bacteria. Most of V alginolyticus isolates showed significant antimicrobial resistance rates to at least ten antibacterial agents. For most isolates, the minimum inhibitory concentration (MIC) data showed that tetracydclin and streptomycin were the most effective antibiotics. Construction of the phylogenetic dendogram showed that studied isolates were in general genetically heterogeneous; however some Vibrio strains were present in different structures of the R. decussatus hatchery. The V cholerae virulence genes investigation showed a wild distribution of toxS (49/54), toxaR (45/54) and toxT (22/54) genes among V alginolyticus strains isolated from the R. decussatus rearing system. Cytotoxic effects of several Vibrio extracellular products (28154) were also observed on HeLa cells.

Photo-induced toxicity of four polycyclic aromatic hydrocarbons (PAHs) to embryos and larvae of the carpet shell clam Ruditapes decussatus.

In this work, we assessed the photo-toxicity of four polycyclic aromatic hydrocarbons (PAHs) to embryos and larvae of the European clam Ruditapes decussatus. The exposure of R. decussatus embryos (24 h) and larvae (96 h) to anthracene, fluoranthene, pyrene and benzo[a]pyrene resulted in reduction of normal D-veliger percentages and high larval mortality, both in darkness and under sunlight conditions. Based on the calculated EC(50) and LC(50) values, the toxicity of the forementioned PAHs was respectively enhanced 72, 35, 60 and 23 times in the embryotoxicity test and 32, 31, 12 and 61 times in the larval mortality test when exposures were performed under sunlight conditions. Simultaneous exposure to sunlight and these PAHs enhanced their toxicity in comparison to dark conditions. The clam embryos and larvae appear to be environmentally relevant life-stages in assessing the toxic and photo-toxic risk of PAHs that enter the marine environment.

Toxicity of Hg, Cu and Zn on early developmental stages of the European clam (Ruditapes decussatus) with potential application in marine water quality assessment.

The toxicity of mercury, zinc and copper on sperm viability, fertilisation and embryogenesis of Ruditapes decussatus was examined. Cu did not affect sperm viability at all the concentrations tested. Conversely, the Zn and Hg significantly (P<0.01) reduced sperm viability only at the highest concentration (respectively 512 and 256 µg/l). Cu caused a significant decrease (p<0.05) of less than 6% in the fertilisation rate at 128 µg/l and Zn of up to 13% at 64 µg/l. Hg significantly (p<0.01) inhibited fertilisation at concentrations as low as 32 µg/l. The median effective concentrations (EC50) reducing rates of embryogenesis by 50% were 21.1 µg Hg/l (0.1 µM), 46.3 µg Cu/l (0.72 µM) and 43.4 µg Zn/l. Therefore, Hg is up to seven times more toxic than Cu (on a molar basis). Ecotoxicity of mercury on larvae survival was also assessed in this work. Result showed a significant (p<0.05) reduction of survival after exposure to 4 and 12 µg/l of Hg. The fertilisation rate and embryogenesis were the most sensitive endpoints, although the latter is more advisable for routine assessment of seawater quality because of its greater sensibility.