When antibacterial activity was detected, a second antibacterial assay in liquid medium was performed to define minimal inhibitory concentrations in standard 96-well microtiter plates (Wiegand et al., 2008; Defer et al., 2013). Briefly, target bacteria in exponential growth state (1 × 106 CFU mL−1) were incubated with serial twofold dilutions (in sterilized Marine Broth) of active cell-free supernatant and incubated for 48 h at optimal growth temperature. Sterile as well as growth and inhibition controls (Polymyxin B at 100 μg mL−1) were carried out. The activity was expressed as a function of protein concentrations (μg mL−1) determined

using BC Assay Kit (Interchim) according to the manufacturer’s instructions and as a function of

the highest dilution factor of cell-free supernatant MS 275 that inhibited 100% of the target strain growth. The target bacteria panel was broadened. Five other strains of Vibrio were included: Vibrio pectenecidae A365, V. coralliilyticus CIP107925, V. tubiashii CIP102760, V. parahaemolyticus and V. harveyi ORM4. The bacterial isolates expressing antibacterial activity were selected for a phylogenetic analysis based on 16S rRNA gene sequences. DNA was Ipilimumab chemical structure extracted as previously described (Godon et al., 1997) and 16S rRNA gene was amplified using two universal primers, W18 : 9F and W20 : 1462R, yielding 1000–1500 pb PCR products (Godon et al., 1997). The PCR mixture was carried out according to the manufacturer’s instructions (PCR Master Mix Promega®). The following PCR conditions were used: initial denaturation at 94 °C for 4 min, followed by 35 cycles at 94 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min and a final elongation step at 72 °C for 10 min. The PCR products were analyzed

on agarose (1.2%) gel electrophoresis and sequenced by GATC Biotech (Germany). Sequences were compared with the GenBank nr/nt database by blastn to identify their closest match. To construct trees, an alignment with the first five hit blast 16S sequences of each strain was made, using clustalw2 (Larkin et al., 2007). Phylogenetic trees were built using mega 5 program package (Tamura et al., 2011). The cytotoxicity activity Carbohydrate was estimated for three active strains isolated from oyster haemolymph. The two antimicrobial compound-producing strains, named hCg-6 and hCg-42, isolated from oyster haemolymph in a previous study (Defer et al., 2013), were also investigated for hemocyte cytotoxic effect. The experimental procedure was as described previously (Delaporte et al., 2003). Briefly, the haemolymph of about 30 C. gigas was withdrawn, pooled and filtered through an 80-μm mesh. A 19-h-long contact was established at 18 °C between hemocytes and bacteria in cytometry tubes. Several concentrations of bacteria were evaluated (ratio bacteria/bivalve hemocytes 25/1, 50/1, 100/1). A control was done using incubated hemocytes in sterile seawater.