On the other hand, despite a high number of isolates, no strain isolated from clam haemolymph demonstrated antibacterial activity. The target bacteria spectrum and/or the growth conditions [nutrients (Spanggaard et al., 2001) and/or temperature (Zhang et al., 2012) or bacterial presence in the surroundings (Mearns-Spragg et al., 1998; Dusane et al.,

2011)] may explain these results. Nonetheless, the potential of bivalve microbiota as a source of antimicrobial compounds is evident, although underexplored. The cryogenic storage resulted in total loss of cultivability for five strains (hMe-15, -22, -82, -119 and -131) and the cell-free supernatant of a further nine strains did not exhibit antibacterial PF-01367338 cost activity (hCg-60, -78, -111 and-114, hPm-100 and -102, hMe-34, -43 and -273). Such loss of cultivability or bioactivity after storage is frequently described with marine bacteria (Gram et al., 2010) and discussed (Hazen et al., 2010; Vynne et al., 2011). The antibacterial activity of the 12 bioactive strains remaining was quantified using a 96-well micro-titration method (Wiegand et al.,

2008). Insofar as the chemical nature of the active compounds was unknown, MICs were expressed as a function of maximal dilution factor of the supernatant that exerted a total EX527 inhibition of pathogen growth. MICs were also expressed in protein concentration (Table 3). All the hCg strains and hMe-187 and -253 supernatants were able to inhibit 100% of bacterial growth of at least one pathogen when diluted at least 64-fold. Moreover, eight haemolymph-associated PD184352 (CI-1040) isolates inhibited at least five different species among the seven Vibrio species included in the panel and one or more other bacteria, suggesting a real potential for antibacterial treatment in aquaculture farming, since Vibrio species are pathogenic for fish (Toranzo et al.,

2005), molluscs (Verschuere et al., 2000) and crustaceans (Wang, 2011). The active haemolymph-associated strains, hCg-23, -48, -51, -108, -109, hPm-26, hMe-95, -223, -253 and -273, were identified by 16S rRNA gene amplification as members of the Gammaproteobacteria class (Fig. 1) belonging to either the Alteromonadales (89%) or the Vibrionales orders (11%). Such affiliation was also observed in antimicrobial screening of marine bacteria and in previously described probiotics used in bivalve hatcheries (Zheng et al., 2005; Gram et al., 2010; Prado et al., 2010; Wilson et al., 2010; Flemer et al., 2012). Vibrio genus has been described to be a natural flora in bivalve and crustacean haemolymph (Faury et al., 2004; Gay et al., 2004; Gomez-Gil et al., 2010). The antibacterial as well as probiotic ability of this genus has been described (Riquelme et al., 1997, 2001; Mansson et al., 2011; Silva-Aciares et al., 2011). Nine strains, hCg-23, -48, -51, -108, -109, hMe-95, -223, -253 and -273, were affiliated with the Pseudoalteromonas genus.