Using mixture design to construct consortia of potential probiotic Bacillus strains to protect gnotobiotic Artemia against pathogenic Vibrio

To evaluate the potential probiotic effect of three Bacillus strains on the survival and growth of an Artemia culture and to obtain the optimal formulation of pure cultures of the bacilli, challenge tests were performed with the pathogenic bacterium Vibrio alginolyticus (S1) using mixture design. According to molecular analyses involving amplified ribosomal DNA restriction analysis (ARDRA), these bacteria corresponded to Bacillus subtilus, Bacillus cereus and Bacillus coagulans. An antimicrobial susceptibility and antagonism assay revealed that these strains were susceptible to most antimicrobial drugs and have an inhibitory effect against tested pathogenic Vibrio. Furthermore, the investigated Bacillus strains were fairly adhesive to polystyrene, with values ranging from 0.10 to 0.32 at 595 nm. Statistical analysis demonstrated that Bacillus strains enhance protection against pathogens, have no impact on survival, but improve the growth of larvae, for which the highest growth rate was obtained when the mixture composition was 32% B. subtilus, 68% B. cereus and no B. coagulans.