Probiotics has been increasingly considered as an alternative to antibiotics in combating disease outbreaks. The use of combined probiotics has been studied to possibly harbor synergistic effects that could provide better protection in aquatic species.
Three potential probionts that had conferred antagonism against Aeromonas hydrophila were Bacillus amyloliquefaciens (L9, isolated from crab), Bacillus sp. (A2, isolated from microalgae) and Enterococcus hirae (LAB3, isolated from seabass) and were combined into a probiotic mixture. The probiotic mixture was able to produce biofilm (2.441±0.346), significantly higher than A. hydrophila (0.578±0.124) at 24 h and showed continuous increased in production of biofilm at 48 h and 72 h. Furthermore, no hemolytic action was presented when the probiotic mixture was streaked on sheep blood agar (5%) whereas A. hydrophila presented α-hemolysis. The lowest concentration of potential multi-strain probiotic mixture, as determined in the minimum inhibitory concentration assay, significantly inhibited the growth of A. hydrophila at 106 CFU mL-1 after 24 h incubation at 107 CFU mL-1 (6.595±0.218) as compared to A. hydrophila only (7.247±0.061). The interaction, in the co-culture assay, between the probiotic mixture at 107 CFU mL-1 and A. hydrophila at 106 CFU mL-1 was 6.883±0.015 as compared to A. hydrophila only was 7.532±0.026, showed significant reduction of pathogen at 12 h of co-incubation. Hence, this research suggests that probiotic mixture of L9, A2 and LAB3 has the potential for further in-vivo research to confer against A. hydrophila infections.