DETECTION OF SECRETED ANTIMICROBIAL PROTEIN ISOLATED FROM CELL FREE-SUPERNATANT OF PROBIOTIC Bacillus spp. AND ITS EFFICACY AGAINST THE FISH PATHOGENS

Due to the increasing numbers of resistant pathogenic bacteria and side effects caused by existing antibiotics, new antimicrobial compounds with effective properties are needed. Bacillus species produce a wide range of antimicrobial protein (bacteriocin) which remarkable variety of antimicrobial spectrum and have the ability to release some antimicrobial substances it responds with bactericidal (toxic) or bacteriostatic (inhibitory) effect on potentially pathogenic bacteria. However, to enhance the aquaculture production, the extensive use of antibiotics has led to drug resistance problems. Therefore, studies are essential to find out the alternative chemotherapeutic agents for sustainable aquaculture production.

This study aimed to isolate an antimicrobial protein from the cell-free culture supernatant (CFCS) of Bacillus spp., screen its antimicrobial activity and partially characterized of the antimicrobial peptide. An antimicrobial substance produced by the Bacillus sp. was detected in Bacillus broth. Subsequently, cell-free culture supernatant (CFCS) was obtained by medium centrifugation and partially purification, and its antimicrobial activity was tested against bacterial fish pathogens. The Bacillus aerophilus of cell free supernatant showed maximum inhibitory activity against the tested bacterial strains. The B. aerophilus strain CFCS was separated with tricine and without tricine SDS-PAGE. A prominent band of at 22 kDa and diffusible bands (12-66 kDa) was observed in tricine SDS-PAGE. SDS-PAGE profile of the possible probiotic organisms and induced with pathogens, B. aerophilus showed the appearance of 22 and 35 kDa protein bands was an induced protein observed with different concentrations (0.5, 1, 2 ml) target pathogens. All the selected probiotic Bacillus strains of partial purified antimicrobial proteins also displayed a bactericidal broad spectrum of targets against fish pathogens. B. aerophilus strain showed reduced survival percentage of all pathogens at low protein concentrations (15 µg) which could be the higher antimicrobial activity against A. hydrophila. Antimicrobial proteins are showed remarkable stability over a wide temperature and enzymes. This study conclude that antimicrobial compounds could potentially be used adjuvant or as vaccine substitutes to antibiotics in fisheries. Further study is needed to understand the mechanism of antibacterial activity of Bacillus spp.