Molecular Identification and Characterization of Probiotic Bacillus Species with the Ability to Control Vibrio spp. in Wild Fish Intestines and Sponges from the Vietnam Sea

Vibriosis in farmed animals is a serious threat to aquaculture worldwide. Using probiotics and anti-Vibrio antimicrobial substances in aquaculture systems can be a means of preventing Vibrio infections. Therefore, we aimed to characterize and compare 16 potential anti-Vibrio probiotics (Vi+) isolated from marine sponges and fish intestines collected from the Vietnam Sea, as well as an anti-Vibrio bacteriocin to fully explore their application potentials. 16S rRNA sequencing confirmed all Vi+ to be Bacillus species with different strain variants across two sample types. An obvious antimicrobial spectrum toward Gram-negative bacteria was observed from intestinal Vi+ compared to sponge-associated Vi+. The reason was the higher gene frequency of two antimicrobial compounds, non-ribosomal peptides (NRPS) and polyketide type-I (PKS-I) from intestinal Vi+ (66.7%) than sponge-associated Vi+ (14.3% and 0%, respectively). Additionally, a three-step procedure was performed to purify an anti-Vibrio bacteriocin produced by B. methylotrophicus NTBD1, including (i) solvent extraction of bacteriocin from cells, (ii) hydrophobic interaction chromatography, and (iii) reverse-phase HPLC. The bacteriocin had a molecular weight of ~2-5 kDa, was sensitive to proteolysis and thermally stable, and showed a broad antimicrobial spectrum, all of which are essential properties for promising feed additives. This study provides necessary information of the potential of probiotic Bacillus species with anti-Vibrio antimicrobial properties to study their further use in sustainable aquaculture.