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

Abstract

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.