Probiogenomic analysis and safety assessment of Bacillus isolates using Omics approach in combination with In-vitro

Abstract

In-vitro and Bioinformatic techniques were applied to three Bacillus strains, isolated from Thai fermented fish, to establish potential probiotic properties of the isolates. 16S and WGS analysis provided more intensive discrimination among Bacillus species, than phenotyping analysis. Two Bacillus strains with over 99% homology to B. subtilis as per 16S, were identified as Bacillus velezensis (6-2) and Bacillus amyloliquefaciens (78-1) by WGS, while the third with 98% and 91% homology as Bacillus infantis (63-11) by16S and WGS. Isolate 6-2 synthesized environmental stress resistant and acid tolerant proteins while isolate 78-1 synthesized acid tolerant and epithelial layer adhesion proteins. Isolate 63-11 synthesized all proteins and exhibited antimicrobial activity with bacteriocin genes. Toxicity and virulence analysis in accordance with WGS and In-vitro cytotoxicity assays, did not identify cytotoxin K, haemolytic enterotoxin, or non-haemolytic enterotoxin genes. Bioinformatics tools predicted haemolysin III gene in isolates 6-2 and 78-1 and a homolog in 63-11. In-vitro analysis verified isolate 78-1 occasionally exhibited beta haemolytic activity. Overall, WGS adequately characterized genes responsible for functional properties of probiotic and isolate 63-11 as the most promising probiotic candidate. However, a case-by-case assessment on expression of the predicted genes and possible mechanism should be conducted.