Isolation and Identification of Bioactive Compounds with AntimicrobialActivity from Marine Facultative Anaerobe, Bacillus subtilis

Introduction: The marine ecosystem contains many microbial species that produce unique, biologically active secondary metabolites with complex chemical structures. We aimed to isolate and identify bioactive compounds with antimicrobial properties produced by a facultative anaerobic strain of Bacillus subtilis (AU-RM-1), isolated from marine sediment. Methodology: We optimized the AU-RM-1 growth conditions, analyzed its growth kinetics and its phenotypic and genotypic characteristics. Extracts of the isolate were studied for antimicrobial activity against three clinically important microorganisms and the structure of the active compound was identified by spectroscopy. Results: Antimicrobial activity of the AU-RM-1 DMSO extract was evaluated by disc diffusion assay and by serial dilution. The AU-RM-1 DMSO extract showed antimicrobial activity against Candida albicans, Escherichia coli, and Klebsiella pneumoniae. The bioactive fraction of the AURM- 1 DMSO extract was separated by TLC-bioautography at Rf = 0.49. We then used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to study the morphological changes in the bacterial cells treated with the isolated compound. It was observed that cells seemed to shrink, and the cell walls appeared to be damaged. A bioactive compound was identified, and its structure was examined by spectroscopic analysis: a LC-MS molecular ion peak (ESI) m/z (% of relative abundance) was calculated for C19H22O3: 298.38, and found to be C19H22O3 +1: 299.51 [M+1]. The chemical structure of the compound (2-(2-{8-methoxy-5aH,6H,7H,8H,9H, 9aH-naphtho[2,1-b]furan-7-yl}ethyl)furan) was determined using 1HNMR and 13CNMR, and its purity was confirmed by HPLC. Fifteen known and previously reported compounds were also identified, in addition to the novel compound; these were lipopeptides, antibiotics and chemical moieties. Conclusion: The facultative anaerobic marine organism Bacillus subtilis (AU-RM-1) produces a novel bioactive secondary metabolite with antimicrobial and antifungal activity.