Optimization, characterization, and mechanistic evaluation of iturin and surfactin like, broad spectrum antimicrobial lipopeptides from Bacillus subtilis RKEA1 isolated from Acanthus ilicifolius.

Antimicrobial resistance (AMR) continues to rise globally, largely due to limited discovery of novel antimicrobial agents capable of overcoming resistance mechanisms associated with conventional antibiotics. This challenge underscores the need to explore alternative bioactive compounds. In the present study, an endophytic Bacillus subtilis strain, RKEA1, isolated from Acanthus ilicifolius collected from an area near the Kadalundi wetland mangrove forest (Kerala, India), was evaluated for its antimicrobial potential. The culture supernatant had broad-spectrum antimicrobial activity against Gram-positive bacteria such as S. aureus, S. pyogenes, C. violatium and L. monocytogenes, and Gram-negative bacteria including K. pneumoniae, E. coli, P. aeruginosa, Shigella spp., Proteus mirabilis, S. enterica and Y. enterocolitica, and also the fungi C. albicans and C. neoformans. Bioactive lipopeptides were extracted and subjected to comprehensive characterization, including determination of molecular weight, Rf value, and mass profiling. Production parameters were optimized, and physicochemical stability of the lipopeptides were examined under varying environmental conditions. Response surface methodology (RSM) based medium optimization enhanced lipopeptide production by 1.8-fold. The minimum inhibitory concentrations (MICs) of the lipopeptides were 31 µg/mL against the tested Gram-positive bacteria and 62.5 µg/mL against Gram-negative bacteria. The minimum bactericidal concentration (MBC) against Gram-positive bacteria was 62.5 µg/mL. Furthermore, mechanistic investigations provided insight into the mode of action, revealing membrane disruptive activity. Importantly, this work offers a detailed characterization of antimicrobial lipopeptides from the endophytic Bacillus subtilis RKEA1 isolated from Acanthus ilicifolius by optimizing lipopeptide production, physicochemical stability studies, antimicrobial activity and membrane-disruptive activity. Overall, the findings highlight Bacillus subtilis RKEA1 as a promising source of antimicrobial lipopeptides with potential applications in combating resistant bacterial infections and contributing to the development of novel therapeutic agents.