Role of C17 fengycin B from Bacillus subtilis DB9011 in enterotoxigenic Escherichia coli inhibition.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of post-weaning diarrhea in piglets, and the rise of antimicrobial resistance underscores the urgent need for antibiotic alternatives in swine production. Bacillus species are promising candidates owing to their ability to produce antimicrobial secondary metabolites, particularly cyclic lipopeptides such as surfactins, iturins, and fengycins. Although Bacillus lipopeptides are known to exhibit antibacterial activity against E. coli, the specific compounds responsible for the inhibition of ETEC in probiotic-relevant Bacillus strains remain undefined. Here, we sought to identify extracellular metabolite(s) associated with the anti-ETEC activity of Bacillus subtilis DB9011. DB9011 culture supernatant strongly inhibited the growth of ETEC and other enteric pathogens, including Salmonella Typhimurium. Bioactivity-guided fractionation of the culture supernatant, followed by liquid chromatography-quadrupole time-of-flight mass spectrometry, identified C17 fengycin B as a major active metabolite enriched in inhibitory fractions, but absent from the non-inhibitory type strain NBRC13719ᵀ. A fengycin-containing fraction enriched in C17 fengycin B exhibited concentration-dependent antibacterial activity against ETEC and enhanced growth inhibition when combined with colistin, whereas incomplete inhibition at native supernatant-equivalent concentrations indicated that additional metabolites contribute to the overall antibacterial phenotype. While fengycins have been primarily recognized for antifungal activity and, more recently, for effects on gram-negative bacteria, our findings identify C17 fengycin B as a major contributor to the ETEC-inhibitory phenotype of B. subtilis DB9011. These findings provide mechanistic insights into how probiotic-relevant Bacillus strains inhibit enteric pathogens; nonetheless, further studies are required to evaluate in vivo relevance and functional performance under intestinal conditions.IMPORTANCEAntimicrobial resistance poses a major threat to livestock production, where antibiotics remain widely used to control post-weaning diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Supplementation with Bacillus subtilis DB9011 is known to reduce pathogenic E. coli colonization in piglets; however, the metabolites responsible for this protective effect remain unidentified. Here, using a bioactivity-guided fractionation approach, we identify C17 fengycin B as a major contributing metabolite associated with the anti-ETEC activity of DB9011 and show enhanced growth inhibition during co-treatment with colistin. These findings clarify the functional contribution of Bacillus-derived metabolites to pathogen inhibition and establish a mechanistic basis for strain-specific antimicrobial activity.