Characterization and Antibacterial Activity of a Low-Molecular-Weight Bacteriocin-like Inhibitory Substance S-2 Produced by Leuconostoc falkenbergense SBL-85-2 Against Aeromonas hydrophila

Aeromonas hydrophila is a globally distributed aquatic pathogen. Antibiotic overuse in traditional management practices has intensified problems such as antimicrobial resistance and environmental pollution, creating an urgent need for safe alternatives. This study characterized a BLIS produced by Leuconostoc falkenbergense SBL-85-2, which shows potential as an antibiotic alternative in aquaculture. L. falkenbergense SBL-85-2 exhibited no hemolytic activity, lacked typical high-risk virulence factors, and only low-identity vancomycin-related intrinsic genes (vanT and vanY) were identified by the CARD database, which may preliminarily indicate its potential biosafety for aquatic applications. The BLIS exhibited marked activity against A. hydrophila, with an inhibition zone of 39.14 ± 0.65 mm, as well as other critical pathogens including Aeromonas rivipollensis, Aeromonas salmonicida, Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus, and Vagococcus fluvialis. Furthermore, the BLIS exhibited remarkable thermal stability (retained 87.18 ± 0.14% activity after treatment at 100 °C for 10 min) and UV stability (92.67 ± 0.75% residual activity after 3 h exposure) coupled with sensitivity to proteases, confirming its proteinaceous nature. Molecular weight determination indicated that the BLIS is a low-molecular-weight peptide (<2.7 kDa). Mechanistically, the BLIS disrupted bacterial cell membrane integrity, ultimately resulting in the leakage of cellular contents and a time-dependent reduction in intracellular ATP levels (reduced by 69.16 ± 4.10% at 2 h), exerting its antimicrobial effect. Collectively, our results indicate that this BLIS combines potent antibacterial efficacy with a favorable biosafety profile, suggesting its significant potential for the biological control of A. hydrophila in aquaculture.