An Amphibious Fish-Derived Antimicrobial Peptide, Boleokidin ₃₉–₆₁ , with Broad-Spectrum Antibacterial Activity and In Vivo Protective Efficacy

The escalating threat of antimicrobial resistance (AMR) has created an urgent need for alternative therapeutic strategies beyond traditional antibiotics. In this study, we identified a new antimicrobial peptide, Boleokidin39–61, encoded by the immune-regulated gene Boleokidin in the amphibious fish Boleophthalmus pectinirostris. Notably, the expression of this gene was significantly upregulated in the liver and spleen following Edwardsiella tarda infection. Boleokidin39–61 exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. Mechanistic analyses showed that the peptide kills bacteria by disrupting membrane integrity, inhibits biofilm formation, and does not induce resistance under prolonged exposure. Cytotoxicity assays revealed no significant toxicity toward mammalian cells or erythrocytes at antimicrobial concentrations. Importantly, in vivo infection studies using both zebrafish and mice models confirmed the peptide’s therapeutic efficacy, demonstrating its ability to reduce bacterial burden and modulate host inflammatory responses. Collectively, these findings highlight Boleokidin39–61 as a promising AMP candidate with potent antimicrobial and immunomodulatory properties, offering the potential for application in aquaculture and biomedical fields.