Engineered Bacillus subtilis secreting piscidin-1/hepcidin enhances resistance against Aeromonas hydrophila in a zebrafish model

Antimicrobial peptides (AMPs) are effective against pathogens; however, their application in aquaculture remains limited due to low stability and poor delivery efficiency. In this study, we designed a xylose-induced-engineered probiotic to enable tightly controlled production and efficient targeted delivery of AMPs. Specifically, the tilapia-derived piscidin-1 and hepcidin were separately cloned into the Bacillus subtilis 168 (BS168) expression vector, resulting in the successful construction of two recombinant engineered strains—BS168-sfGFP-piscidin and BS168-sfGFP-hepcidin. These two engineered strains could significantly inhibit the growth of Aeromonas hydrophila compared with BS168 transformed with an empty vector (pSTOP1622) in vitro. To investigate the additive bacteriostatic activity, piscidin-1 and hepcidin were fused in tandem within the same expression vector, generating BS168-sfGFP-PH. These three engineered strains were orally administered to zebrafish to evaluate their antimicrobial efficacy in vivo. The results showed that zebrafish fed with BS168-sfGFP-PH exhibited the highest survival rate following A. hydrophila challenge. This protection was attributed to the strain's ability to reduce intestinal pathogen load, thereby suppressing inflammatory responses and improving intestinal integrity. Collectively, this work established an orally deliverable probiotic for secreting AMPs, serving as a promising strategy against bacterial infections in aquaculture.