Impact of antimicrobial peptide Hidefensin5 as a feed additive on the aquatic environment and fish gut antibiotic resistant microbes

• Hidefensin5 was found to be the ideal green alternative to antibiotic. • Hidefensin5 enhanced alpha-diversity of intestinal microbiota in zebrafish. • Hidefensin5 lowered the total abundance of resistome across risk rank I to IV. • Hidefensin5 cut the joint presence of resistance gene, mobile element and pathogen. Antibiotic resistance has arisen as a formidable challenge to global health governance. Antimicrobial peptides (AMPs) have garnered considerable attention as highly potent alternative to antibiotic for preventing resistant pathogens. In this study, the AMP Hidefensin5 (Hi5), derived from Hermetia illucens, was designed and exhibited antibacterial activity against gram-negative pathogenic bacteria at a concentration of 100 μg/mL. Furthermore, the ecological effects of applying crude Hi5 fermentation and purified Hi5 as feed additives on the structure of the gut microbiota and the antibiotic resistance gene (ARG) profile of zebrafish were assessed. The findings indicated that compared with unpurified Hi5, purified Hi5 markedly enhanced the microbial α diversity and simultaneously reduced the accumulation of pathogenic bacteria (PBs) and the prevalence of drug resistance-related metabolic pathways, as well as the energy metabolic burden within the gut microbiota. With respect to the ARG resistome, purified Hi5 application reduced the absolute ARG abundances across risk ranks I to IV and the co-association patterns of ARGs-mobile genetic elements-PBs, whereas unpurified Hi5 increased the risk of pathogens developing drug resistance. This study confirmed that purified AMP application contributed to hindering the development of drug-resistant PBs, providing a valuable reference for antimicrobial resistance risk management in antibiotic-free aquaculture.