Metagenomic screening of antimicrobial peptide candidates and isolation of two active peptides from bat gut bacteria.

Bacterial antibiotic resistance has intensified the need to identify new antimicrobial molecules from underexplored microbial systems. Wild mammalian gut microbiota may harbor antimicrobial peptide (AMP) candidates and candidate bacteriocins, but these systems remain poorly investigated as sources for antimicrobial discovery. Here, we used parallel metagenomic and culture-dependent approaches to explore candidate AMP sequences and candidate bacteriocins from the gut bacteria of the Asian particolored bat Vespertilio sinensis. Machine-learning screening of 553,401 short non-redundant ORF protein sequences identified 12,907 candidate AMP sequences. Of these, 31 were prioritized after in silico safety and structural filtering. In parallel, culture-dependent screening yielded two antagonistic bacterial isolates, CQJ and LYS. Activity-guided purification followed by LC-MS/MS identified two active peptides, CQJ01 and LYS01, with no exact matches in public databases. Both peptides exhibited broad in vitro antibacterial activity against 16 pathogenic strains, with minimum inhibitory concentration (MIC) values as low as 8 µg/mL against selected Gram-positive and Gram-negative bacteria. CQJ01 retained activity across pH 2-9 and after heat treatment up to 80 °C, whereas LYS01 retained activity from - 20 °C to 100 °C. Both peptides remained active after catalase, trypsin, papain, and proteinase K treatments but were sensitive to pepsin. They showed low hemolytic activity and limited cytotoxicity in preliminary assays. These findings support bat gut bacteria as an underexplored source of AMP candidates and candidate bacteriocins.