Exploration of Novel Antimicrobial Peptides from Gut Probiotics Enterococcus spp. Against Extensively drug-resistant Pathogens Through cutting-edge Computational Discovery.

Antimicrobial peptides (AMPs) are promising candidates against antimicrobial resistant pathogens due to their lower likelihood of generating resistance. Enterococcus spp., a common group of gut probiotics, are essential sources of bacteriocins that make them a valuable source for novel AMPs discovery. In this study, we aimed to systematically identify AMPs by analyzing the transcriptomes of Enterococcus species using computational methods. Briefly, the transcriptomes of diverse Enterococcus species were downloaded, processed, de novo assembled, and translated into open reading frames. AMPs were predicted and filtered based on physicochemical and structural criteria. The antibacterial activity of the selected candidate has been evaluated against drug-resistant bacteria. We identified 14 candidate AMPs with net positive charge ≥ + 3, lengths of 10-25 residues, non-toxicity, non-hemolytic properties, antibiofilm activity, and stable secondary structure, capable of interacting with bacterial membranes, using computational tools. Among these, EM_4 exhibited optimal characteristics and was selected for further evaluation. In vitro, EM_4 demonstrated potent inhibition of both susceptible and extensively drug-resistant (XDR) Staphylococcus aureus and Acinetobacter baumannii strains (MIC 2.5-20 ± 0.0 µM; MBC 5.0-20.0 µM), retained activity under various temperature and salt conditions, showed significant antibiofilm effects (40-80 ± 0.0 µM), low hemolytic activity (3.2%), and induced dose- and time-dependent bacterial membrane disruption confirmed by DNA-release assays. In conclusion, our research highlights computational AMP discovery efficacy and presents EM_4 as a promising candidate for the development of next-generation antimicrobials targeting pathogens.