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

Behnam Hasannejad-Asl; Kamran Pooshang Bagheri; Mojgan Bandehpour; Azam Bolhassani; Ali Hashemi; Farkhondeh Pooresmaeil; Bahram Kazemi

doi:10.1007/s12602-026-10919-w

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

Probiotics Antimicrob Proteins. 2026 Feb 13. doi: 10.1007/s12602-026-10919-w. Online ahead of print.

Authors

Behnam Hasannejad-Asl¹, Kamran Pooshang Bagheri², Mojgan Bandehpour³, Azam Bolhassani⁴, Ali Hashemi⁵, Farkhondeh Pooresmaeil⁶, Bahram Kazemi⁷

Affiliations

¹ Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
² Venom and Biotherapeutics Molecules Lab, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
³ Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
⁴ Department of Hepatitis, AIDS and Blood-borne Diseases, Pasteur Institute of Iran, Tehran, Iran.
⁵ Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
⁶ Department of Medical Biotechnology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
⁷ Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. bahram_14@yahoo.com.

PMID: 41686421
DOI: 10.1007/s12602-026-10919-w

Abstract

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.

Keywords: Enterococcus species; Acinetobacter baumannii; Antimicrobial peptide; Antimicrobial resistant bacteria; Computational peptide discovery; Probiotics; Staphylococcus aureus.