Novel Leech Antimicrobial Peptides, Hirunipins: Real‐Time 3D Monitoring of Antimicrobial and Antibiofilm Mechanisms Using Optical Diffraction Tomography

Abstract Antimicrobial peptides (AMPs) are promising agents for treating antibiotic‐resistant bacterial infections. Although discovering novel AMPs is crucial for combating multidrug‐resistant bacteria and biofilm‐related infections, their clinical potential relies on precise, real‐time evaluation of efficacy, toxicity, and mechanisms. Optical diffraction tomography (ODT), a label‐free imaging technology, enables real‐time visualization of bacterial morphological changes, membrane damage, and biofilm formation over time. Here, a computational analysis of the leech transcriptome using an advanced AI‐based peptide screening strategy with ODT to identify potential AMPs is employed. Among the 19 potential AMPs identified, hirunipin 2 demonstrates potent antibacterial activity, low mammalian cytotoxicity, and minimal hemolytic effects. It demonstrates efficacy comparable to melittin, resistance to physiological salts and human serum, and a low likelihood of inducing bacterial resistance. Microscopy and 3D‐ODT confirm its disruption of bacterial membranes and intracellular aggregation, leading to cell death. Notably, hirunipin 2 effectively inhibits biofilm formation, eradicates preformed biofilms, and synergizes with antibiotics against multidrug‐resistant Acinetobacter baumannii (MDRAB) by enhancing membrane permeability. Additionally, hirunipin 2 significantly suppresses pro‐inflammatory cytokine expression in LPS‐stimulated macrophages, highlighting its anti‐inflammatory properties. These findings highlight hirunipin 2 as a strong candidate for developing novel antibacterial, anti‐inflammatory, and antibiofilm therapies, particularly against multidrug‐resistant bacterial infections.