Reducing bacterial resistance evolution through antimicrobial peptide modifications

Antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics, yet bacteria can evolve resistance toward them through mechanisms that are poorly understood. Here, we selected a well-characterized antimicrobial peptide, melittin, as a model peptide that was shown to induce high levels of resistance in the human pathogen Staphylococcus aureus. We hypothesize that the physicochemical properties of AMPs might play a role in resistance development. To explore this hypothesis, we used computational approaches to design melittin derivatives with diverse physicochemical characteristics. A data set of 191 melittin-derived sequences (8-26 amino acids long) was analyzed to identify representative peptides spanning diverse physicochemical clusters. Selected peptides were synthesized and tested in an experimental evolution assay. Resistance dynamics, cross-resistance patterns, and cytotoxicity were evaluated in parallel. Full-length melittin with an amide or carboxylic acid C' terminus and highly hydrophobic derivatives [e.g., Mel(2-22)] showed high resistance occurrence, whereas Mel(5-24), a truncated variant, maintained antimicrobial activity while eliciting minimal resistance development. Whole-genome sequencing of endpoint populations revealed that melittin-evolved strains accumulated convergent mutations most prominently in pmtR, whereas Mel(5-24)-evolved populations converged only on ydbT, with no mutations in canonical resistance loci, suggesting fewer accessible adaptive pathways against the truncated peptide. Our results indicate that peptide hydrophobicity and chain length correlated with resistance fold change, whereas coil propensity showed a negative correlation. Notably, Mel(5-24) displayed reduced cross-resistance to other peptides and lower cytotoxicity toward mammalian cells than melittin, suggesting a broader therapeutic window. Overall, our findings indicate that tuning peptide hydrophobicity and chain length along with structural propensity can mitigate the evolutionary pressure driving resistance while maintaining antibacterial potency. Rational modulation of such physicochemical properties may provide a practical route for designing AMPs with improved selectivity and lower resistance potential.