Antimicrobial resistance (AMR) poses a critical global health challenge, prompting the exploration of antimicrobial peptides (AMPs) as alternatives. Here, we investigated the genetic mechanisms of resistance evolution in Staphylococcus aureus against single and combined AMPs (temporin, melittin, and pexiganan). Whole-genome sequencing of evolved populations revealed that combination therapy significantly reduced the overall number of mutations, and importantly, did not typically lead to broad multi-AMP resistance. Instead, resistance likely focused on one component of the combination. While mutations in pmtR (toxin transport) and tagO (wall-teichoic acid biosynthesis) were common across treatments, AMP-specific mutations, such as dagK and msrR, were also identified. Notably, mutations in a hypothetical membrane protein operon (SAOUHSC_02307–02309) imply a potential pexiganan resistance pathway. The findings suggest that AMP combinations might limit mutation accumulation, while constraining the development of general AMP resistance. The genetic mechanism of resistance is complex, thus careful selection is required for designing effective AMP-based therapies.
