Uncovering the genetic basis of resistance to single antimicrobial peptides and their combinations.

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 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 (toxin transport) and (wall-teichoic acid biosynthesis) were common across treatments, AMP-specific mutations, such as and , 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.