Antibiotic-resistant microbial pathogens are becoming a major threat to human health. Therefore, there is an urgent need to develop new alternatives to conventional antibiotics. One such promising alternative is antimicrobial peptides (AMPs), which are produced by virtually all organisms and typically inhibit bacteria via membrane disruption. However, previous studies demonstrated that bacteria can rapidly develop AMP resistance. Here, we study whether combination therapy, known to be able to inhibit the evolution of resistance to conventional antibiotics, can also hinder the evolution of AMP resistance. To do so, we evolved the opportunistic pathogen Staphylococcus aureus in the presence of individual AMPs, AMP pairs, and a combinatorial antimicrobial peptide library. Treatment with some AMP pairs indeed hindered the evolution of resistance compared with individual AMPs. In particular, resistance to pairs was delayed when resistance to the individual AMPs came at a cost of impaired bacterial growth and did not confer cross-resistance to other tested AMPs. The lowest level of resistance evolved during treatment with the combinatorial antimicrobial peptide library termed random antimicrobial peptide mixture, which contains more than a million different peptides. A better understanding of how AMP combinations affect the evolution of resistance is a crucial step in order to design "resistant proof" AMP cocktails that will offer a sustainable treatment option for antibiotic-resistant pathogens. IMPORTANCE The main insights gleaned from this study are the following. (i) AMP combination treatment can delay the evolution of resistance in S. aureus. Treatment with some AMP pairs resulted in significantly lower resistance then treatment with either of the individual AMPs. Treatment with a random AMP library resulted in no detectable resistance. (ii) The rate at which resistance to combination arises correlates with the cost of resistance to individual AMPs and their cross-resistance. In particular, combinations to which the least resistance arose involved AMPs with high fitness cost of resistance and low cross-resistance. (iii) No broad-range AMP resistance evolved. Strains that evolved resistance to some AMPs typically remained sensitive to other AMPs, alleviating concerns regarding the evolution of resistance to immune system AMPs in response to AMP treatment.
Keywords: antibiotic resistance; antimicrobial combinations; antimicrobial peptides; experimental evolution.