Synergistic Activity of Antimicrobial Peptides with Orthogonal Mechanisms of Action

Abstract Objective: The spread of multidrug-resistant (MDR) strains significantly limits the efficacy of existing antibiotics and necessitates the development of new therapeutic approaches. This problem is particularly relevant for Gram-negative ESKAPE pathogens, especially MDR strains of Klebsiella pneumoniae. Antimicrobial peptides (AMPs) can be considered promising alternative antimicrobial agents with high activity against MDR pathogen strains. Furthermore, the development of approaches based on the combined action of multiple AMPs or combinations of AMPs with classical antibiotics will allow a multiple potentiation of their antimicrobial action due to the synergistic effect of antimicrobial agents. We aimed to evaluate the synergistic action of AMPs with orthogonal mechanisms of action against MDR clinical isolates of K. pneumoniae, and to explore the translation of these synergistic principles to potentiate the clinically relevant glycopeptide antibiotic vancomycin. Methods: We utilized a screening approach based on the recombinant production of a panel of AMPs in the methylotrophic yeast Pichia pastoris to identify synergistic interactions. Promising synergistic combinations were subsequently validated in vitro using the checkerboard assay against clinical MDR isolates of K. pneumoniae. Results and Discussion: While individual recombinant AMP producers showed limited activity against clinical K. pneumoniae isolates, combining AMPs with distinct molecular targets resulted in pronounced synergistic effects. Unlike combinations of two membranolytic AMPs, pairing the membrane-disrupting peptide cecropin P1 with thanatin (an inhibitor of lipopolysaccharide transport) demonstrated true antimicrobial synergy, reducing individual minimum inhibitory concentrations fourfold. Furthermore, cecropin P1 effectively potentiated vancomycin (an antibiotic naturally inactive against Gram-negative bacteria), sensitizing MDR K. pneumoniae strains to this agent. Conclusions: Our findings demonstrate that combining orthogonal antimicrobial agents targeting at the membrane and cell wall components yields a universal synergistic effect. This approach provides a robust framework for developing highly effective antimicrobial regimens based on peptide synergy and the sensitization of MDR pathogens, thereby expanding the therapeutic spectrum of clinically vital antibiotics against drug-resistant infections.