Repurposing azithromycin and rifampicin against Gram-negative pathogens by combination with peptide potentiators

Gram-negative pathogens are intrinsically resistant to several antibiotics that are not able to penetrate the envelope barrier. The objective of this study was to identify peptides that at low concentrations induce susceptibility to these antibiotics in multidrug-resistant (MDR) Gram-negative strains of clinical relevance. A pairwise screening of 34 diverse peptides and four antibiotics (erythromycin, linezolid, rifampicin and vancomycin) with primary activity against Gram-positive bacteria identified four peptides that at sub-micromolar concentrations conferred susceptibility to rifampicin or erythromycin in Escherichia coli ATCC 25922. The identified peptides exhibited synergy with azithromycin and potentiated clindamycin in MDR E. coli ST131 and Klebsiella pneumoniae ST258. The low cytotoxicity toward eukaryotic cells (IC50 >50 µM) observed for two peptides (KLWKKWKKWLK-NH2 and GKWKKILGKLIR-NH2) prompted synthesis and evaluation of the corresponding all-D analogs (D1 and D2), which retained similar synergistic antibacterial profiles. Low concentrations of D1 and D2 in combination with azithromycin and rifampicin inhibited growth of most clinical E. coli, K. pneumoniae and Acinetobacter baumannii strains tested. Our data demonstrate that combinatorial screening at low concentrations constitutes an efficient approach to identify clinically relevant peptide-antibiotic combinations. In vivo PK/PD and toxicity studies are needed to further validate the use of the peptides identified by this study for repurposing azithromycin and rifampicin against Gram-negative pathogens.