Synergistic activity of rifampicin and polymyxin B against intracellular Gram-negative ESKAPE pathogens involves bacterial membrane alterations and enhanced oxidative damages

Antibiotic-resistant bacteria, particularly the ESKAPE ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens, pose a major public health threat. Their ability to reside inside cells contributes to their persistence and resistance. Combining rifampicin with polymyxins is a much-discussed approach against multidrug-resistant Gram-negative bacterial infections. We therefore evaluated a combination of polymyxin B and rifampicin against Gram-negative clinical isolates in extracellular and intracellular in vitro models of infection. The combination was synergistic against intra- and extra-cellular forms of P. aeruginosa, A. baumannii, E. coli , and K. pneumoniae . This synergy was enhanced in an acidic environment resembling the host vacuole where intracellular bacteria reside. The combination remained synergistic against rifampicin and polymyxin B-resistant P. aeruginosa . To reveal the molecular underpinnings of the synergy, we used reverse genetics to identify and describe P. aeruginosa mutants more susceptible to the combination. They show altered membrane properties and more pronounced oxidative damage when exposed to the combination. This work sheds a new light on the mechanisms of the synergy between rifampicin and polymyxins, demonstrates its applicability to Gram-negative ESKAPE pathogens, including when residing intracellularly. Overall, the data suggest that repurposing rifampicin with polymyxin B can effectively target hard-to-eradicate intracellular bacteria.