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 (<i>Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,</i> and <i>Enterobacter</i> 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 <i>in vitro</i> models of infection. The combination was synergistic against intra- and extra-cellular forms of <i>P. aeruginosa, A. baumannii, E. coli</i>, and <i>K. pneumoniae</i>. 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 <i>P. aeruginosa</i>. To reveal the molecular underpinnings of the synergy, we used reverse genetics to identify and describe <i>P. aeruginosa</i> 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.