Boosting the Antibacterial Activity of Azithromycin on Multidrug-Resistant Escherichia coli by Efflux Pump Inhibition Coupled with Outer Membrane Permeabilization Induced by Phenylalanine-Arginine β-Naphthylamide

Abstract

The global spread of multidrug-resistant (MDR) bacteria increases the demand for the discovery of new antibiotics and adjuvants. Phenylalanine-arginine β-naphthylamide (PAβN) is an inhibitor of efflux pumps in Gram-negative bacteria, such as the AcrAB-TolC complex in Escherichia coli. We aimed to explore the synergistic effect and mechanism of action of PAβN combined with azithromycin (AZT) on a group of MDR E. coli strains. Antibiotic susceptibility was tested for 56 strains, which were screened for macrolide resistance genes. Then, 29 strains were tested for synergy using the checkerboard assay. PAβN significantly enhanced AZT activity in a dose-dependent manner in strains expressing the mphA gene and encoding macrolide phosphotransferase, but not in strains carrying the ermB gene and encoding macrolide methylase. Early bacterial killing (6 h) was observed in a colistin-resistant strain with the mcr-1 gene, leading to lipid remodeling, which caused outer membrane (OM) permeability defects. Clear OM damage was revealed by transmission electron microscopy in bacteria exposed to high doses of PAβN. Increased OM permeability was also proven by fluorometric assays, confirming the action of PAβN on OM. PAβN maintained its activity as an efflux pump inhibitor at low doses without permeabilizing OM. A non-significant increase in acrA, acrB, and tolC expression in response to prolonged exposure to PAβN was noted in cells treated with PAβN alone or with AZT, as a reflection of bacterial attempts to counteract pump inhibition. Thus, PAβN was found to be effective in potentiating the antibacterial activity of AZT on E. coli through dose-dependent action. This warrants further investigations of its effect combined with other antibiotics on multiple Gram-negative bacterial species. Synergetic combinations will help in the battle against MDR pathogens, adding new tools to the arsenal of existing medications.