Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections. Bacteria evolved numerous mechanisms to stop the action of antibiotics. Efflux pumps are such vital molecular machinery that constantly exports antibiotics out of the bacterial cell. As a result, the antibiotics fail to accumulate sufficiently, thereby decreasing the bacteria's susceptibility to antibiotics. One excellent strategy to tackle Multi-drug resistant (MDR) pathogens is to inhibit these efflux pumps and thereby increase the pathogen sensitivity to existing/obsolete antibiotics. This study identified pyrrole derivatives as efflux pump inhibitors and highlights their potential to increase the susceptibility of MDR E. coli, P. aeruginosa, and K. pneumoniae to multiple antibiotics in vitro. Moreover, we observed that the combination displayed remarkable efficacy in reducing bacterial burden in vivo. Additionally, this study provides a glance at the accessory anti-virulence properties of the EPIs as a secondary effect of efflux pump inhibition. Altogether, the present study provides relevant clues to consider combination therapy as a go-to solution for MDR bacterial infections.