Pseudomonas aeruginosa (P. aeruginosa) poses a grave clinical challenge due to its multidrug resistance (MDR) phenotype, leading to severe and life-threatening infections. This bacterium exhibits both intrinsic resistance to various antipseudomonal agents and acquired resistance against nearly all available antibiotics, contributing to its MDR phenotype. Multiple mechanisms, including enzyme production, loss of outer membrane proteins, target mutations, and multidrug efflux systems, contribute to its antimicrobial resistance. The clinical importance of addressing MDR in P. aeruginosa is paramount, and one pivotal determinant is the resistance-nodulation-division (RND) family of drug/proton antiporters, notably the Mex efflux pumps. These pumps function as crucial defenders, reinforcing the emergence of extensively drug-resistant (XDR) and pandrug-resistant (PDR) strains, which underscores the urgency of the situation. Overcoming this challenge necessitates the exploration and development of potent efflux pump inhibitors (EPIs) to restore the efficacy of existing antipseudomonal drugs. By effectively countering or bypassing efflux activities, EPIs hold tremendous potential for restoring the antibacterial activity against P. aeruginosa and other Gram-negative pathogens. This review focuses on concurrent MDR, highlighting the clinical significance of efflux pumps, particularly the Mex efflux pumps, in driving MDR. It explores promising EPIs and delves into the structural characteristics of the MexB subunit and its substrate binding sites.

Aims: Efflux pumps, particularly resistance-nodulation-division family such as Mex pumps in Pseudomonas aeruginosa, are the major contributors to multidrug resistance in Gram-negative bacteria. Since the well-known efflux pump inhibitor (EPI), phenylalanine-arginine β-naphthylamide (PAβN), is a substrate of cathepsin C (a proteolytic enzyme), inhibitory effects of similar substrates of this enzyme, proline-arginine 4-methoxy-β-naphthylamide (PA4MβN) and N_α-Benzoyl-DL-arginine β-naphthylamide (BAN), were investigated as possible new EPIs. Additionally, the probable EPI activities of three different alkaloids (noscapine, caffeine and ergotamine) against Mex-pump expressing strains were also evaluated mainly based on their structural similarities to major EPIs. Methodology: MPC₈ (minimum potentiating concentrations) of mentioned compounds in combination with levofloxacin (LVX) were screened against a wild type (PAO1) and two different Mex-pump overexpressing P. aeruginosa strains (nalB and nfxB). The most synergizing compound was further validated by the checkerboard assay for its EPI potency against strains overexpressing or lacking different Mex-pumps. The frequencies of emergence of LVX-resistance strains when it was used either alone or in combination with the suggested EPI were also compared against each other. Results: PA4MβN caused significant increase in LVX activity against PAO1 and different Mex-pump overexpressing P. aeruginosa strains showing a significant decrease in the intrinsic (8-fold) and acquired (16-fold) resistances. Interestingly, PA4MβN fully prevented further emergence of P. aeruginosa mutants highly resistant to fluoroquinolones. Conclusion: A new compound from peptidomimetic family (PA4MβN), which is also a substrate of cathepsin C, was introduced in the current study as an effective EPI against Mex pumps from P. aeruginosa.