Inhibition of the Mex Pumps of Pseudomonas aeruginosa with a Newly Characterized Member of Peptidomimetic Family

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 Nα-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.