Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: Novel agents for combination therapy

Whole-cell assays were implemented to search for efflux pump inhibitors (EP Is) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isol ates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum E PI which is active against all three known Mex efflux pumps from P. aerugin osa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was d iscovered. When this compound, MC-207,110, was used, the intrinsic resistan ce of P. aeruginosa to fluoroquinolones was decreased significantly (eightf old for levofloxacin). Acquired resistance due to the overexpression of eff lux pumps was also decreased (32- to 64-fold reduction in the MIC of levofl oxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC -207,110 were observed for strains with overexpressed efflux pumps and vari ous target mutations that confer resistance to levofloxacin (e.g., gyrA and parC). We also compared the frequencies of emergence of levofloxacin-resis tant variants in the wild-type strain at four times the MIC of levofloxacin (1 mug/ml) when it was used either alone or in combination with EPI. In th e case of levofloxacin alone, the frequency was similar to 10(-7) CFU/ml. I n contrast, with an EPI, the frequency was below the level of detection (<1 0(-11)). In summary, we have demonstrated that inhibition of efflux pumps ( i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emerge nce of P. aeruginosa strains that are highly resistant to fluoroquinolones.