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

Abstract

Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g., gyrA and parC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (1 microg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was approximately 10(-7) CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10(-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 emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones.

FIG. 1

Efflux pump inhibitors used in this study.

FIG. 2

(A) A linear increase in fluorescence with time due to intracellular hydrolysis of MC-005,556 (Ala-Nap) is seen when Ala-Nap (32 μg/ml) is added to intact cells of P. aeruginosa. Open and filled triangles, cells of PAM1723 that overexpress MexAB-OprM and that were not treated or treated with CCCP, respectively; open and filled circles, cells of PAM1626 (the pump deletion mutant) that were not treated or treated with CCCP, respectively. Much less fluorescence is produced in the case of PAM1723 compared to the amount produced in the case of PAM1626, apparently due to MexAB-OprM-mediated efflux of Ala-Nap. As a result of CCCP treatment, efflux is abolished and both cell types produce the same amount of fluorescence. (B) The relationship between the change in fluorescence and the concentration of Ala-Nap for different cell types was determined. The amount of fluorescence produced by intracellular Ala-Nap hydrolysis in 30 min versus the concentration of Ala-Nap used is shown. Differential rates of uptake of Ala-Nap between cells overexpressing each of the three efflux pumps (MexAB-OprM [filled diamonds], MexCD-OprJ [filled triangles], MexEF-OprN [filled circles]) and the cells of PAM1626 (open circles) are seen over a wide range of Ala-Nap concentrations. The linear increase in fluorescence in PAM1626 in the presence of up to 128 μg of externally added Ala-Nap per ml indicates that the protease hydrolyzing Ala-Nap is not yet saturated at this concentration.

FIG. 3

An increase in the rates of fluorescence production due to hydrolysis of MC-055,556 (64 μg/ml) by intact cells of P. aeruginosa overexpressing Mex pumps is seen in the presence of different concentrations (in micrograms per milliliter, as indicated below each panel) of MC-002,595, a close analog of MC-207,110. No effect of compound was observed for cells of PAM1626. (A) PAM1626 lacking three Mex pumps; (B) PAM1723 overexpressing MexAB-OprM; (C) PAM1738 overexpressing MexCD-OprJ; (D) PAM1753 overexpressing MexEF-OprN.

FIG. 4

(A) Cells of E. coli ECM1642 overexpressing AcrAB-TolC were treated with CCCP at 100 μM and preloaded with 9 μM NPN for 10 min. Extrusion was initiated by the addition of either glucose alone (filled squares) or by the addition of glucose mixed with different concentrations of MC-002,595 to give final concentrations of 16 to 128 μg/ml. Extrusion is visualized as the decay of NPN fluorescence in the presence of glucose versus that for the control (no glucose added) (open boxes). MC-002,595 inhibits extrusion of NPN in a dose-dependent manner. (B) Glucose alone or in combination with 128 μg of MC-002,595 per ml is added to CCCP-treated, NPN-loaded cells of ECM1668 (in which AcrAB-TolC is nonfunctional) or ECM1642. The similar level of NPN fluorescence in both cell types after MC-002,595 addition indicates complete inhibition of the AcrAB-TolC-mediated efflux of NPN. a.u., arbitrary units.

FIG. 5

The pH gradient is visualized as two peaks of the ³¹P NMR spectrum corresponding to intracellular and extracellular phosphorus, respectively. (A) Glucose alone; (B) glucose and MC-207,110 (0.4 mM); (C) glucose and CCCP (0.4 mM). The addition of MC-207,110 did not affect the transmembrane proton gradient.

FIG. 6

The outer membrane-permeabilizing activity of either PMBN or EPI is visualized as an increase in initial rates of nitrocefin hydrolysis by intact cells of P. aeruginosa in the presence of increasing concentrations (in micrograms per milliliter, as indicated beside each panel) of these compounds. PMBN was used at 2 to 32 μg/ml, and MC-207,110 was used at 2 to 128 μg/ml. Nitrocefin hydrolysis was monitored over time by monitoring the increase in absorbance at 490 nm. (A) The effect of PMBN was studied with PAM2005; (B) the effect of MC-207,110 was studied with PAM2005; (C) the effect of MC-207,110 on intact PAM2035 was studied; (D) the effect of MC-207,110 on CCCP-treated PAM2005 was studied.

FIG. 7

The activity of the combination of MC-207,110 with levofloxacin against 50 clinical isolates of P. aeruginosa was determined with a fixed concentration of MC-207,110 (10 μg/ml). Open circles, levofloxacin alone; filled circles, levofloxacin in combination with EPI.