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Antimicrobial Agents and Chemotherapy, January 2001, p. 105-116, Vol. 45, No. 1
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.1.105-116.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents for Combination Therapy

Olga Lomovskaya,1,* Mark S. Warren,1 Angela Lee,1 Jorge Galazzo,1 Richard Fronko,1 May Lee,1 Johanne Blais,1 Deidre Cho,1 Suzanne Chamberland,1 Tom Renau,1 Roger Leger,1 Scott Hecker,1 Will Watkins,1 Kazuki Hoshino,2 Hiroko Ishida,2 and Ving J. Lee1

Microcide Pharmaceuticals, Inc., Mountain View, California 94043,1 and Daiichi Pharmaceutical Co., Ltd., Tokyo 134, Japan2

Received 24 July 2000/Returned for modification 9 September 2000/Accepted 9 October 2000

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 µg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was ~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.

* Corresponding author. Mailing address: Microcide Pharmaceuticals, Inc., 850 Maude Ave., Mountain View, CA 94043. Phone: (650) 428-3548. Fax: (650) 428-3550. E-mail: olga{at}microcide.com.

Antimicrobial Agents and Chemotherapy, January 2001, p. 105-116, Vol. 45, No. 1
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.1.105-116.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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