title: Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa.
abstract: Drug efflux pumps in Pseudomonas aeruginosa were evaluated as potential targets for antibacterial therapy. The potential effects of pump inhibition on susceptibility to fluoroquinolone antibiotics were studied with isogenic strains that overexpress or lack individual efflux pumps and that have various combinations of efflux- and target-mediated mutations. Deletions in three efflux pump operons were constructed. As expected, deletion of the MexAB-OprM efflux pump decreased resistance to fluoroquinolones in the wild-type P. aeruginosa (16-fold reduction for levofloxacin [LVX]) or in the strain that overexpressed mexAB-oprM operon (64-fold reduction for LVX). In addition to that, resistance to LVX was significantly reduced even for the strains carrying target mutations (64-fold for strains for which LVX MICs were >4 microg/ml). We also studied the frequencies of emergence of LVX-resistant variants from different deletion mutants and the wild-type strain. Deletion of individual pumps or pairs of the pumps did not significantly affect the frequency of emergence of resistant variants (at 4x the MIC for the wild-type strain) compared to that for the wild type (10(-6) to 10(-7)). In the case of the strain with a triple deletion, the frequency of spontaneous mutants was undetectable (<10(-11)). In summary, inhibition of drug efflux pumps would (i) significantly decrease the level of intrinsic resistance, (ii) reverse acquired resistance, and (iii) result in a decreased frequency of emergence of P. aeruginosa strains highly resistant to fluoroquinolones in clinical settings.
authors.affiliation: Microcide Pharmaceuticals Inc., Mountain View, California 94043, USA. olga@microcide.com
authors.forename: O
authors.initials: O
authors.lastname: Lomovskaya
authors.forename: A
authors.initials: A
authors.lastname: Lee
authors.forename: K
authors.initials: K
authors.lastname: Hoshino
authors.forename: H
authors.initials: H
authors.lastname: Ishida
authors.forename: A
authors.initials: A
authors.lastname: Mistry
authors.forename: M S
authors.initials: MS
authors.lastname: Warren
authors.forename: E
authors.initials: E
authors.lastname: Boyer
authors.forename: S
authors.initials: S
authors.lastname: Chamberland
authors.forename: V J
authors.initials: VJ
authors.lastname: Lee
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004352:Drug Resistance, Microbial; D064704:Levofloxacin; D009154:Mutation; D015242:Ofloxacin / Q000494:pharmacology*; D009876:Operon; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D064704:Levofloxacin; D015242:Ofloxacin; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II