Characterization of the MexC-MexD-OprJ Multidrug Efflux System in Δ mexA-mexB-oprM Mutants of Pseudomonas aeruginosa

Abstract

Expression of the multidrug efflux system MexC-MexD-OprJ innfxB mutants of Pseudomonas aeruginosacontributes to resistance to fluoroquinolones and the “fourth-generation” cephems (cefpirome and cefozopran), but not to most β-lactams, including the ordinary cephems (ceftazidime and cefoperazone). nfxB mutants also express a second multidrug efflux system, MexA-MexB-OprM, due to incomplete transcriptional repression of this operon by the mexR gene product. To characterize the contribution of the MexC-MexD-OprJ system to drug resistance in P. aeruginosa, a site-specific deletion method was employed to remove the mexA-mexB-oprM region from the chromosome of wild-type and nfxB strains ofP. aeruginosa. Characterization of mutants lacking themexA-mexB-oprM region clearly indicated that the MexC-MexD-OprJ efflux system is involved in resistance to the ordinary cephems as well as fluoroquinolones and the fourth-generation cephems but not to carbenicillin and aztreonam. Rabbit polyclonal antisera and murine monoclonal antibody against the components of the MexA-MexB-OprM system were prepared and used to demonstrate the reduced production of this efflux system in the nfxB mutants. Consistent with this, transcription of the mexA-mexB-oprM operon decreased in an nfxB mutant. This reduction appears to explain the hypersusceptibility of the nfxB mutant to β-lactams, including ordinary cephems.

Functional replacement of OprJ by OprM in the MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa

“…Plasmid pKMM002 [8], carrying a part of mexR and mexAB-oprM on an 8.4-kb SacI^HindIII fragment from pPV20 [2], was digested at the NruI-site in mexA and the SphI-site in mexB, and then bluntended with T4 DNA polymerase. A 9-base XbaI linker, 5P-GCTCTAGAG-3P, was inserted into the excised region and used to construct pKMM128.…”

“…2, lane 5). Preparation of total membranes and analysis by SDS-PAGE and immunoblot assay were performed as described [8]. Table 2 shows the susceptibility of the constructed mutants and their parent strains to antimicrobial agents.…”

Alterations of susceptibility of Pseudomonas aeruginosa by overproduction of multidrug efflux systems, MexAB-OprM, MexCD-OprJ, and MexXY/OprM to carbapenems: Substrate specificities of the efflux systems

“…1), for those strains were examined by an agar dilution method using L-agar as described previously. 11 The results are shown in Table 1. The MICs of BO2727, panipenem, meropenem, S4661, and DU6681a for KG5002, which lacked the three efflux systems, were increased by each constitutive expression of the efflux systems: those of panipenem, DU6681a, meropenem, and S4661 by overexpression of MexAB-OprM (refer to KG5004); those of BO2727, panipenem, meropenem,…”

Interplay between Three RND Efflux Pumps in Doxycycline-Selected Strains of Burkholderia thailandensis

“…This paradoxical decrease in MICs has remained unexplained in B. pseudomallei , as reported by Dance et al in 1989 [40]. A similar phenomenon is also observed in other Gram-negative bacteria, such as P. aeruginosa , and hypersusceptibility to ß-lactams and aminoglycosides in multidrug-resistant strains has been attributed to a decrease in the levels of MexAB-OprM, a homolog of BpeAB-OprB [41]. Aminoglycosides are extruded principally by the AmrAB-OprA pump, but may also act as substrates of BpeAB-OprB in B. pseudomallei .…”