Meropenem potentiation of aminoglycoside activity against Pseudomonas aeruginosa: involvement of the MexXY-OprM multidrug efflux system.

Objectives: To assess the ability of meropenem to potentiate aminoglycoside (AG) activity against laboratory and AG-resistant cystic fibrosis (CF) isolates of Pseudomonas aeruginosa and to elucidate its mechanism of action. Methods: AG resistance gene deletions were engineered into P. aeruginosa laboratory and CF isolates using standard gene replacement technology. Susceptibility to AGs ± meropenem (at ½ MIC) was assessed using a serial 2-fold dilution assay. mexXY expression and MexXY-OprM efflux activity were quantified using quantitative PCR and an ethidium bromide accumulation assay, respectively. Results: A screen for agents that rendered WT P. aeruginosa susceptible to a sub-MIC concentration of the AG paromomycin identified the carbapenem meropenem, which potentiated several additional AGs. Meropenem potentiation of AG activity was largely lost in a mutant lacking the MexXY-OprM multidrug efflux system, an indication that it was targeting this efflux system in enhancing P. aeruginosa susceptibility to AGs. Meropenem failed to block AG induction of mexXY expression or MexXY-OprM efflux activity, suggesting that it may be interfering with some MexXY-dependent process linked to AG susceptibility. Meropenem potentiated AG activity versus AG-resistant CF isolates, enhancing susceptibility to at least one AG in all isolates and susceptibility to all tested AGs in 50% of the isolates. Notably, meropenem potentiation of AG activity was linked to MexXY in some but not all CF isolates in which this was examined. Conclusions: Meropenem potentiates AG activity against laboratory and CF strains of P. aeruginosa, both dependent on and independent of MexXY, highlighting the complexity of AG resistance in this organism.

mexEF-oprN multidrug efflux operon of Pseudomonas aeruginosa: regulation by the MexT activator in response to nitrosative stress and chloramphenicol.

A null mutation in the mexS gene of Pseudomonas aeruginosa yielded an increased level of expression of a 3-gene operon containing a gene, xenB, whose product is highly homologous to a xenobiotic reductase in Pseudomonas fluorescens shown previously to remove nitro groups from trinitrotoluene and nitroglycerin (D. S. Blehert, B. G. Fox, and G. H. Chambliss, J. Bacteriol. 181:6254, 1999). This expression, which paralleled an increase in mexEF-oprN expression in the same mutant, was, like mexEF-oprN, dependent on the MexT LysR family positive regulator previously implicated in mexEF-oprN expression. As nitration is a well-known result of nitrosative stress, a role for xenB (and the coregulated mexEF-oprN) in a nitrosative stress response was hypothesized and tested. Using s-nitrosoglutathione (GSNO) as a source of nitrosative stress, the expression of xenB and mexEF-oprN was shown to be GSNO inducible, although in the case of xenB, this was seen only for a mutant lacking MexEF-OprN. In both instances, this GSNO-inducible expression was dependent upon MexT. Chloramphenicol, a nitroaromatic antimicrobial that is a substrate for MexEF-OprN, was shown to induce mexEF-oprN but not xenB, again dependent upon the MexT regulator, possibly because it resembles a nitrosated nitrosative stress product accommodated by MexEF-OprN.

Protein modulator of multidrug efflux gene expression in Pseudomonas aeruginosa.

nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [K(D)], 158.0 +/- 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative "noninteracting" mutant MexR protein, MexR(I104F), was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; K(D), 853.2 +/- 151.1 nM). Consistent with this, MexR(I104F) repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal alpha-helix of the protein that alone is sufficient for interaction with MexR.