We investigated the regulation of the MexEF-OprN multidrug efflux system of Pseudomonas aeruginosa, which is overexpressed innfxC-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim. Sequencing of the DNA region upstream of the mexEF-oprN operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT. By using T7-polymerase, a 34-kDa protein was expressed in Escherichia coli from a plasmid carrying the mexT gene. Expression of amexE::lacZ fusion was 10-fold higher in nfxC-type mutants than in the wild-type strain; however, transcription of mexT as well as the mexT DNA region was unchanged. Located adjacent to mexT but transcribed in opposite direction, the beginning of an ORF termedqrh (quinone oxidoreductase homologue) was identified. Expression of a qrh::lacZ fusion was also found to be activated by MexT. Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of nfxC-type mutants to carbapenem antibiotics.

While meropenem MICs were strongly influenced by the presence or absence of the MexAB-OprM efflux pump in both OprD-proficient and -deficient strain backgrounds, MICs of imipenem and of ER-35786 remained unchanged, demonstrating that meropenem is a substrate of MexAB-OprM but not imipenem and ER-35786. In vitro, all three carbapenems selected loss of OprD as a first mechanism of resistance. However, in an OprD-deficient background, meropenem was able to select MexAB-OprM overproducers as a secondary resistance mechanism, while ER-35786 selected a mutant cross-resistant to sparfloxacin and cefpirome.

We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 g/ml) but susceptible to meropenem (<1 g/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-type oprD gene were cloned and expressed in a defined oprD mutant. The meropenem MICs for strains carrying the oprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprD gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid L-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.Pseudomonas aeruginosa is an opportunistic organism causing difficult-to-treat infections. The high intrinsic resistance of this bacterium to antibiotics results from the complex interaction of several mechanisms, among which the rather impermeable porin pathway plays a key role (40). Substrate-specific transport systems in the outer membrane allow the diffusion of essential nutrients present at low concentrations in the vicinity of the cells and compensate for this low nonspecific permeability. The outer membrane porin OprD facilitates the uptake of basic amino acids (36), small peptides, and carbapenem antibiotics, such as imipenem and meropenem (35). All of these molecules share common binding sites inside the OprD channel (6).Carbapenems are potent inhibitors of P. aeruginosa because of their high stability to the chromosomally encoded AmpC ␤-lactamase produced by this species (15). However, resistance of clinical strains to both imipenem and meropenem is increasingly observed as the result of the loss of protein OprD (18, 29), alone or associated with the stable overexpression of the AmpC ␤-lactamase (16). Overproduction of the MexABOprM active efflux system in nalB mutants may also increase the resistance to meropenem but has no effect on the susceptibility of P. aerugi...

Outer membrane (OM) proteins of ß-lactam-susceptible and -resistant strains of Pseudomonas aeruginosa were analyzed by 2-D polyacrylamide gel electrophoresis. Carrier ampholytes, pH 4-8, and immobilized pH gradient (IPG), pH 3.5-10.0, procedures were used. An acidic-protein spot (pi = 5.2) detected in susceptible but not in an imipenem-resistant strain was sequenced and twenty-five N-terminal amino acids had total homology with the OM protein D, the imipenem-specific porin of P. aeruginosa. A basic-protein spot (pI = 9.0) detected in ceftazidime-resistant, but not in a susceptible strain was sequenced and fourteen N-terminal amino acids had homology with a ß-lactamase encoded by the ampC gene of P. aeruginosa. The IPG procedure allows identification of more than one hundred proteins of the OM fraction from a single gel. Detection of ß-lactamase in OM fractions might reflect a periplasmic contamination, but its anchorage within the OM cannot be ruled out.

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