C-Terminal Region of Pseudomonas aeruginosa Outer Membrane Porin OprD Modulates Susceptibility to Meropenem

Abstract

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...

Development and Persistence of Antimicrobial Resistance in Pseudomonas aeruginosa : a Longitudinal Observation in Mechanically Ventilated Patients

“…All imipenemresistant isolates showed decreased oprD expression. However, the n-fold decrease in oprD expression did not correlate well with imipenem susceptibility levels, suggesting that additional factors, such as posttranscriptional regulation of oprD (9) or interplay with AmpC expression, might play a role, as suggested previously (28).…”

Carbapenems for the treatment of immunocompetent adult patients with nosocomial pneumonia

“…This propensity, related to the decreased expression of an outer membrane porin channel [79], was not reduced by the simultaneous use of an aminoglycoside in one study [77], which was not included in the current metaanalysis. The development of resistance to imipenem/cilastatin in P. aeruginosa strains could explain the fewer pathogen eradications and, consequently, the lower clinical success of this antibiotic in patients with P. aeruginosa HAP.…”

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

“…both clinical and in vitro studies have reported that aberrant or modified P. aeruginosa OprD porin is linked to carbapenem resistance, with or without the production of a carbapenem hydrolyzing enzyme 58,59 . In addition, several mutations that confer reduced carbapenem susceptibility have been reported in the oprD gene 60 . The OprD crystal structure has recently been solved 61 .…”

“…several OprD residues belonging to loops 3 and 7 and a number of backbone residues create an asymmetric charge distribution and participate in the formation of the pore-constriction zone. some mutated residues have previously been reported in carbapenem-resistant isolates and are located in or near loops 6 and 7 (rEFs 60,61 [63][64][65] . A small aliquot of liposomes is rapidly mixed in a polymer-free solution that contains the molecule of interest and that has the same osmotic pressure.…”