title: High-level resistance to meropenem in clinical isolates of Pseudomonas aeruginosa in the absence of carbapenemases: role of active efflux and porin alterations.
abstract: High-level carbapenem resistance is worryingly increasing in clinical isolates and is often attributed to carbapenemase expression. This study aimed to determine the mechanisms leading to high-level meropenem resistance in six carbapenemase-negative Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients and seven carbapenemase-positive isolates from patients suffering from hospital-acquired pneumonia (HAP). MICs were determined in the absence or presence of l-arginine or glycine-glutamate as competitive substrates for OprD (OccD1) or OpdP (OccD3), respectively, or the efflux pump inhibitor Phe-Arg β-naphthylamide (PAβN). β-Lactamases were screened by phenotypic tests and/or PCR. The oprD gene and its promoter were sequenced; protein expression was evidenced by SDS-PAGE. mexA, mexX, mexC and mexE transcripts were evaluated by real-time and semiquantitative PCR. Meropenem/imipenem MICs were 64-128/16-32 mg/L and 128/128-256 mg/L in CF and HAP isolates, respectively; PAβN reduced meropenem MICs to 4-16 mg/L only and specifically in CF isolates; porin competitors had no effect on MICs. All isolates showed an increase in transcription levels of mexA, mexX and/or mexC and mutations in oprD leading to production of truncated proteins. AmpC-type cephalosporinases were overexpressed in CF isolates and VIM-2 was expressed in HAP isolates. Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is sufficient to confer high-level resistance to meropenem in isolates overexpressing AmpC-type cephalosporinases. As efflux is preponderant in these isolates, it confers a paradoxical phenotype where meropenem is less active than imipenem. Concomitant susceptibility testing of both carbapenems and rapid elucidation of the most probable resistance mechanisms is thus warranted.
authors.affiliation: Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium.
authors.forename: Hussein
authors.initials: H
authors.lastname: Chalhoub
authors.affiliation: Centro de Investigación Biomédica de La Rioja (CIBIR), Logroño, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Sáenz
authors.affiliation: Laboratoire de microbiologie, Cliniques universitaires Saint-Luc, Brussels, Belgium.
authors.forename: Hector
authors.initials: H
authors.lastname: Rodriguez-Villalobos
authors.affiliation: Laboratoire de microbiologie, Hôpital Erasme, Université libre de Bruxelles, Brussels, Belgium.
authors.forename: Olivier
authors.initials: O
authors.lastname: Denis
authors.affiliation: University Hospital Münster, Münster, Germany.
authors.forename: Barbara C
authors.initials: BC
authors.lastname: Kahl
authors.affiliation: Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Tulkens
authors.affiliation: Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium. Electronic address: francoise.vanbambeke@uclouvain.be.
authors.forename: Françoise
authors.initials: F
authors.lastname: Van Bambeke
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D000077731:Meropenem; D008826:Microbial Sensitivity Tests; D018410:Pneumonia, Bacterial / Q000382:microbiology; D016133:Polymerase Chain Reaction; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013845:Thienamycins / Q000494:pharmacology*; D001618:beta-Lactamases / Q000032:analysis / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D018272:Porins; D013845:Thienamycins; D001618:beta-Lactamases; D000077731:Meropenem
keywords: Carbapenemase; Cystic fibrosis; Hospital-acquired pneumonia; MexAB–OprM; OprD