High-level resistance to meropenem in clinical isolates of Pseudomonas aeruginosa in the absence of carbapenemases: role of active efflux and porin alterations

Chalhoub, Hussein [UCL] Sáenz, Yolanda Rodriguez-Villalobos, Hector [UCL] Denis, Olivier Kahl, Barbara C. Tulkens, Paul [UCL] Van Bambeke, Françoise [UCL]

High-level carbapenem resistance is worryingly increasing in clinical isolates and often attributed to carbapenemase expression. This study aims at determining the mechanisms leading to high-level resistance to meropenem in six carbapenemase-negative Pseudomonas aeruginosa isolated from cystic fibrosis patients (CF), compared with seven carbapenemase-positive isolates from patients suffering from hospital-acquired pneumonia (HAP). MICs were determined in the absence or in the presence of L-Arginine or Glycine-Glutamate as competitive substrates for OprD (OccD1) or OpdP (OccD3), respectively, or of the efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN). Beta-lactamases were screened by PCR and/or phenotypic tests. 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 semi-quantitative PCR, respectively. Meropenem/imipenem MICs were 64-128/32 mg/L and 128/128-256 mg/L in CF and HAP isolates, respectively; PAβN reduced only meropenem MICs to 4-16 mg/L specifically in CF strains; 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 porin leading to the production of truncated proteins. AmpC was derepressed in CF strains and VIM-2 expressed in HAP strains. Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is as effective as carbapenemases in conferring high-level resistance to meropenem in strains expressing AmpC. As efflux is preponderant in these strains, 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.