Pseudomonas aeruginosa carbapenem resistance mechanisms in Spain: impact on the activity of imipenem, meropenem and doripenem

Objectives: To investigate the mechanisms of carbapenem resistance in the 175 Pseudomonas aeruginosa isolates (39%; 175/448) showing non-susceptibility (European Committee on Antimicrobial Susceptibility Testing breakpoints) to imipenem (35%), meropenem (33%) and/or doripenem (33%) recovered in 2008-09 from 16 Spanish hospitals during the Comparative Activity of Carbapenem Testing (COMPACT) surveillance study. Methods: MICs (Etest), clonal relatedness (PFGE) and metallo-beta-lactamase (MBL) production (Etest-MBL, PCR and sequencing) were determined. Mutation-driven resistance was studied in 60 non-MBL producers according to the doripenem MICs (15 isolates from each of four MIC groups: <= 1, 2-4, 8-16 and >= 32 mg/L). The expression of ampC, mexB, mexY, mexD and mexF was determined by real-time reverse transcription-PCR and the presence of mutations in oprD by PCR and sequencing. Isogenic mutants expressing combinations of mutation-driven carbapenem resistance were constructed. Results: Twelve (6.9%) isolates were MBL (VIM-20, VIM-2 or VIM-13) producers and all showed high-level resistance (MIC 32 mg/L) to all three carbapenems. Regarding mutation-driven resistance, all but 1 of the 60 isolates were non-susceptible (MIC >32 mg/L) to imipenem, linked to oprD inactivation. In addition, 50% of the isolates overexpressed ampC, 33% mexY, 32% mexB and 15% mexF, while none overexpressed mexD. Increasing prevalence of ampC overexpression correlated with increasing doripenem MICs (<= 1, 13%; 2-4, 53%; 8-16, 60%; and >= 32, 73%) while overexpression of efflux pumps correlated only with moderate resistance. Doripenem showed slightly higher activity than meropenem against isolates overexpressing ampC, especially mexB or mexY. The analysis of a collection of isogenic laboratory mutants supported this finding. Conclusions: Although the prevalence of MBL producers is increasing, mutation-driven resistance is still more frequent in Spain. Imipenem resistance was driven by OprD inactivation, while additional AmpC and particularly efflux pump hyperproduction had a lower impact on the activity of doripenem compared with meropenem.