title: Pseudomonas aeruginosa carbapenem resistance mechanisms in Spain: impact on the activity of imipenem, meropenem and doripenem.
abstract: OBJECTIVE
  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-β-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.
authors.affiliation: Servicio de Microbiología and Unidad de Investigación, Hospital Universitari Son Espases, Palma de Mallorca, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Riera
authors.forename: Gabriel
authors.initials: G
authors.lastname: Cabot
authors.forename: Xavier
authors.initials: X
authors.lastname: Mulet
authors.forename: María
authors.initials: M
authors.lastname: García-Castillo
authors.forename: Rosa
authors.initials: R
authors.lastname: del Campo
authors.forename: Carlos
authors.initials: C
authors.lastname: Juan
authors.forename: Rafael
authors.initials: R
authors.lastname: Cantón
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
mesh_terms: D000900:Anti-Bacterial Agents / Q000627:therapeutic use*; D015780:Carbapenems / Q000494:pharmacology* / Q000627:therapeutic use; D000077726:Doripenem; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D015378:Imipenem / Q000627:therapeutic use*; D000077731:Meropenem; D008826:Microbial Sensitivity Tests; D017720:Molecular Epidemiology; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011159:Population Surveillance; D018272:Porins / Q000235:genetics / Q000378:metabolism; D011552:Pseudomonas Infections / Q000453:epidemiology* / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013030:Spain / Q000453:epidemiology; D013845:Thienamycins / Q000627:therapeutic use*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D018272:Porins; D013845:Thienamycins; C077157:OprD protein, Pseudomonas aeruginosa; D015378:Imipenem; D000077726:Doripenem; D001618:beta-Lactamases; D000077731:Meropenem