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Pseudomonas aeruginosa with a novel blaVIM-4/blaP1b and a second class-1 integron, efflux pumps overexpression and repressed porin OprD

Abstract number: 1732_329

Maniati M., Ikonomidis A., Mantzana P., Maniatis A., Pournaras S.

Objectives: To investigate the resistance mechanisms in a multi-drug resistant Pseudomonas aeruginosa isolate which was positive for metallo-b-lactamase (MBL).

Methods: Strain P. aeruginosa 140 was studied. Identification and susceptibility testing to antipseudomonal antimicrobials was performed by the VITEK 2 automated system. MICs of imipenem, meropenem, ceftazidime, aztreonam, gentamicin and piperacillin/tazobactam were determined by agar dilution. Etest MBL and the imipenem–EDTA double disc synergy test (DDST) were done to detect MBL production. PCR for gene blaVIM and conserved segments of class-1 integrons was performed. Mating experiments were performed by mixed broth mating using P. aeruginosa PU21 (rif r) as recipient and plasmid isolation with alkaline lysis. Quantitative real-time reverse transcription-PCR (QRT-PCR) was performed in order to detect transcripts of genes mexB, mexY and ampC. Relative expression levels in strain 140 were compared against those in a susceptible isolate. Outer membrane proteins (OMPs) were analysed by SDS-PAGE.

Results: Isolate PA140 was resistant to all tested antimicrobials except aztreonam. Agar dilution MIC of imipenem was 512 and that of meropenem 128 mg/L, while MICs of both gentamicin and piperacillin/tazobactam were >512 mg/L. MICs of ceftazidime and aztreonam were 32 and 8 mg/L, respectively. Etest MBL exhibited marked decrease in MICs of imipenem compared to imipenem-EDTA (256 and 1.5 mg/L respectively) and DDST was positive. PCR revealed two class-1 integrons. The MBL-associated integron was sequentially consisted of genes blaVIM-4 and blaP1b coding for b-lactamases VIM-4 and PSE-1/CARB-2 respectively. The second integron comprised sequentially of genes aac(6')-Ib and blaOXA-35. Plasmid extraction did not reveal any visible plasmids and no transcojugants were produced by mating experiments. QRT-PCR revealed overexpression of genes mexB and mexY as well as a fairly detectable expression of gene ampC in strain PA140 relatively to the susceptible strain. Also, OMP analysis revealed a lower intensity of a protein band of MW » 46 kD in strain 140, consistent with OprD.

Conclusions: A P. aeruginosa clinical isolate harboured a novel class-1 integron with two (blaVIM-4 and blaP1b) b-lactamase gene cassettes and a second integron. The isolate was highly carbapenem- and multidrug-resistant, had reduced production of porin oprD and overexpressed efflux pumps MexAB-OprM and MexXY-OprM.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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