The first metallo-b-lactamase producing clinical isolate of Pseudomonas aeruginosa in Norway

Abstract number: 1733_861

Samuelsen Ø., Toleman M.A., Hermansen N.O., Aasnæs B., Haldorsen B., Sundsfjord A., Walsh T.R.

Objectives: MBL are of great clinical significance as they are able to hydrolyse virtually all b-lactams limiting therapeutic options. Here we describe the first clinical isolate of MBL-producing Pseudomonas aeruginosa in Norway and its subsequent genetic characterisation.

Methods: A clinical isolate of P. aeruginosa (K34–7) was recovered from a patient recently admitted from an African country with a high-level resistance to carbapenems and was examined for MBL production. Susceptibility testing was performed using disc diffusion, VITEK and Etest. The presence of MBL was confirmed by PCR (custom oligonucleotides for all major blaMBL genes) and by spectrophotometric analysis of crude cell extracts (with and without EDTA – 25mM for 1 hr) for the ability to hydrolyse imipenem. Characterisation of the MBL gene and its genetic support was evaluated by its association with integrons, transposons and insertion elements (ISCR) using PCR and sequencing.

Results: Susceptibility testing showed that P. aeruginosa K34–7 was multi-drug resistant, highly resistant to imipenem and meropenem (MIC > 32 mg/L) and positive on the MBL Etest (ratio MIC imipenem/imipenem + EDTA ≥8). MBL production was also verified by hydrolysis/inhibition assays on crude cell extracts ± EDTA. Sequencing analysis of the positive blaVIM amplicon revealed it was 100% identical to blaVIM-2. PCR and sequencing analysis revealed that K34–7 contained 2 very different integrons. The class 1 integron contains 4 antibiotic resistant genes including blaOXA31 and genes encoding novel multi-drug resistant pumps and aminoglycoside modifying enzymes. The second integron contained TniCR at the 3' end instead of qac/sul and the gene cassette sequence: aacA7-blaVIM-2-aacA5. Although standard transposons was not detected adjacent to the integrons, we detected a new ISCR element, designated ISCR10, and the first to be found in P. aeruginosa.

Conclusion: This is the first report of an MBL-producing P. aeruginosa identified in Norway. Genetic analysis revealed the ``new'' TniCR integron associated with the blaVIM-2 MBL gene and the first ISCR10 found in P. aeruginosa.