Characterization of carbapenem-resistant Pseudomonas aeruginosa clinical isolates, carrying multiple genes coding for this antibiotic resistance

Background: Carbapenemase genes are one of the most frequent mechanisms reported in carbapenem-resistant P. aeruginosa; however, description of P. aeruginosa co-harbouring two or more carbapenemases is unusual. Methods: In this study we evaluated the presence of carbapenemase genes and the clonality of P. aeruginosa isolates obtained from a hospital over a 12-year period. A total of 127 isolates of carbapenem-resistant P. aeruginosa recovered from 109 patients feces (four samples), rectal swab (three samples), nasal swab (one sample) and anal abscess (one sample), were evaluated. Minimum inhibitory concentrations of the following antibiotics imipenem, meropenem and polymyxin E were determined by broth microdilution. The molecular profile of isolates was evaluated by pulsed field gel electrophoresis (PFGE). PCR for the following carbapenemase genes bla(IMP); bla(SPM); bla(VIM); bla(SIM); bla(NDM); bla(KPC); bla(GES) and nucleotide sequencing to confirm the enzyme gene types were performed and compared with the database available on the Internet (BLAST-http://www.ncbi.nlm.nhi.gov/blast/). Results: All isolates were carbapenem-resistant, their MIC50 and MIC90 were respectively 64 mu g/mL and 256 mu g/mL to imipenem and 32 mu g/mL and 256 mu g/mL to meropenem, all isolates except one (MIC = 8 mg/L) were susceptible to polymyxin E. The most frequent carbapenemase genes identified were bla(SPM) identified in 41 isolates (32%), followed by 10 with bla(kpc) and 5 with bla(VIM) (3.9%). All belonged to the class SPM-1 and VIM-2. In 2011, one isolate harbouring three carbapenemase genes (SPM-1, VIM-2 and KPC-2) that belonged to a new clone was identified in a hematopoietic stem cell transplanted patient. Then, 19 carbapenem-resistant P. aeruginosa were identified in an outbreak that occurred in the bone marrow transplant unit, all positive for SPM-1 gene, and 9 (47.3%) harbored both SPM-1 and KPC. Conclusion: Our findings showed that PCR for KPC gene should be performed to evaluate carbapenem resistance in P. aeruginosa and that this agent can harbor more than one carbapenemase gene. Attention should be focused on the possible rapid spread of KPC in P. aeruginosa isolates and for the fact that P. aeruginosa may become a reservoir of this transmissible resistance mechanism.