Carbapenem resistance mechanisms and molecular epidemiology of Pseudomonas aeruginosa in a Croatian hospital

Since carbapenems are important agents for the therapy of infections caused by multi-drug-resistant Pseudomonas aeruginosa (MDRPA), the development of carbapenem resistance is concerning as efficacious antimicrobial options are severely limited. This study was conducted to determine the underlying mechanisms that conferred the carbapenem resistance phenotype, and to examine the clonal relationships of pseudomonas isolates collected from hospitalised patients at Clinical Hospital Center Rijeka, over a period of 6 months from July to December 2009. A total of 38 non-repetitive P. aeruginosa isolates were identified, and serotyped by slide agglutination. Susceptibility to antimicrobials was carried out by the disc-diffusion method and E-test according to the EUCAST guidelines. Antimicrobials testing were meropenem, imipenem, doripenem, amikacin, gentamicin, tobramycin, ciprofloxacin, cefepime, ceftazidime, and colistin. The definition of MDRPA was established as an isolate intermediate or resistant to at least three drugs in the following classes: β-lactams, carbapenems, aminoglycosides, and fluoroquinolones. The transcription of the genes coding for the efflux proteins MexB, MexF, MexY, as well as outer membrane protein OprD, was analysed using real-time PCR. The whole genomic typing was performed by pulsed-field gel electrophoresis (PFGE using SpeI). The predominant serotypes were O11 (47%), and O1 (29%). Interestingly, isolates belonging to serotype Ol were more susceptible to antibiotics. However, all isolates were resistant to meropenem and doripenem, and in general, intermediary resistant to imipenem. MDRPA rate was 82%. Production of metallo-beta-lactamases (MBL), screened by Etest® MBL commercial strips was not detected. PCR analyses did not detect any MBLs or KPC genes. Significant down-regulation of oprD expression was observed in majority (82%) of the isolates. However the MexAB-OprM efflux system was overexpressed in 44%, MexCD-OprJ in 11% and MexEF-OprN in 26% of the isolates. Some isolates have increased expression of two efflux systems. Genotyping by PFGE revealed 47 distinct profiles which were grouped in 6 groups showing correlation with the hospital departments where the isolates were collected. According to our findings carbapenem resistance of P. aeruginosa in our hospital was due to combination of several mutation-driven mechanisms leading to OprD inactivation and overexpression of efflux systems. PFGE results imply that most of the infections are due to spread of the epidemic strains of the P. aeruginosa within the hospital.