engleski

VIM-2 beta-lactamase in clinical isolates of Pseudomonas aeruginosa from Zagreb, Croatia

VIM-2 BETA-LACTAMASE IN P. AERUGINOSA ISOLATES FROM ZAGREB, CROATIA BACKGROUND AND AIM The worldwide spread of acquired metallo-beta-lactamases (MBLs) in gram-negative bacilli has become a great concern. MBLs possess a broad hydrolysis profile that includes carbapenems and almost all extended-spectrum beta-lactams. The aim of this investigation was to characterize MBLs in P. aeruginosa isolates from Zagreb, Croatia. MATERIALS AND METHODS 100 P. aeruginosa isolates with reduced susceptibility to either imipenem or meropenem were tested for the production of MBLs by E test MBL. The strains were isolated during 2002 to 2005 at the Clinical Hospital Center Zagreb and University Clinic Merkur in Zagreb. The susceptibility to a wide range of antibiotics was determined by broth microdilution method The strains which gave a positive results in the test were chosen for the further investigation. The presence of blaVIM and blaIMP genes was tested by PCR. The amplicons were sequenced from both sides. Hydrolysis of 0.1 mM imipenem by crude enzyme preparations of beta-lactamases was monitored by UV spectrophotometer at 294 nm. Inhibition of enzyme activity was determined by measuring the residual carbapenemase activity after incubation of the crude extracts with 10mM EDTA. Outer membrane proteins were prepared and analysed by isoelectric focusing on polyacrilamide gel. RESULTS Six out of 100 isolates were positive for MBLs by E test. All six strains were resistant to imipenem and five of them were resistant to meropenem as well. Resistance to ciprofloxacin was detected in 5 and to aztreonam in 4 of those strains. Four strains were identified as VIM MBLs producers by PCR. Sequencing of blaVIM genes revealed the production of VIM-2 beta-lactamase in all four strains. No IMP MBLs producers were detected by PCR. The four VIM beta-lactamase producing isolates and 2 negative for VIM beta-lactamases hydrolyzed imipenem. The enzyme activity ranged from 1.2 to 42 nmol/imipenem/min/mg of protein. Carbapenemase activity was almost completely inhibited by 10mM EDTA. Four of the tested strains possessed an OmpD2 protein. CONCLUSIONS This investigation proved the occurence of VIM-2 beta-lactamase among P. aeruginosa strains from Zzagreb, Croatia. The strains harbouring VIM-2 beta-lactamase were resistant to all beta-lactam antibiotics, aminoglycosides and fluoroquinolones and pose a serious therapeutic problem in our hospitals. VIM-2 beta-lactamase with similar properties was previously described in Italy, Germany, United Kingdom, France and it can be concluded that it is widespread in Europe. MBLs do not hydolyze aztreonam and thus the resistance to this antibiotic is probably due to other resistance mechanisms such as loss of porins or efflux.

metallo-beta-lactamases; VIM-2 beta-lactamase; Pseudomonas aeruginosa; resistance

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