VIM-2 metallo-beta-lactamase in Pseudomonas aeruginosa strains from Zagreb, Croatia
Abstract number: p928
Bedenic B., Mazzariol A., Jarza-Davila N., Plecko V., Cornaglia G., Fontana R.
The worldwide spread of acquired metallo-beta-lacctamases (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 metallo-beta-lactamases (MBLs) in P. aeruginosa isolates from Zagreb, Croatia.
Materials and methods:
Hundred 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 Hospital 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 E 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 298 nm. Inhibition of enzyme activity was determined by measuring the residual carbapenemase activity after incubation of the crude extracts with 2 mM EDTA. Outer membrane proteins were prepared and analysed by SDS-PAGE. Pulsed field gel-electrophoresis (PFGE) was performed to determine if the strains were clonally related.
Eight out of 100 isolates were positive for MBLs by E test. Six strains were resistant to imipenem and meropenem. Resistance to ciprofloxacin was detected in 4 and to aztreonam in 3 of these strains. Six strains were identified as VIM MBLs producers by PCR. Sequencing of blaVIM genes revealed the production of VIM-2 beta-lactamase in all six strains. No IMP MBLs producers were detected by PCR. All VIM beta-lactamase producing isolates hydrolyzed imipenem. The enzyme activity ranged from 1.2 to 42 nmol/imipenem/min/mg of protein. Carbapenemase activity was almost completely inhibited by 2 mM EDTA. Loss of OprD2 protein was found in 4 strains. The strains were not clonally related by PFGE.
This investigation proved the occurrence of VIM-2 beta-lactamase among P. aeruginosa strains from Zagreb, 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.
|Session name:||XXIst ISTH Congress|
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