Evaluation of Imipenem/Imipenem+EDTA Disk Method for Detection of Metallo- β -Lactamase-producing Klebsiella pneumoniae Isolated from Blood Cultures

The objective of this study was to evaluate the imipenem (IMP) and IMP+EDTA (IMP/IMP+EDTA) disk method for the detection of metallo- β -lactamases (MBLs) in clinical isolates of Klebsiella pneumoniae with various MIC levels to IMP. Forty-one blood isolates of K. pneumoniae with MIC to IMP ranging from ≤0.5 to ≥16 µ g/ml were examined. The MICs were determined by VITEK-2 (bioMerieux Vitek two, France). Disks of 10 µ g IMP with and without the addition of 0.5 M EDTA were used for the IMP/IMP+EDTA disk method. The E-test (AB Biodisk, Solna, Sweden) for MBL detection was also used. All isolates were examined for the bla VIM-1 gene by PCR and for clonality of VIM-1-producing isolates by pulsed-field gel electrophoresis (PFGE). All isolates with MIC values of IMP ≤0.5 µ g/ml exhibited no differences in inhibition zone diameters (IZD) produced by IMP and IMP+EDTA disks, whereas the isolates with MICs ≥1 µ g/ml showed an increase in IZD, ranging from 8 to 26 mm. All isolates with MIC values of ≥1 µ g/ml were found positive for the bla VIM-1 gene by PCR and for MBL production by the E-test, whereas none of isolates with MICs <0.5 µ g/ml was found positive by any of the tests. DNA restriction fragments generated by PFGE of VIM-1-producing isolates were classified in four main types. The IMP/IMP+EDTA disk method is simple to perform, sensitive, and specific for detection of MBL-producing K. pneumoniae clinical isolates. K. pneumoniae isolates with MICs of IMP ≥1 µ g/ml and/or IZD produced by IMP disk <19 mm should be tested for MBL production.