Evaluation of Imipenem/Imipenem+EDTA Disk Method for Detection of Metallo-β-Lactamase-producingKlebsiella pneumoniaeIsolated from Blood Cultures

Abstract

The objective of this study was to evaluate the imipenem (IMP) and IMP+EDTA (IMP/IMP+EDTA) disk method for the detection of metallo-beta-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 < or =0.5 to > or =16 microg/ml were examined. The MICs were determined by VITEK-2 (bioMerieux Vitek two, France). Disks of 10 microg 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 < or =0.5 microg/ml exhibited no differences in inhibition zone diameters (IZD) produced by IMP and IMP+EDTA disks, whereas the isolates with MICs > or =1 microg/ml showed an increase in IZD, ranging from 8 to 26 mm. All isolates with MIC values of > or =1 microg/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 microg/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 > or =1 microg/ml and/or IZD produced by IMP disk <19 mm should be tested for MBL production.

Metallo-β-Lactamase Detection: Comparative Evaluation of Double-Disk Synergy versus Combined Disk Tests for IMP-, GIM-, SIM-, SPM-, or VIM-Producing Isolates

“…Many authors have published distinct phenotypic techniques for screening MBL-producing isolates; however, these studies have important limitations, such as the following: (i) the inclusion of a small number of MBL-producing isolates, sometimes harboring the same type of enzyme (2, 36); (ii) the absence of molecular typing to exclude the influence of a single clone in the interpretation of their results (28); (iii) a lack of tests for evaluating the inhibitory effect of IMBL activity on bacterial growth and ␤-lactam hydrolysis (2,11,26,34,37); (iv) no inclusion of SPM-or GIM-producing P. aeruginosa isolates or SIM-producing Acinetobacter spp. isolates (11,14,17,21,25); (v) a lack of results stratified according to pathogen/species, in which the SN and SP values reflect the overall performance of all isolates (2,14,17); and (vi) accurate statistical analysis usually is not carried out to interpret and validate their results (2,11,21,37,38).…”

“…Most previous studies that evaluated MBL phenotypic detection were performed under distinct experimental conditions, jeopardizing the comparison of their results to those of others (2,11,14,15,21,25,26). The sizes of inhibition zones produced by ␤-lactam/ IMBL combinations may vary according to the way that the IMBL is incorporated into the ␤-lactam disks (1).…”

“…Although they are simple to perform and cheaper than genotypic methods, they have shown discordant results depending on the employed methodology, ␤-lactam substrates, MBL inhibitors (IMBL), and bacterial genus tested (11,14,17,21,26). In addition, SPM-, GIM-, and SIM-producing pathogens rarely have been evaluated by these studies.…”

Carbapenemases: the Versatile β-Lactamases

“…In a set of 19 K. pneumoniae isolates with imipenem MICs in the susceptible range of 1 to 4 g/ml, a metallo-␤-lactamase was suggested on the basis of disk testing with imipenem in the presence and absence of EDTA and was then confirmed by PCR as VIM-1 (161). A collection of five related K. pneumoniae strains with the VIM-1 gene demonstrated imipenem MICs ranging from 2 to 64 g/ml (susceptible to high-level resistance) (117).…”

Influence of Disk Preparation on Detection of Metallo-β-Lactamase-Producing Isolates by the Combined Disk Assay

“…Several phenotypic tests have been developed for MBL detection, such as the MBL Etest (AB Biodisk, Solna, Sweden) (9,14,18), double-disk synergy tests (1,7,9), combined disk (CD) assay (14,15,20), microdilution (11), and the Hodge test (7). All of these tests are based upon the ability of chelating agents, EDTA and thiol-based compounds, to inhibit the MBL activity.…”