Imipenem-EDTA Disk Method for Differentiation of Metallo-β-Lactamase-Producing Clinical Isolates of Pseudomonas spp. and Acinetobacter spp

Abstract

Rapid detection of metallo-␤-lactamase (MBL)-producing gram-negative bacilli is necessary to prevent their dissemination. The method using a disk with imipenem plus 750 g of EDTA differentiated all MBL-producing pseudomonads, and the sensitivity and specificity for acinetobacters were 95.7 and 91.0%, respectively. The imipenem-EDTA disks were stable for 12 and 16 weeks at 4 and ؊20°C, respectively.An increasing prevalence of carbapenem resistance mediated by acquired metallo-␤-lactamases (MBLs) is being reported, particularly for Pseudomonas aeruginosa clinical isolates in several countries (4, 6, 8, 9, 11-14, 17, 18). In Korea, approximately 10 and 50% of imipenem resistance in P. aeruginosa (8) and Acinetobacter spp. (19), respectively, are due to MBL production. The resistance may spread rapidly to various species of gram-negative bacilli, as the MBL genes reside in mobile gene cassettes inserted in integrons (3). The rapid detection of MBL-positive gram-negative bacilli is necessary to aid infection control and to prevent their dissemination (5). A PCR method was simple to use in detecting MBL-producing isolates initially (16), but it became more difficult with the increased number of types of MBLs.MBL activity is inhibited by chelating agents. Double-disk synergy tests using a ceftazidime disk and a 2-mercaptopropionic acid disk (1), or an imipenem disk and an EDTA disk (7), have been reported as a simple method to detect MBL-producing clinical isolates. However, occasional adjustment of the distance between the two disks is required to obtain optimal results (1, 7), as is the case with the double-disk test for the detection of extended-spectrum ␤-lactamase-producing isolates (2). For the phenotypic confirmation of extended-spectrum-␤-lactamase-producing isolates, inhibition zones are compared by using both ceftazidime and cefotaxime disks with and without clavulanic acid (10). The aim of this study was to determine the feasibility of using an imipenem disk with added EDTA to confirm MBL-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp.The MBL-producing gram-negative bacilli used in this study were 102 isolates of P. aeruginosa, 14 of Pseudomonas putida, 20 of Acinetobacter baumannii, and 3 of Acinetobacter genomospecies 3. All of the isolates were VIM-2 MBL producers except for one isolate of P. aeruginosa and five isolates of acinetobacters, which were IMP-1 MBL producers. MBL genes were detected by PCR, and MBL production was detected by the imipenem-EDTA double-disk synergy test as described previously (8). Imipenem-resistant or -intermediate but non-carbapenemase-producing isolates were included for comparison.Test organisms were inoculated onto plates of Mueller-Hinton agar (Becton Dickinson, Cockeysville, Md.) as recommended by the National Committee for Clinical Laboratory Standards (10). A 0.5 M EDTA solution was prepared by dissolving 186.1 g of disodium EDTA ⅐ 2H 2 O (Junsei Chemical, Tokyo, Japan) in 1,000 ml of distilled water and adjusting it to pH 8.0 by using NaOH. The m...

Distribution and phenotypic and genotypic detection of a metallo-β-lactamase, CphA, among bacteraemic Aeromonas isolates

“…Isolates that did not fit the criteria of 'susceptible', and belonged to the categories of 'intermediate' and 'resistant', were classified as being 'non-susceptible'. A .7 mm increase in the inhibition zone diameter in the presence of imipenem associated with EDTA in comparison with that of imipenem alone was interpreted as evidence of MBL production (Yong et al, 2002). By MBL Etest, the expression of MBL was reflected by a reduction of imipenem MIC by at least eightfold, the appearance of a phantom zone or deformation of the imipenem ellipse in the presence of EDTA.…”

Prevalence of ESBL and MBL Producing Pseudomonas aeruginosa in a Tertiary Care Hospital in Tirunelveli

“…Class B or the metallo-enzymes (MBL) are the most clinically threatening carbapenemases because they are capable of hydrolyzing all β lactams except aztreonem (Yong et al, 2002). Metallo beta lactamase is a binuclear zinc-dependent enzyme whose activity is susceptible to EDTA, where it is not inhibited by clavulanic acid, tazobactam, and sulbactam (Nordmann and Poirel, 2002).…”

“…To one of the discs 5 μL of EDTA solution was added. After 24 hours of incubation at 37°C, an increase of ≥7 mm in the zone diameter of Imipenem with EDTA compared to imipenem alone was considered to be a positive test for the presence of an MBL in imipenem resistant strains (Yong et al, 2002).…”

An evaluation of four different phenotypic techniques for detection of metallo-β-lactamase producing <i> Pseudomonas aeruginosa</i>

“…The IMP-EDTA combined disk test was performed as described by Yong et al [10] Test organisms were inoculated on to plates with Mueller Hinton agar as recommended by the CLSI. [9] Two 10 μg imipenem disks (Becton Dickinson)…”